Although thoracic pedicle (TP) screw has gained increasingly popularity in the surgical treatment of adolescent idiopathic scoliosis (AIS) patients, questions remain about the accurate selection of entry point for TP screw placement in these patient. The main objective of the present study was to evaluate the accuracy of TP screw placement in AIS patients using the entry point identified by new landmarks.Thirty-four thoracic AIS patients treated with posterior TP screw instrumentation were included. All these TP screws were inserted through the entry point identified by new landmarks with free-hand technique. Postoperative CT scans were obtained to evaluate the screw position. The perforations of the pedicle were classified as grade 0 (no perforation), grade 1 (≤ 2 mm), grade 2 (2.1–4 mm), grade 3 (4.1–6 mm) and grade 4 (6.1–8.0 mm). Screws in grade 0, displaced either medially or anteriorly in grade 1 and displaced laterally in grades 1 to 2 were considered acceptable.Of the 495 TP screws inserted, 34 (6.9%) screws were displaced with 7 screws (1.4%) displaced medially, 20 screws (4.1%) displaced laterally and 7 screws (1.4%) displaced anteriorly (P < 0.05). Among the 34 displaced screws, 11 screws (32.4%) were considered as grade 1, 14 screws (38.2%) as grade 2 and 9 screws (29.4%) as grade 3 (P < 0.05). The overall rate of acceptable screws was 97.8%. No screw-related complication was noted.Our new method for selecting the entry point of TP screw in AIS patients is convenient and can achieve high accuracy of screw placement, which is worthy of being widely popularized. Nowadays, the application of thoracic pedicle (TP) screw has gained increasingly popularization in the surgical treatments of adolescent idiopathic scoliosis (AIS) patients due to its advantages such as better correction rate, shorter instrumentation length and less loss of correction as compared to the conventional internal fixations –3. HowevTo improve the accuracy of TP screw placement, various screw placement methods, such as free hand technique, funnel technique, fluoroscopy and computer-assisted surgery had been described –13. HoweTo facilitate the TP screws placement, we proposed an ideal pedicle entry point which can be easily identified by new landmarks. In this current study, we evaluate the accuracy of TP screws placements in AIS patients using this entry point with free-hand technique to verify the effectiveness and superiority of this novel method.A total of 34 consecutive thoracic AIS patients underwent posterior correction surgery using TP screws in our institution from August 2018 to June 2019 were included. There were 25 female and 9 male patients with a mean age of 15.0 years at the time of surgical treatment. There were 30 Lenke type 1 curves and 4 Lenke type 2 curves with the average main thoracic curve of 50.7°. All the TP screws were inserted by the first author (J.J.) using a new method of selecting entry point. All these patients had postoperative CT scans of the thoracic spine. This study was approved by the institutional review board in our hospital.After a standard midline incision, the posterior parts of the thoracic spine were exposed with sub-periosteal dissection laterally to the transverse process. The facet joints were meticulously cleaned of soft tissue to show the bony landmarks better. After cleaning the soft tissue, a curved border connecting lateral border of the superior facet and the transverse process can be found. The entry point was at the junction of the base of superior facet and the line through the intersection of the curved border and inferior facet, paralleling to the supraspinous ligaments Fig. . After iPostoperative CT scan was performed in all the patients. The screw perforations were evaluated on PACs image system . The perforations of the bony cortex were measured in millimeters and were divided into five grades: grade 0 (fully contained within the pedicle), grade 1 (perforation ≤ 2 mm), grade 2 (perforation 2.1–4.0 mm), grade 3 (perforation 4.1–6.0 mm) and grade 4 (perforation 6.1–8.0 mm). Screws in grade 0, displaced either medially or anteriorly in grade 1 and displaced laterally in grades 1 to 2 were considered acceptable.Extrapedicular technique of screw placement was used in the thoracic vertebra with extremely small pedicles. For the patients with dysplastic pedicle, the lateral wall of pedicle was intentionally breached and the screw passed through the pedicle-rib junction with the tip of the screw within the vertebral body. Therefore, the screw was fully contained in bony structures and was not considered as laterally displaced although the lateral wall of the dysplastic pedicle was perforated Fig. .Fig. 2ExStatistical analysis was performed by SPSS software for Windows . Qualitative variables was analyzed by chi-square test. Significance was established at the P < 0.05 level.A total of 495 TP screws were placed in all these cases. The average operation time was 290.1 min and the average blood loss was 870.6 ml. There were 34 screws displaced with the overall perforation rate of only 6.9%. Of these 34 screws, 7 (20.6%) were displaced medially, 20 (58.8%) were displaced laterally and 7 (20.6%) were displaced anteriorly. There were 11 screws (32.4%) in grade 1, 14 screws (41.2%) in grade 2 and 9 screws (26.5%) in grade 3 respectively. All the 9 screws in grade 3 were laterally displaced . Previous studies had demonstrated that the morphology of proximal thoracic spine was smaller than that of distal thoracic spine and the smaller morphology of proximal thoracic spine was most likely associated with an increased risk of anterior cortical perforation , 36. JiaThere were several limitations which should be mentioned in this study. Firstly, although our new method for selecting the entry point achieved high accuracy of screw placement, the perforation rate in the upper thoracic spine was as high as 15.7%, indicating that this method might be less useful in upper thoracic region. Secondly, the sample size was relatively small. Further study with large sample size is need to verify the accuracy of TPS insertion with our new method. Thirdly, as we know, the accurate TP screw placement depends not only the selection of entry point but also other important steps, such as proper use of awl, the convergence of the screw, and so on. All the TP screws were placed by an attending surgeon (J.J) who was experienced in screw placement. Whether the residents can achieve the same accuracy of screw placement with this method needs further study to evaluate.The intersection of the curved border and inferior facet is a reliable landmark for TP screw placement. Using this landmark, high accuracy of screw placement was obtained in AIS patients. We believe that this method is easy to learn for beginners and is worthy of being widely popularized.


There is a triple A-sitecation ordering with some degrees of anti-site disorder among sitesoccupied by 3d transition metals: [Y2]A[Cu0.8Mn0.2]A′[Ga0.8Mn0.2]A″[Mn3.6Cu0.2Ga0.2]BO12. It has the space group P42/nmc (no. 137) between 1.5and 873 K with a = 7.33884 Å and c = 7.66251 Å at 297 K. Despite anti-site disorder, it exhibitsa long-range ferrimagnetic order at TC = 115 K with the ordered moment of 2.19 μB at eachB site and 0.89 μB at the A′ or A″site. Magnetic moments are aligned along the c axis;all moments are ordered ferromagnetically at the B sites, and themoments at the A′ or A″ site are ordered in the oppositedirection. Cu2+ doping drastically changes magnetic propertiesas “parent” Y2MnGaMn4O12 just shows spin-glass magnetic properties without long-range ordering.Anisotropic thermal expansion was observed in Y2CuGaMn4O12: the lattice parameter a almostlinearly decreases from 1.5 K to TC andthen monotonically increases up to 873 K ; the parameter c monotonically increases from1.5 to 300 K and then decreases up to 600 K.A new member of A-site columnar-ordered A 2A′A″B4O12 family,Y2CuGaMn4O12, was prepared at highpressure and high temperature, with triple A-site ordering and ferrimagneticproperties.A new member of the A-site columnar-orderedquadruple perovskiteA The main driving forces of cation orderingare differences in cation sizes, oxidation states, and electronicstructures.2 In the case of B-site ordering, B cationshave basically the same octahedral coordination. Therefore, oxidationstate differences (and resulting differences in cation sizes) becomethe primary driving force. In the case of B cations, the charge differencecan reach as high as 6 . For example, cation ordering has significant effects on structural,magnetic, and magnetoresistance properties of Sr2FeMoO6.5Cation ordering in ABO+ and Th4+), but in most cases, it is just2  or 1 . Therefore, the number of examples ofthe oxidation-state-driven A-site cation ordering is significantlysmaller than the number of examples of such B-site cation ordering.2 In most cases, layered A-site cation ordering is realized when oneof the A cations is Li+ or even a vacancy.1 In some cases, layered A-site cation ordering is promotedby rock-salt B-site cation ordering .6For A cations, the charge difference does notexceed 3 in the Glazer notation.7 The coordinationenvironments of A cations can change drastically for large tilts ofthe a+a+a+ and a+a+c– types.The a+a+a+ system with large tilts produces A-site-orderedquadruple perovskites, AA′3B4O12,10 where the A′ site has a square-planar coordination as thefirst coordination sphere with eight much longer A′–Odistances. The a+a+c– system with large tiltsproduces A-site columnar-ordered quadruple perovskites, A2A′A″B4O12,11 where the A′ site has a square-planar coordination(similar to AA′3B4O12) andthe A″ site has a tetrahedral coordination as the first coordinationsphere. Therefore, when the electronic structures of the A, A′,and A″ cations are different, A-site quadruple perovskitescan be stabilized even for cations with the same oxidation states. Different electronic structures mean that some cations  show a very strong tendency for the first-orderJahn–Teller effect and square-planar coordination.In perovskites(if we consider just oxides and fully oxygen stoichiometricones), the coordination environment of B cations is fixed to the octahedralone. On the other hand, coordination environments of A cations aremore flexible. The coordination number of A cations can change from12 to 7 even for the usually observed 3B4O12 perovskitesare intrinsically fully ordered at the A-sites. Also, the number ofexamples of the AA′3B4O12 perovskitescould even be larger than the number of examples of fully ordereddouble A2BB′O6 perovskites, even thoughit is generally believed that B-site ordering is more common in thelatter.12 The coordination environments of the A2A′A″B4O12 perovskitessuggest that they have intrinsic triple A-site ordering. However,the triple ordering by different cations has been experimentally realizedjust in few examples so far, in R2MnGa(Mnx4–Gax)O12 (R= Ho and Y) solid solutions,14 R2CuMnMn4O12 (R = Dy and Y),15 and Ca(Mn0.5Cu0.5)FeReO6.16 In few cases of Sm2MnMn(Mn3Ti)O12 and RMn3O6,18 the A′and A″ sites are occupied by the same element in differentoxidation states . In the majority of cases, the A′ and A″sites are occupied by the same cation .21TheAA′2CuGaMn4O12, where theA, A′, and A″ sites are occupied by different cations,or in other words, with triple A-site ordering. This was achievedby selecting cations (Y3+/Cu2+/Ga3+) with different electronic properties . Y2CuGaMn4O12 demonstratescompletely different magnetic properties in comparison with the “parent”Y2MnGaMn4O12, which shows spin-glassmagnetic properties.14 Ferromagnetic (FM)ordering at the B sites and antiparallel ordering at the A′or A″ sites take place in Y2CuGaMn4O12 at TC = 115 K.In this work, we could prepare a new A-sitecolumnar-ordered quadrupleperovskite, Y22CuGaMn4O12 was prepared froma stoichiometric mixture of h-YMnO3, Mn2O3, Ga2O3 (99.9%), CuO (99.9%),and MnO1.839 (99.995%) at 6 GPa and about 1500 K for 2h in Au capsules. After annealing at 1500 K, the samples were quenchedto room temperature (RT) by turning off the heating current, and pressurewas slowly released. Single-phase Mn2O3 wasprepared from commercial “MnO2“  by heating in air at 923 K for 24 h. MnO1.839 was (another) commercial “MnO2“,whose oxygen content was determined by the thermal analysis and whichdid not contain any carbonate impurities. Single-phase h-YMnO3 was prepared from a stoichiometric mixture of Y2O3 (99.9%) and Mn2O3 by annealingin air at 1430 K for 60 h with several intermediate grindings. Theuse of h-YMnO3 (instead of Y2O3) allowed increasing the sample amount prepared in onehigh-pressure high-temperature experiment.Y1 radiationat 45 kV and 200 mA; 2θ range of 15–105°, a stepwidth of 0.02°, and a scan speed of 1°/min) from 293 to873 K and from 873 to 293 K with a step of 20 K . Synchrotron XRPD datawere collected from 100 to 850 K  and then at 297 K (without cooling/hearing guns)on the beamline BL02B2 of SPring-8 .22 High statistics data with the measurement time of 300 s were collectedat 100, 297, and 700 K; at all other temperatures, the measurementtime was 10 s . The sample wasplaced into an open Lindemann glass capillary tube (inner diameter:0.2 mm), which was rotated during measurements. The Rietveld analysisof all XRPD data was performed using the RIETAN-2000 program.23X-ray powder diffraction(XRPD) data were collected at RT on aRigaku MiniFlex600 diffractometer using Cu Kα radiation . High-temperature XRPD data were measured inair on a Rigaku SmartLab instrument  was loaded into a cylindrical 3 mm diametervanadium can and measured at 1.5 K from 5 to 150 K with a step of5 K and at 200 K. The Rietveld analysis of neutron data was performedusing the FullProf program25 against the data measured in detector banks at average 2θvalues of 58, 90, 122, and 154°, each covering 32° of thescattering plane.Neutron powder diffraction data were collectedat the ISIS-pulsedneutron and muon spallation sources at the Rutherford Appleton Laboratory(UK) using the WISH diffractometer located at the second target station.Cp at magnetic fieldsof 0 Oe and 90 kOe was recorded from 300 to 2 K by a pulse relaxationmethod using a commercial calorimeter (Quantum Design PPMS).Magnetic measurements were performed on SQUIDmagnetometers  between 2 and 400 K in applied fieldsof 100 Oe and 10 kOe under both zero-field-cooled (ZFC) and field-cooledon cooling (FCC) conditions. Isothermal magnetization measurementswere performed between −70 and 70 kOe at different temperatures.The specific heat Figure S1a); energy-dispersiveX-ray (EDX) spectra were taken on the same instrument . The cation ratio was 2:1:1:4 forY/Cu/Ga/Mn within 2σ.Scanning electron microscopy (SEM) images of fractured surfacesof pellets were obtained on a Hitachi Miniscope TM3000 (operatingat 15 kV)  into starting oxide mixtures containing R2O3 and Mn2O3 often resultedin the formation of the RMn2O5 impurity duringthe high-pressure synthesis of such perovskites.18 However in the case of Y2CuGaMn4O12, the formation of the YMn2O5 impuritywas suppressed, probably due to a specific combination of other cations.The introductionof Mn2+ (27 electrons) andGa3+ (28 electrons) are nearly the same. The same is truefor coherent neutron scattering lengths, 7.718 fm for Cu and 7.288for Ga. Therefore, it is very difficult to distinguish Cu and Ga (bynon-resonant X-ray diffraction) and refine their distribution at onesite, even with neutron diffraction. However, considering that theA′ and A″ sites in A2A′A″B4O12 perovskites are fundamentally different, wecan assume that Ga3+ cations cannot occupy the square-planarA′ site and would strongly prefer the tetrahedral A″site.26 On the other hand, Cu2+ cations would strongly prefer a square-planar site instead of atetrahedral site.26 X-ray scattering factorsof Mn differ enough from those of Cu2+ and Ga3+ to check cation distributions from synchrotron XRPD data. The existenceof anti-site disorder could be seen from reduced occupation factorsof the Cu and Ga sites if refined: g(Cu) = 0.943(7)and g(Ga) = 0.969(7) . The occupation factor of the Mn site was just slightlyhigher than 1: g(Mn) = 1.017(3). The anti-site disorderwas refined assuming the full occupation of the cation sites . The cation distributions wererefined to 0.72(4)Cu + 0.28 Mn for the A′ site and 0.90(3)Ga+ 0.10 Mn for the A″ site at 100 K, 0.72(3)Cu + 0.28 Mn and0.90(3)Ga + 0.10 Mn at 297 K, and 0.72(4)Cu + 0.28 Mn and 0.87(3)Ga+ 0.13 Mn at 700 K from the synchrotron data . The occupation factorof the Y site was unity within 2σ .X-ray scattering factors of Cug(Cu) = 0.692(7), g(Ga) = 0.688(8), and g(Mn) = 0.721(8)) and a more precise distribution of Mnand Cu/Ga atoms. The cation distributions were refined to 0.798(5)Cu+ 0.202 Mn for the A′ site and 0.800(5)Ga + 0.200 Mn for theA″ site at 200 K using the neutron diffraction data. To keepthe total chemical composition, anti-site disorder was assumed withthe B site. Therefore, this cation distribution, [Y2]A[Cu0.8Mn0.2]A′[Ga0.8Mn0.2]A″[Mn3.6Cu0.2Ga0.2]BO12, was fixed inall other refinements using both synchrotron and neutron diffractiondata. Refined structural parameters from neutron diffraction at 200K, primary bond lengths, and bond-valence sums (BVS)27 are summarized in Table S1. Experimental, calculated, and differencesynchrotron patterns of Y2CuGaMn4O12 at 100 K are shown in Figure S2. The crystal structure of Y2CuGaMn4O12 is plotted in The neutron scattering length of Mn (−3.73fm) differs significantlyfrom that of Cu and Ga. Therefore, neutron diffraction can give strongerevidence about anti-site disorder .6 octahedron appears as a Jahn–Tellerdistorted one with an unusual −Q3 distortion1 28 as the Mn–O1 distance is much shorter than the Mn–O2and Mn–O3 distances. However, the analysis of symmetry-adaptedatomic displacive modes30 showed that none ofthem individually can be associated with the cooperative Jahn–Tellerdistortion. This implies that the above-mentioned anisotropy of theMnO6 octahedra is an accidental effect of the superpositionof several distortion modes unrelated directly to the lifting of degeneracybetween the eg electrons of Mn3+.3+ cations are not found in tetrahedral sites.26 However, the ionic radius of Mn2+ cations (rIV(Mn2+) = 0.66 Å) is noticeablylarger than that of Ga3+ cations (rIV(Ga3+) = 0.47 Å).31 Therefore,the presence of Mn2+ cations increases the observed Ga–Obond lengths and decreases the resulting BVS values.The BVS values (at 200 K) of +3.10 for the Y sitewere close tothe ideal value of +3. The BVS values of +2.01 for the Cu site werealso close to the ideal value of +2. The BVS values of +3.33 for theMn site were close to the average oxidation state of +3.25 for cationsat this site. On the other hand, the BVS values of +2.60 for the Gasite noticeably deviated from the expected value of +3. The existenceof anti-site disorder can explain such an underbonded value. The oxidizationstate of manganese at the tetrahedral Ga site should be +2 as Jahn–Telleractive MnTC = 115 K resulted ina significant increase in intensity for some of the diffraction peaksin neutron diffraction patterns (k = 0. The absence of magnetic scatteringto the (00l) reflections points to a simple collinearmagnetic order with the spins polarized along the c-axis. Indeed, refinement of the magnetic structure assuming threecollinear sublattices for A′, A″, and B sites provideda very good fitting quality. The refinement yields the moment sizeof 2.19(1)μB for the B site and −0.89(3)μB and −0.05(3)μB for the A′and/or A′′ sites. The latter two values cannot be unambiguouslyassigned to the A′ and A″ sublattices either based onthe refinement quality or on the obtained site occupancies. Althoughthe A″ site is supposed to be non-magnetic in the fully orderedstructure, the presence of a small amount of Mn2+ (S = 5/2) can result in a finite ∼(5*0.2 = 1μB) moment for this sublattice, polarized by the A–Bexchange.Coolingbelow 2+ and Mn3+ in the A′ site, then only a smallnet moment ∼(4*0.2 – 1*0.8 = 0μB) shouldbe expected and it can be assigned to the nearly zero-moment (0.05μB) sublattice obtained in the refinement procedure. The reducedmoment size of the B-site Mn can be naturally attributed to the presenceof non-magnetic Ga3+ cations as well as magnetic Cu2+ with negative Cu–O–Mn superexchange interactions.Experimental, calculated, and difference neutron patterns of Y2CuGaMn4O12 at 1.5 K and the magneticstructure are shown in 27 values are summarized in The net magnetization of the A′ sublatticecan also be stronglydependent on the sign of the A–B exchange between Cu/Mn inthis position and the B-site Mn. If the sign is different for Cu2CuGaMn4O12 is shown in a almost linearly decreases from 1.5 K to TC = 115 K and then monotonically increases up to 873 Kwith almost linear behavior above 300 K. The c latticeparameter monotonically increases from 1.5 to 300 K and then graduallydecreases up to 600 K. The temperature behavior of parameter c above 600 K was slightly different for the synchrotronand laboratory XRPD data sets. Parameter c continuesto decrease for the laboratory XRPD data set but demonstrates a tinyincrease for the synchrotron XRPD data set. This difference couldoriginate from a completely different timescale of the measurements:the synchrotron data above 600 K were collected in about 20 min ,while the laboratory data above 600 K were collected in about 15 h,where larger changes in the oxygen content or stronger structuralrelaxation could take place. After heating, the lattice parameters(at RT) showed the same qualitative changes for both the laboratoryand synchrotron XRPD data sets—parameter a increases and parameter c decreases. The absolutevalues were again different because of different time scales. Suchchanges in the lattice parameters should be intrinsic and not relatedto zero shifts, as no changes in zero shift parameters were expectedfor the synchrotron data set, and the sample height was adjusted ateach temperature point for the laboratory data set. Irreversible changesin the lattice parameters during heating and cooling were also observedin another A-site columnar-ordered perovskite, NaSmMn2Ti4O12.20Temperature dependenceof the lattice parameters in YTC = 115 K due to the development of a strongFM moment  between about 250 and400 K. The Curie–Weiss temperature θ was positive [θ= +133.5(7) K], indicating a predominantly FM interaction betweenmagnetic ions. This is consistent with the FM structure at the B sitesfound from neutron diffraction.Temperature-dependent magnetic measurements ina small magneticfield of 100 Oe showed sharp rises in magnetic susceptibilities above M moment 6. A diffT = 2, 100, 130, 150, and 200 K , suggesting the presenceof short-range magnetic correlations. Above 200 K, the M versus H curves were nearly linear, as expectedfor paramagnetic states. Specific heat measurements .Magnetic-field-dependent magnetic measurementsat nd 200 K 7 showed urements 8 showed 2CuGaMn4O12 are significantly different from those of the“parent”Y2MnGaMn4O12 compound.14 No long-range magnetic ordering was found inY2MnGaMn3GaO12 and Ho2MnGaMn4O12 using neutron diffraction, and onlydiffuse magnetic scattering was observed.14 Magnetic measurements also suggested spin-glass-like magnetic propertiesof Y2MnGaMn4O12.14 This behavior was quite unexpected considering the highconcentration of magnetic Mn atoms at the B sites. All Mn atoms areformally trivalent in Y2MnGaMn4O12. Cu2+ doping introduces Mn4+ cations intothe B sites, and the double-exchange interaction between Mn3+ and Mn4+ cations at the B sites could be responsiblefor an FM structure observed at the B sites of Y2CuGaMn4O12 similar to ferromagnetism observed in Lax1–CaxMnO3 and Lax1–SrxMnO3.32 However,we note that ferromagnetism was only observed in Rx1–CaxMnO3 andRx1–SrxMnO3 with large R3+ cations  and, therefore, large Mn–O–Mn bond angles. Inthe case of small R3+ cations  andsmaller Mn–O–Mn bond angles, Rx1–CaxMnO3 compoundsdemonstrate spin-glass or antiferromagnetic transitions.38 Y2CuGaMn4O12 has very small Mn–O–Mnbond angles .35 Therefore,the presence of magnetic cations at the A′ and A″ sitesin Y2CuGaMn4O12 should also playan important role for the establishment of the FM structure at theB sites. This is consistent with the conclusion by Vibhakar et al.39 that the A–B exchange is the key ingredientfor understanding magnetic ground states of such columnar-orderedperovskites.The magnetic properties of Yd angles 2, even s2CuGaMn4O12 was realized through the selection of appropriatecationswith different electronic properties. Ga3+ has the 3d10 configuration, and it strongly prefers tetrahedral and octahedralenvironments.26 Ga3+ has neverbeen found in a square-planar coordination. Cu2+ has the3d9 configuration, and it shows a very strong tendencyfor the first-order Jahn–Teller effect and prefers distortedoctahedral environments, with extreme cases reaching square-planarand pyramidal coordinations.26The triple A-site ordering in Y42CuGaMn4O12. Triple A-site cation ordering was achievedby selecting Y3+, Cu2+, and Ga3+ cationswith different electronic properties—such a combination hadnot been reported before. The crystal structure was studied by synchrotronpowder X-ray diffraction and neutron diffraction. A small anti-sitedisorder was observed for the Cu, Ga, and Mn sites but not for theY site. Y2CuGaMn4O12 exhibits FMordering at the B sites with an antiparallel arrangement of magneticmoments at the A′ or A″ site.In conclusion, a new memberwas added to the A-site columnar-orderedquadruple perovskite family with the composition of Y


The use of dental implants in oral rehabilitations has become increasingly common, thanks to the safety and predictability of these rehabilitations. Unfortunately, dental implants, being alloplastic devices, are not free from biomechanical complications, especially in the case in which the connections are complex and involve several components. The aim of the study is to highlight what could be surface alterations using different screwing torques, or by repeating the screwing process several times. In this study, 40 passant screws  were examined under a Zeiss EVO LS 10 scanning electron microscope (SEM), operating with an accelerating voltage of 20 kV. Passant screws were subdivided into 4 groups: 30 Nmm tightening torque; maximum tightening torque; 2 times 30 Nmm tightening torque; no screwing, new ones (control group). There are no significant differences in the surfaces of the passant screws in SEM images, and the 100% of the passant screws is free of defects or fractures. Surely, further studies and investigations will certainly be needed to allow improvement of these devices. The use of dental implants has several advantages in dentistry. Additionally, the efficiency and clinical success of osseointegration have been reported by numerous articles, reporting that dental implants have become a common treatment for tooth/teeth loss , 2. GoinMeanwhile, over the last decade, an evolution of dental implants has been observed due to the development and research for improving of patient care . As a suInterestingly, there have been indicated different complications after several years of follow-up on a single implant prosthesis. Despite the high clinical success of the implant, various complications should be considered, such as fracture of the abutment screw, inflammation of the mucosa, and penetration of soft tissues, and the most common complication was abutment screw loosening –12. ShorAs well known, removable and fixed prostheses have been—and still are—used to restore chewing function and aesthetics as well. For this, dental implants have become an additional treatment option to replace missing tooth/teeth and the respective treatment concepts have reported high success rates of 99.7% and 89.5% in 10  and 20 ySurely, these success rates, especially in recent years, tend to be higher. This is because there has been an evolution in the materials and biomechanics of dental implants. It is enough to consider, for example, the large differences between dental implants, which provided for cemented abutments, and those which provided for screwed. Already in this there are big differences from a clinic and survival point of view. Also, with regard to the passing screws themselves, the subject of this study, it is said that rescrewing of a loosened abutment should not be done because it can increase the risk of a fracture of the abutment –18.In addition, early failures can have multiple causes, i.e., overheating of the bone during implant site preparation, lack of primary stability due to excessive site preparation or poor bone quality, overload, or parafunctions , reporteWith the evolution of implant types, different designs of the implant-abutment connection have been introduced . AccordiTo exceed some design limitations and bone loss of the external hex connection, internal connections were introduced, with the aim to provide better esthetics, joint strength, microbial sealing, and long-term stability –24. One Notwithstanding by the geometric configuration type, the prosthetic abutment will be fixed to the implant through a screw, leading an interface between the implant-abutment junction, while the inevitable gap that can be created between implant and abutment can cause biological complications by the bacteria infiltration and/or their metabolic products towards the connection . In addiMostly, the filtration degree between the implant and its prosthetic components depends on several factors, such as the geometry connection, an accurate fit between the components, the freedom of abutment rotation on the implant, the applied torque load to tighten the abutment, and the micromovements between the components of the implant-abutment complex , 26, 29.Group 1 with 30 Nmm tightening torqueGroup 2 with max tightening torqueGroup 3 with 2 times 30 Nmm tightening torqueGroup 4 the control groupAll samples were subdivided into 4 groups of 10 screws each  for fixing the prosthesis on the abutment. On the other hand, in the cemented prosthesis, the connection screw is used only to fix the abutment on the implant and then the prosthesis is cemented on the abutment.In any case, the problem that often arises in the use of the connection screw is to guarantee the reliability of the tightening, preventing it from breaking in use or unscrewing over time. The tightness of the screw depends on many mechanical aspects. Firstly, the screw is initially inserted into its housing and then screwing is initiated during which the rotation motion of the screw is transformed into a feed motion, which allows the screw to enter the threaded hole. This phase takes place without significant friction and therefore applying a very low torque. As for Figures 1Substantially, the accuracy of fit at the implant-abutment interface is an important key for long-term osseointegration and the success of dental implants. Mainly, maladjustment at the implant-abutment interface can produce biological  and mechanical complications (screw loose/fracture and abutment fracture) , 13, 15.In the literature, dental implant has been described in a lot the implant fracture, including, here, possible causes, such as bruxism, large occlusal forces, mechanical trauma, small implant diameter, material fatigue, and advanced bone loss, leading to reduced mechanical support of the implant. Other authors have described the risk of implant fractures with a low prevalence of 1%, thus considering a risk especially in the molar region .Recently, dental implant was published on implant fractures, evaluating 19,087 implants in 8,501 patients and observing fractures in 70 implants (0.4%), whereas, for zirconia implants, less data is available, and for the latter, some authors evaluated the fracture rate of zirconia implants in a systematic review . PrincipIn the daily clinic, different methods of implant removal are encountered, including, here, the use of the implant ratchet , piezoelFrequently, connecting screw is discussed in the fracture of prosthetic connecting screw that years ago was considered a fairly common complication in implantology , 12. WhiIn general, however, the ideal load is the one that allows to obtain the average effort and this depends on the material, the section of the screw, and the initial length of the screw.pitch errorseccentricityerrors in the profile Gear errors must also be considered. These usually are in relation to the manufacturing that can be classified intoA single pitch error consists of incorrect spacing between two consecutive teeth. For high-performance gears produced with advanced technologies, the maximum pitch error between adjacent teeth can be quantified in the order of a deviation of few microns of the pitch from the nominal value.μm. In practice, the effect of eccentricity is to generate, in the absence of “real” pitch errors, an “apparent” pitch error which varies cyclically at each rotation. The magnitude of the maximum “apparent” pitch error can be easily evaluated geometrically and is equal to 2ecsin (π/Z).The eccentricity of a gear determines a behavior similar to that of a series of pitch errors. Eccentricity for high performance gears should not exceed 20 In the errors in the geometry of the profile, we can say that with the current technologies for high performance gears, there are deviations from the “drawing” geometry of the order of the measurable quantities of the verification tools; therefore, in a study that neglects the effects of profile errors, it can be considered exhaustive (at least in the context of high-level design). How to solve and overcome the inaccuracy between thread and threaded pitch in order to reach a window that engages the whole screw and is not expressed only in a “traction” between the head and the first turn of the thread passing through the shaft of the screw itself?A first hypothesis is to increase the thickness of the thread, making fewer turns and, therefore, a larger pitch. This would increase the tightness of the screw as in reality it is only the first thread to enter into traction and guarantee the seal between the abutment and fixture and/or abutment and prosthesis (crown). Having a long screw with many turns, if “precision” is not guaranteed, does not actually favor sealing.A second hypothesis is to increase the precision between the thread of the screw and the threaded cable of the implant or abutment, by inserting “thread-locking” pastes or other gasket materials inside the threaded cable or on the screw thread before screwing. These could also play an important occluding function.In the third hypothesis, the tightening torque varies according to the engagement of the screw, increasing from 15 N to 35 N. Furthermore, the screw, once tightened to a maximum of 35 N, should be replaced at each check-unscrewing, because it loses its original capacity.In practice, they should be disposable so always change at each unscrewing or disassembly in the various controls. Furthermore, the screw should be tightened to the torque imposed by the manufacturers, on average between 65 and 75% of the yield strength of the screw, which must be reached in tightening, but never exceeded.It is also important to wait 10 minutes after the first tightening and repeat the locking operation. This is at least three times.Thus, Kreissl et al. found that the incidence of screw fracture over a 5-year period was up to 3.9%.Additionally, a screw fractures before any irreversible damage to the implant or prosthesis, serving as a safety system. Anyway, several studies have described techniques to save or remove fractured screws. About this issue, various dental implant companies have their own screw recovery kits, but most of these systems use aggressive motorized devices that increase the risk of implant damage, achieving a nonrestorable implant. The first step is the removal of the coronal portion of the fractured screw, gaining a visual access to see the remaining screw fragment. In cases when the fracture is sufficiently coronal, an explorer and a hemostat can be used; otherwise, an ultrasonic scaler with a blunt tip, without damaging the internal threads of the implant. It is important to use a flathead screwdriver to insert the created groove and attempt to rotate the screw fragment counterclockwise. Unfortunately, in cases when the internal threads of the implant are damaged during the removal of the fractured screw, the implant will become useless and not restorable, with the fate to be removed.Knowing in depth the behavior of these materials will certainly help to optimize their performance and improve their duration and therefore the predictability over time of rehabilitations in the biomedical field. This study aims to be preliminary for further analysis of this kind. Precisely for this reason, a fracturographic analysis was not carried out, which will be the result of subsequent studies. It could be interesting to test these screws under dynamic masticatory loads.This study brings to light a series of information regarding implant components, especially prosthetic ones. Once, it is possible to clarify what the protocols are as follows: they are clear protocols on how an implant failure or failures linked to prosthetic components that could be derived from materials. This observational study did not show any defect or fracture through SEM analysis in different groups, stating a 100% success rate. Surely, thanks to this study and to others who follow. It will be possible to optimize these materials, so as to make them more performing and guarantee greater safety and predictability in implant-prosthetic oral rehabilitation.


Engystomops pustulosus), albeit in different ways. Males arrived later in the night at their breeding sites in response to anthropogenic noise. ALAN, on the other hand, led to an increase in calling effort. We found no evidence that noise or light pollution either attracted frogs to or repelled frogs from breeding sites. Thus, anthropogenic noise may negatively affect calling males by shifting the timing of sexual signaling. Conversely, ALAN may increase the attractiveness of calling males. These changes in breeding behavior highlight the complex ways that urban multisensory pollution can influence behavior and suggest that such changes may have important ecological implications for the wildlife that are becoming increasingly exposed to urban multisensory pollution.Increasing urbanization has led to large-scale land-use changes, exposing persistent populations to drastically altered environments. Sensory pollutants, including low-frequency anthropogenic noise and artificial light at night (ALAN), are typically associated with urban environments and known to impact animal populations in a variety of ways. Both ALAN and anthropogenic noise can alter behavioral and physiological processes important for survival and reproduction, including communication and circadian rhythms. Although noise and light pollution typically co-occur in urbanized areas, few studies have addressed their combined impact on species’ behavior. Here, we assessed how anthropogenic noise and ALAN can influence spatial and temporal variation in breeding activity of a wild frog population. By exposing artificial breeding sites inside a tropical rainforest to multiple sensory environments, we found that both anthropogenic noise and ALAN impact breeding behavior of túngara frogs ( Urbanization exposes animal populations to sensory pollutants, including anthropogenic noise and artificial light, possibly affecting survival and reproductive success. We examined the effects of noise and light pollution on breeding behavior in a wild population of frogs. Frogs called later in the night with anthropogenic noise, potentially increasing predation and reducing reproductive success. Light increased the amount of frog calling, suggesting that light might play an important role in altering communication in urban frog populations. Urbanization has led to the rapid transformation of land across the globe, and the expansion of urban areas is expected to continue . These l(Zonotrichia leucophrys)  . Most wocophrys) . As senscophrys) .Sexual communication can directly be influenced by sensory pollutants, either resulting in changes in signal production or signal perception  breeding site use, 2) timing of breeding activity, and 3) male signaling traits. In addition, we recorded receiver activity, including mate attraction and predator attraction, to examine any potential fitness consequences.All experiments were performed at the Smithsonian Tropical Research Institute (STRI) facilities on Barro Colorado Island, Panamá, between July and August 2019. All experiments were performed under the approval of STRI (IACUC permit: 2019-0301-2022) and Autoridad Nacional del Ambiente de Panamá (SE/A-47–19). We placed 32 artificial breeding sites, which were plant dishes (Ø 50 cm), in the forest that were at least 10 m from the nearest trail to reduce any human-related disturbances and > 75 m from each other. Additionally, no calling activity was heard from areas surrounding the artificial breeding sites, indicating that breeding sites were isolated from any natural choruses. As this species requires water to call and to breed, we filled these artificial breeding sites with water collected from another active breeding site. All the artificial breeding sites were filled with water from the same source. After a total of 32 breeding sites were placed in the field, we conducted nightly surveys of these sites and noted any túngara frog activity . Once frog activity was documented, which occurred in 19 breeding sites, these sites were monitored either with video (Reconyx Ultrafire RX9 Trail Camera) or audio recorders (Wildlife Acoustics SM4 Songmeter). Frogs occupied breeding sites an average of 12.11 days after they were placed in the field.After four artificial breeding sites had become active, we installed setups to broadcast noise and light stimuli at each site and monitored these sites with our video recorders. One of four treatments was then presented to a puddle on a given night: Control (C), Light (L), Noise (N), and Light + Noise (L + N). Treatments were randomized and balanced within a block, ensuring that all the treatments were presented at sites within a single block on the same night. Treatments were changed each day at each site, so that over the course of a round, all sites in a block received all treatments. We included 16 of the 19 sites with breeding activity in our experiments. Experiments lasted between 18:00 and 23:00, starting approximately 40 minutes before sunset, culminating in 533.5 hours of video and audio data. Each block of sites received either one round (4 days) or two rounds (8 days) of treatment exposure. In one block, equipment failure caused us to repeat a night of trials.seewave (version 2.1.3) (tuneR (version 1.3.2) . We simulated urban anthropogenic noise by recording urban noise from four sites surrounding Panama City, Panama. We then selected two 5-minute segments from each site. In order to create a standardized, but still ecologically relevant noise stimuli, we synthesized “urban white noise”. This was accomplished with R packages n 2.1.3)  and tunen 1.3.2)  by analyn 1.3.2) .Videos were scored primarily by one experimenter (A.D.C.). The scorer was blind to the sensory treatment except for a small subset of videos in which frogs were out of view, in which case the audio file was checked for calling behavior. We scored breeding site use as the presence/absence of frogs within the breeding site for each recording period. Each recording period was a 10-minute interval, resulting in a maximum of 30 recording periods per night per site. Additionally, we counted the maximum number of frogs in the puddle at a given time. For the presence of calling, we scored the presence/absence of any calling within a recording period, along with the total number of calling frogs. If a frog left the view within a recording period and another frog entered, it was assumed to be the same frog unless there was strong evidence to assume it was a new individual . Mating behaviors (such as amplexus or nest building) were also scored, in addition to any predation attempts on the frogs. For the timing of breeding activity, we documented two distinct measures. Breeding site arrival time was calculated as the number of minutes from the start of the experiment (18:00 h) until the first adult frog entered the puddle. Calling start time was measured as the difference between when the first frog started calling and when the frog arrived at the breeding site. This measure was therefore independent of arrival time at the breeding site. In two instances, frogs were already present and calling when the experimental period began, and these data points for both breeding site arrival and calling start time were removed.For call analyses, we randomly selected and scored five recording periods in which calling was present per night per site. Call analysis was conducted by one experimenter (A.D.C.) and J. Mariën. If five or fewer recordings were available for a site on a given night, we analyzed all period. Because there were limited instances when more than one male called, and male calling behavior is highly influenced by calling competitors , we limiggplot2 (version 3.3.5) (lme4 package (version 1.1.27.1) (glmmTMB package (version 1.1.3) (dHARMA package (version 0.4.4)  . Models .1.27.1)  or the gn 1.1.3) . Becausen 0.4.4) . Call bop < 0.05, and additionally report all trends (p < 0.1).For model comparisons, we utilized a backward selection approach, and evaluated the influence of our fixed effects with likelihood ratio tests. For significant interactions, we examined if a combination of light and noise acted synergistically or antagonistically by comparing interaction estimates with the expected additive effects of light and noise . When thThe number of frogs within the breeding site at a given moment ranged from 0 to 7, with an overall average of 0.43 frogs/recording period. In intervals where at least one frog was present, calling occurred 59% of the time, with several instances of two males calling at the same time (4%). There were 14 instances of amplexus, split almost evenly among treatments. We documented no predation attempts throughout the duration of this experiment. Due to the low occurrence of amplexus and the lack of predation attempts, we were unable to further analyze either of these responses to ALAN or anthropogenic noise.p > 0.18) . The nature of this interaction was antagonistic, as the estimated additive effects of light and noise (+62 minutes) were 29 minutes higher than the observed interaction term (+33 minutes) , delaying the arrival time relative to the control (estimated effect: +33 minutes). Light as an independent factor also showed a trend to delay breeding site arrival time (estimated effect: +29 minutes), although this was not statistically significant  (p = 0.73). There was an additional trend showing a delay in call timing due to noise . We found no significant effect of light on calling start time (p = 0.41)  , Table 1 = 0.09) , Table 1 = 0.41) , Table 1n = 186 observations from 11 sites, χ2 = 1.13, p = 0.29), and noise in isolation did not alter calling effort . However, light did significantly increase calling effort  (p > 0.17) (p > 0.49)  , Table 2 > 0.17) , Table 2 > 0.49) , Table 2Both ALAN and anthropogenic noise showed effects on the breeding behavior of túngara frogs, although each sensory pollutant altered different aspects of breeding activity and sexual signaling. Anthropogenic noise was found to delay when frogs arrived at breeding sites, whereas ALAN exposure increased calling effort. Due to low sample sizes, we were unable to assess the effects of ALAN or anthropogenic noise on mate or predator attraction.Our study experimentally demonstrates that anthropogenic noise, and possibly ALAN, can alter the timing of breeding activity of anuran amphibians. Under control conditions, males always arrived within an hour after start of the experiment, whereas at noise polluted sites, males would on average arrive + 33 minutes later compared with the control, and sometimes up to three hours later. The presence of ALAN showed a similar trend, delaying the timing of breeding site arrival by + 29 minutes, although this was non-significant. In avian species, where most previous research has focused, the relative importance of specific sensory pollutants on the timing of breeding activity has shown mixed results and suggests that the effects of sensory pollutants on spatio-temporal breeding behaviors is highly species-specific . SeveralTrachops cirrhosus, eavesdrops on frog vocalizations to localize and capture calling male túngara frogs . Longer-term field studies will therefore provide not only a more ecologically relevant scenario, but may also reveal differences between short-term and long-term exposures that may be caused by different behavioral, physiological, or ecological mechanisms. Our results do, however, suggest that urban sensory pollutants can immediately alter a variety of breeding behaviors, which may in turn negatively impact animals through a mismatch in optimal timing of signaling, although this could be negated by potential positive effects of increased signaling effort. Understanding the overall fitness consequences of multisensory pollution in relation to breeding behavior remains a relatively open field, and one that must be further studied in order to properly grasp the evolutionary and ecological ramifications of urbanization.


WS), solar radiation, and temperature, at two dairy farms located on Hawai`i Island, Hawai`i, in Ookala named “OK DAIRY” and in Upolu Point named “UP DAIRY.” Temperature–humidity index (THI) and WS variations in the hottest four months (June to September) were analyzed to determine when critical thresholds that affect animal health are exceeded. Rainfall data were used to estimate the capacity of forage production in 6-mo wet (November to April) and dry (May to October) seasons. Future projections of temperature and rainfall were assessed using mid- and end-century gridded data products for low (RCP 4.5) and high emissions (RCP 8.5) scenarios. Our results showed that the “OK DAIRY” site received higher rainfall than the “UP DAIRY” site, favoring grass growth and forage availability. In addition, the “UP DAIRY” site was more stressful for animals during the summer (THI 69 to 73) than the “OK DAIRY” site (THI 67 to 70) as the THI exceeded the critical threshold of 68, which is conducive for high-lactating cattle. On the “UP DAIRY” site, the THI did not drop below 68 during the summer nights, which created fewer opportunities for cattle to recover from heat stress. Future projections indicated that air temperature would increase 1.3 to 1.8 °C by mid-century and 1.6 to 3.2 °C by the end-century at both farms, and rainfall will increase at the “OK DAIRY” site and decrease at the “UP DAIRY” site by the end-century. The agriculture and livestock industries, particularly the dairy and beef subsectors in Hawai`i, are vulnerable to climate changes as higher temperatures and less rainfall will have adverse effects on cattle. The findings in this study demonstrated how both observed and projected changes in climate support the development of long-term strategies for breeding and holistic livestock management practices to adapt to changing climate conditions.Proper knowledge and understanding of climatic variability across different seasons are important in farm management. To learn more about the potential effects of climate change on dairying in Hawaii, we conducted a study on site-specific climate characterization using several variables including rainfall, wind speed ( Global increase in surface air temperature have been predicted to reach +1.5 °C higher between 2030 and 2052 at the current rate of warming . As a reA previous analysis of rainfall in Hawai‘i showed a 5% to 10% decrease in the wet season (November to April) and a 5% increase in the dry season (May to October) over the past decades  = G (Genetics) + E (Environment) + G × E, where environmental factors include feeding, housing, ranch management, and ambient climates. Rainfall and temperature have a significant role in livestock management, particularly in dairy farming, as these factors directly affect feed availability and maintain homeostasis in animals . These cHawai`i has a long history of livestock farming. Published articles and reports indicated that dairy farming started in 1793, right after the beef cattle were introduced to the island . Beef anThe arrival of imported milk in the Islands initiated a sharp decline in cattle operations that continues today. There were 12,000 heads in the 1980s, which dropped down to only 2,000 heads in 2018 . AlthougThe limitation of land and the scarcity of feeds are the hindering forces that oppose the growth/revitalization of the dairy industry in Hawai‘i, which further advances the understanding and application of climates in precision livestock farming. However, future projections of climate change at local farms and how conducive it will be for livestock by the mid- and end-century are yet to be analyzed or discussed. Also, limited research has been conducted on farm-specific microclimatic predictions and their potential implications for developing long-term strategies to cope with the change. Therefore, this research focuses on two farms on the Hawai‘i island to characterize historical climate observations and future climate projections in the contexed dairy farm operations in the State. The overarching objective is to examine the suitability of dairy production in Hawai‘i in the context of future changing climatic conditions for those sites. This article highlights how additional heat stress and forage scarcity due to elevated temperature and reduced rainfall challenge animals’ production and health, forage growth, and ranch management.The dairy farm in Ookala (“OK Dairy” here forward) is located on Hawai‘i island, Hawai‘i, at a mean elevation of 399 m in the range of 47 to 686 m with a total of 2,500 acreages of land. The dairy farm in Upolu Point (“UP Dairy” here forward), also on Hawai‘i island, is located at a mean elevation of 135 m in the range of 0 to 323 m with 880 acreages of land . These tWS) data were downloaded from the open weather map (https://openweathermap.org/).A hundred years (1920 to 2019) of monthly rainfall maps , which wFuture rainfall projections were obtained using two available downscaled climate products with two emission scenarios (low emissions RCP 4.5 and high emissions RCP 8.5). Note that downscaling is a method used to relate information obtained from Global Circulation Models to a local scale that takes topography and microclimatic variability into account. In Hawaiʻi, two types of downscaled projections are available: Statistical downscaling (StDs) products  are avaiThe equation  to calcuwhere T is the ambient or dry-bulb temperature in °C, and RH is relative humidity expressed as a proportion .Average temperature and relative humidity data over the past 100 yr (1920 to 2019) were used to calculate monthly THI for the research sites. A THI of 68 is commonly used as a critical threshold to characterize heat stress for high-lactating cattle, while the THI of 72 is used for low-lactating cattle . The diuDFPQ) was used to calculate available forage. The DFPQ is defined as the available dry matter per acre per day per inch of rain. The DFPQ varies from place to place based on soil characteristics, grass type, and climate conditions. Dry matter produced in pasture per month is calculated as follows:Rainfall is positively correlated to forage production . TherefoIt is assumed that half of the forage remains in the ground, while the other half is grazed by cattle.2) than the “OK Dairy” site (211 to 229 W/m2), while the cloud ratio in “UP Dairy” site was lower (0.42) compared with “OK Dairy” (0.52). Other differences between the two farms included lower soil moisture ratio (0.43 to 0.87) and lower evapotranspiration  at the “UP Dairy” site than in the “OK Dairy” site, where the soil moisture ratio ranged from 0.66 to 0.71, and the evapotranspiration ranged from 763 to 1,457 mm. These climatic parameters are directly associated with moisture availability in soil and plants. Although rainfall and temperature are primarily considered in the climatic analysis, the above factors, directly and indirectly, affect the micro-climatic conditions at these locations, thus ultimately influencing livestock and agricultural management practices.In Hawai‘i, instep climatic gradients can occur over relatively short distances due to elevation, topography, and orientation to the prevailing winds. Climate characteristics over the past 100 years (1920 to 2019) were analyzed at the “UP Dairy” and “OK Dairy” sites to examine the suitability for dairy cow production now and in the future. The “UP Dairy” and “OK Dairy” sites were shown to have extreme climatic diversity , althougA comprehensive analysis of rainfall and temperature was conducted in the two sites with the data from the past 100 yr. Results indicated that average monthly rainfall and temperature patterns varied over the years at the “OK Dairy” site. The highest monthly rainfall at the “OK Dairy” site was received in March (521 mm), while the lowest monthly rainfall was in June 191 mm;  and S3. To assess the risk of heat stress on cattle production, monthly THIs were calculated for both locations using the average monthly temperature and humidity data between 1920 and 2019. Results showed that the THI ranged from 64.6 to 70.1 at the “OK Dairy” site, while it ranged from 67.8 to 73.5 at the “UP Dairy” site . The fouIt is known that WS is another important factor affecting the animal body’s thermal comfort. Thus, we analyzed how WS varied every hour during the four hottest months  and d anThe temperature was projected to have an increase of 1.3 to 1.8 °C by the mid-century and an increase of 1.6 to 3.1 °C by the end-century at the “OK Dairy” site . SimilarForage production is estimated for both wet and dry seasons based on a total amount of six-month rainfall. Results indicate that the dry season rainfall is 1.5 m, and the wet season rainfall is 2.4 m at the “OK Dairy” site. At the “UP Dairy” site, the dry season average rainfall was 450 mm, and the wet season rainfall was 716 mm . As the Rainfall at the “OK Dairy” site is expected to increase over time, while the “UP Dairy” site can be even dryer by the mid-century and the end-century. Empirical results for future forage production indicated that the monthly forage production in the “OK Dairy” site is projected to increase by 6% to 8% by mid-century and 13% to 19% by the end-century. Whereas, the forage production in the “UP Dairy” site is projected to decrease 5% to 8% by mid-century and 10% to 11% by the end-century . These pLivestock farming in Hawai‘i, particularly in lower altitudes, could suffer severe heat stress and forage scarcity due to observed increases in near-surface air-temperature elevated temperatures  and decrMilk production decreases sharply for every 1 °C increase in air temperature above 21 to 27 °C . AlthougWS plays a vital role in thermal regulation in dairy cows despite the air temperature rising above the critical zone . The hig2 concentration can positively bolster photosynthetic activity in C3 plants (The grass production was estimated based on monthly rainfall, and we concluded that the “OK Dairy” site was more productive than the “UP Dairy” site. Complex interactions between the frequency and intensity of rainfall govern the response of grazing land toward rainfall . Increas3 plants  with a c3 plants . TherefoThe animals’ pressure on pasture increases with a decrease in rainfall; therefore, the daily dry matter requirement of dairy cows in dry areas can be met by allowing the animals to graze in more acreages of land; however, the land is limited in Hawai‘i. For example, 100 acres of pasture in dry areas can supply forage to fewer cattle than the pastures in wet areas. Ranchers may have to reduce their herds, particularly in the farms that are in dry belts during the dry season, to balance the forage demand and supply. The grass production at the “UP Dairy” site may be severely affected during dry seasons due to a decline in rainfall patterns and elevated temperature by the end-century. In contrast, forage availability in the “OK Dairy” site perhaps is bolstered by increased rainfall. Thus, ranchers in Hawai‘i and tropical regions need to plan early to balance the animal population, cultivate hardy, drought-tolerant grass varieties, and manage groundwater supply to irrigate existing pastures to ensure continuous forage supply. In addition, adaptive response to climate changes involves selecting superior breeds with higher adaptation and implications of breeding strategies to develop genetically resistant breeds against diseases, parasites, and heat stress. Options are already available for dairy cattle as studies had identified the “slick hair gene” in cattle . AnimalsA 3 °C increase in annual temperature in Hawai‘i can significantly increase the THI, resulting in heat stress in dairy cows. Those areas suitable for operating dairy farms at present might not be appropriate after a few decades without proper modifications in housing, management, and other cooling interventions to dissipate additional heat loads. Those dairy farms in the upper critical temperature 20 °C;  can turnUnderstanding the animal and grassland responses to different environmental conditions is essential to successfully implementing strategies to alleviate the adverse effects brought by climate change. Although the “OK Dairy” site is more conducive to dairy cows than the “UP Dairy” site, both locations are vulnerable to the warmer world, where heat stress and periodic forage scarcity are projected to be more common in the future. The temperature rise by the mid- and end-century will likely cause a decline in animal productivity, reproductivity, and increased mortality unless mitigation is made. Above all, results demonstrated that the environment in the “OK Dairy” site is conducive for low-lactating cattle throughout the year, while the potential for animals to experience heat stress at the “UP Dairy” site is much higher. Therefore, to meet the needs of the cattle industry, it is critical to develop strategies in climate change adaptation to address housing, breeding, feeding, and pasture management so that sustainable dairy industries in Hawai‘i can be preserved for future generations. Thus, the findings from this article can be used as a guiding tool to rethink and develop long-term strategies to cope with the inevitable climate change and carry out sustainable farming practices in Hawai‘i and other tropical areas worldwide.txac064_suppl_Supplementary_Figure_S1Click here for additional data file.txac064_suppl_Supplementary_Figure_S2Click here for additional data file.txac064_suppl_Supplementary_Figure_S3Click here for additional data file.txac064_suppl_Supplementary_Figure_S4Click here for additional data file.txac064_suppl_Supplementary_Figure_S5Click here for additional data file.txac064_suppl_Supplementary_TablesClick here for additional data file.


Cyt bc1 and bcc complexes are constituents of the electron transport chain (ETC) of cellular respiration, and cyt b6f complex is a component of the photosynthetic ETC. Cyt bc complexes share in general the same Mitchellian Q cycle mechanism, with which they accomplish proton translocation and thus contribute to the generation of proton motive force which drives ATP synthesis. They therefore require a quinol oxidation (Qo) and a quinone reduction (Qi) site. Yet, cyt bc complexes evolved to adapt to specific electrochemical properties of different quinone species and exhibit structural diversity. This review summarises structural information on native quinones and quinone-like inhibitors bound in cyt bc complexes resolved by X-ray crystallography and cryo-EM structures. Although the Qi site architecture of cyt bc1 complex and cyt bcc complex differs considerably, quinone molecules were resolved at the respective Qi sites in very similar distance to haem bH. In contrast, more diverse positions of native quinone molecules were resolved at Qo sites, suggesting multiple quinone binding positions or captured snapshots of trajectories toward the catalytic site. A wide spectrum of inhibitors resolved at Qo or Qi site covers fungicides, antimalarial and antituberculosis medications and drug candidates. The impact of these structures for characterising the Q cycle mechanism, as well as their relevance for the development of medications and agrochemicals are discussed.Cytochrome (cyt)  Isoprenoid quinones are a family of natural electron and proton carriers present in prokaryotic cellular membranes, in the mitochondrial inner membrane and in the chloroplast thylakoid membrane . The varbc1 complex  oxidises quinol and transfer the electrons to cytochrome c oxidase  via the electron carrier protein cytochrome (cyt) c. The cyt c oxidase catalyses the reduction in dioxygen to water. NADH dehydrogenase and cyt bc1 complex couple quinone redox chemistry to proton translocation across the inner mitochondrial or bacterial cellular membrane to generate an electrochemical proton gradient and thereby power ATP synthesis  and ando sitaeroides  69]. In. Ino sit haem bH  in the d species . That thdb 1ppj) .i site of crystallised cyt bc1 complex. The power of cryo-EM to better cope with global or local protein dynamics brought forward a higher variety of quinone binding modes at the Qi site. In cryo-EM structures of mitochondrial respiratory chain supercomplexes, of the yeast supercomplex III2/IV . Th. Thi sit complex . The isoi site ubiquinone in mitochondrial cyt bc1 complexes, cryo-EM structures more recently provided additional information of ubiquinone positions in the context of supercomplexes, and previously unavailable structures of plastoquinone and menaquinone-occupied Qi sites which show considerably different architecture as compared with mitochondria cyt bc1 complexes. We anticipate that alternate reaction mechanisms will be required to accomplish quinone reduction and protonation at the Qi site in these complexes.Whereas high-resolution X-ray structures revealed detailed binding modes of the Qo and Qi site inhibitors was instrumental in studies of cyt bc1 complexes in order to explore the molecular basis of the Q cycle mechanism and to elucidate electron transfer pathways [o and Qi site, in particular that of stigmatellin [bc1 complexes were determined  are Qi site inhibitors -pyridones GSK 932121 and GW844520 [bc1 complex with bound compounds SCR0911 and GSK 932121 [Mycobacterium cyt bcc-aa3 complex with the tuberculosis drug candidate telacebec (Q203) [o site demonstrated this approach for bacterial cyt bc complexes and supercomplexes [bc complexes to develop novel human medications as well as agrochemicals [The use of Qpathways . Their bmatellin ,17, myxomatellin  were alltermined . Stigmat complex  provide ystrobin ,73 belonystrobin , a groupystrobin  which acystrobin . The Qo or crops . Atovaquor crops  is used or crops , and is or crops . Note thvailable ,83–85. Cis issue ,87. Intehibitors . This inGW844520 , MJM170 GW844520 , and a 2GW844520 . Althougc (Q203)  and withomplexes ,65. Cryoomplexes . In thishemicals .Thermus thermophillus cyt caa3 oxidase  permits the determination of higher order assemblies of protein complexes as well as structural dynamics directly in cells [Owing to the nature that membrane proteins are located in the lipidic compartments of the cell , structu, L.C.P. ), cryo-Eanodiscs ) and theanodiscs ; all resin cells . Althougin cells  will evebc complexes will contribute to an in-depth understanding of redox-driven proton translocation via the Q cycle and its regulation as well as support the design of fungicides, anti-malarial and anti-tuberculosis drugs.Structural biology research of cyt bc complexes from a wide spectrum of species as well as in different types of supercomplexes is important to expand the knowledge on conserved and species-specific binding modes of native substrates, drugs, and inhibitors at the quinone binding sites.Structural characterisation of cyt bc complexes is still lacking. We encourage that the cryo-EM specimens or crystals should be prepared in lipid environment.Structural information on the enzyme-substrate complex and defined catalytic states of cyt


This inhibitory effect was comparable to the effect produced by a standard AChE inhibitor physostigmine. One of the known fenugreek constituents, diosgenin, was also tested, and it also showed an AChE inhibitory effect in a concentration-dependent manner (11-190 μg/ml). Interaction between diosgenin and AChE was further investigated by molecular docking and molecular dynamics simulations for 100 ns, which showed that diosgenin interacted with the active-site gorge of AChE through hydrophobic, pi-pi stacking, and hydrogen bonds with various amino acids of the AChE enzyme.  When tested on the baseline of isolated tissues, Tfg.Cr was devoid of any activity (stimulant or relaxant) till 10 mg/ml. This is an interesting finding, keeping in mind that the fenugreek seeds are used to alleviate constipation and diarrhoea. When Tfg.Cr was tried for any potential AChE inhibitory activity, it did show an inhibitory effect in increasing concentrations (47-380  The results show that the fenugreek extract does not possess any GI stimulant or relaxant activity even though it is used traditionally in GI motility disorders. The extract and diosgenin could inhibit the AChE enzyme pointing towards their benefit to enhance the memory. Trigonella foenum-graecum Linn. (family: Fabaceae or Leguminosae), or “fenugreek” as commonly known in English, is a widely used medicinal plant. In Urdu or Hindi, it is called “methi.” An annual herb , 2, fenuFor centuries, fenugreek has popularly been used by herbalists and traditional healers of China and South Asia in several medical conditions, specifically for gastrointestinal (GI) and neurological issues. The seeds are consumed as is or processed (boiled or roasted) for their benefit in dyspepsia, colic, flatulence, diarrhoea, dysentery, anorexia , 2, 4, gMany of the benefits of this plant are linked to the chemicals that have been isolated from it. Fenugreek is known to contain steroidal saponins like diosgenin, tigogenin, trigogenin, and fenugreekine; alkaloids like choline and trigonelline; amino acids like histidine, arginine, tryptophan, and lysine; and vitamins like nicotinic acid , 4, 16. As noted above, several experimental and clinical studies on fenugreek scientifically elucidate its traditional uses. But still, a lot more work needs to be done to discover the many hidden benefits of this herb. This was the idea behind undertaking this endeavour. A 70% aqueous methanolic crude extract was prepared. This extract was tested pharmacologically on gastrointestinal (GI) smooth muscle preparations from rabbits and rats to see if there is any smooth muscle tone modulatory activity in the GI system, a potential acetylcholine- (ACh-) like effect that would lead us to believe in its memory-enhancing effects too. A substance acting like ACh can potentially have both GI stimulant and memory-enhancing effects due to an action similar to ACh on the cholinergic receptors in the GI tract and CNS. The extract was also tested for activity against the acetylcholinesterase (AChE) enzyme in vitro. This is because fenugreek is used to enhance the memory. Although the extract did not exhibit any effect on GI smooth muscle preparations, it did show an inhibitory effect on the AChE enzyme. The AChE inhibitory activity was traced back to its known chemical ingredient, diosgenin . InteracCare was taken to avoid any suffering to the animals used in this study. Experiments were performed ethically in strict accordance with lab animal handling specifications of European Community guidelines, EEC Directive 86/609/EEC. Local rabbits  and Sprague-Dawley rats  were used in this project. These were kept in the animal quarter at the Aga Khan University. The air was pathogen-free, and the temperature was controlled at around 23°C. The rabbits and rats had free access to water, although the food was withheld a day before the experiments. Food given to the animals was made of the following: fiber, table salt, white flour, sweetener, Nutri-vet L, potassium metabisulfite, grease, seafood, and powdered milk.2, 11.90 NaHCO3, 0.42 NaH2PO4, 1.80 CaCl2, and 5.55 glucose.Standard chemicals and reagents were obtained from Sigma Company, USA. These included acetylcholine (ACh), acetylthiocholine (ATCh), 5,5-dithiobis (2-nitro), benzoic acid (DTNB), diosgenin, electric eel AChE (type VI-S), histamine, nicotine, and physostigmine. The solutions and dilutions of these were made on the day of the experiment. To make Tyrode's physiological salt solution, chemicals were purchased from Sigma, USA, and Merck, Germany. Tyrode's was constituted as follows (mM): 2.68 KCl, 136.90 NaCl, 1.05 MgClFenugreek seeds (around a kg) were acquired from a supplier in Karachi, Pakistan. This was botanically identified by Mr. S. Ahmad. A specimen was kept in the herbarium of Natural Products Research Unit, Aga Khan University, for identification and cataloguing (# TF-SE-05-04-59). For making the crude extract, methodology described previously  was used2 and 5% CO2 at physiologic temperature. All changes in contractility were noted isotonically using Harvard equipment (oscillographs and force transducers). The tissues were left to stabilize for half an hour. The advantage of using rabbit jejunum is its ability to contract spontaneously , Tfg.Cr extract (20 μl and diluted in 5% ethanol), and AChE (20 μl) were dissolved and kept for 15 min at 25°C. Then, DTNB (10 μl) was added. The interaction of chemicals initiated with the addition of 10 μl of ATCh (0.71 mM). Breakdown of ATCh was quantified by determining synthesis of 5-thio-2-nitrobenzoate anion  using SpectraMax microplate spectrophotometer . The test for the substances under study was performed at least thrice. The preliminary difference was calculated as the difference in optical density/min and used in the following determination. The test reaction contained test samples, while the control lacked test substance. For comparison, the standard AChE inhibitor was physostigmine.The enzyme inhibitory tests were done in 96-well microtiter plates. Briefly, sodium phosphate buffer  module implemented in Desmond Schrödinger package to record the protein deviation, fluctuation, compactness, and hydrogen bond contacts with their occupancies, while ligand root means square deviation (RMSD), the radius of gyration (rGyr), torsional angle, molecular surface area (MolSA), solvent accessible surface area (SASA), and polar surface area (PSA) were also calculated during the production run of MD simulated time.The investigated docked AChE-diosgenin complex was further expedited to spurt molecular dynamics (MD) production run via the Desmond simulation package. MD simulations of the AChE-diosgenin docked complex were accomplished to reveal the capability of the diosgenin inhibition in the acetylcholinesterase. Receptor topology was generated, and the SPC water model with the specified periodic boundary conditions at a distance of 1.0 nm was set to create an aqueous environment. Furthermore, the solvated receptor charges were neutralized by the addition of required Nan” is observations) and the effective concentration producing 50% inhibition (EC50) with 95% confidence intervals (CI). The graphs were constructed and analyzed utilizing the GraphPad program . Statistical comparisons were made via two-way analysis of variance (ANOVA), and unpaired Student's t-test (p < 0.05) was taken as statistically different (GraphPad program).Data are shown as mean ± standard error of the mean  and nicotine (10 μM) both elicited an immediate stimulant effect on the resting spontaneous contractions  and histamine (10 μM) both exhibited a sharp spasmogenic response on the baseline of rat ileum tissues , inhibited the ACh-degrading enzyme AChE . Tfg.Cr showed a maximum of 48.8 ± 1.3% (n = 4) inhibition of the enzyme . This action of fenugreek aligns with its folkloric consumption to enhance memory [Another reason for using the isolated tissue preparations was to see if the extract can show an ACh-like spasmogenic effect. This is because ACh is a GI stimulant and a major neurotransmitter implicated in the pathophysiology of Alzheimer's disease . Chemicayme AChE . The EC5e enzyme . This ine memory . One stue memory  reports e memory  and cogne memory .μg/ml), inhibited the AChE enzyme . The EC50 of the suppressive action of diosgenin was 27.9 μg/ml . Diosgenin showed a maximum of 53.0 ± 0.4% (n = 4) inhibition of the AChE enzyme . We have earlier reported this AChE inhibitory effect of diosgenin [To determine the responsible compound for this AChE inhibitory action, we tested diosgenin , a steroE enzyme . This acE enzyme . The max/ml 24.9-.1, n = 4ΔG binding energy threshold of −10.7 kcal/mol was observed among all the ranked poses based on the estimated interaction energy. The top ranked conformation of diosgenin according to the highest ΔG binding energy was elected for molecular investigation. Auto-Dock Vina top most ranked pose output revealed that diosgenin established hydrogen-mediated, hydrophobic, and pi-pi stacking contacts with the aromatic amino acid residues of TcAChE of the peripheral anionic site , an anionic subsite (Phe330), catalytic site (Phe331), and acyl pocket residues (Phe288 and Phe290) as illustrated in A molecular modelling study was executed to interpret the binding mode analysis of diosgenin into the possible binding cavity of the TcAChE receptor 7B2W. The docking output with putative interaction site with the highest Molecular dynamic simulation data analysis is the basic quantitative evaluation parameter to analyze the stability, instability, deviations, fluctuations, compactness, and molecular interaction contact analysis of the protein and ligand via conformational geometries and dynamic vibrations and motions.RMSD was used to record the dislocation of the atoms present within the protein and ligand during MD simulated time with the initially generated frame of the pre-MD production run. RMSD plot analyzed for the protein backbone carbon-alpha atoms and the heavy atoms of the ligand during 100 ns of MD executed production runs. RMSD plot of TcAChE receptor 7B2W-diosgenin complex as depicted in The stability of the TcAChE-diosgenin was estimated via a protein-ligand contact histogram plot to check the overall protein-ligand contacts with the active-site residues in terms of interaction fraction pattern. These protein-ligand contacts were also inspected on the simulated trajectories of 100 ns of the production run. The interaction patterns follow H-bonds, hydrophobic, ionic, and water bridging-mediated interactions. H-bonding interactions play a crucial role in drug designing, but in this contrast, hydrogen bonding occurred as 0.74% with Asp72 and <0.1% with the Tyr121 and Tyr334. Hydrophobic contacts were established with Tyr70, Phe75, Trp279, Phe331, and Tyr334 while 0.55% hydrophobic contact with Trp279 and 0.3% and 0.35% fraction contact with Phe331 and Tyr334 were noted. Trp84, Tyr121, and Tyr334 possessed weak hydrogen bonding interactions. Asp72 showed maximum H-bond occupancy throughout the simulation, but in the last 25 ns, MD run. Its contact with diosgenin was diminished and converge into the water-mediated bridging interactions. At the same time, other residues which are mediating interactions with negligible hydrogen bond occupancies were observed negligible as implemented in A timeline representation of the above-mentioned receptor-ligand histogram was further analyzed to check the total number of interaction or molecular contacts in each trajectory record frame of all amino acid residues of the TcAChE, further clarified by the timeline exhibit of active-site residues of the TcAChE-diosgenin complex. The top panel of the timeline representation, as depicted in The ligand properties were studied carefully to understand the conformational repositions utilizing the reference of pre-MD production data. For this purpose, all atoms of the diosgenin used for the detailed analysis such as RMSD, compactness or extendedness, MolSA determination via 1.4 Å probe radius, SASA, the PSA were estimated as shown in 2 to 388Å2, and the equilibrium achieved around 385Å2 is visualized in 2O solvent was also noted. The SASA denoted that the raised value from 80 to 320 Å2 until 20 ns simulation and then afterward became persistent until the end of the simulation with the range of 200Å2 (2 and N atoms. The PSA was in the range of 50Å2-60Å2 throughout the simulation, but there were visible fluctuations occurred at 46 ns to 65 ns simulation period, while the equilibrium attained around 56Å2 inspected in The RMSD of the ligand showed stability throughout the simulation, while minor fluctuations were seen at 50-55 ns after reaching the equilibrium position after 5 ns. The ligand RMSD lay around 0.25 Å to 0.8 Å, while RMSD attained around 0.6 Å during the whole simulation shown in of 200Å2 . The PSADial plot illustrated that the torsion angle variations persisted throughout the whole MD production runs. The bar plot emphasized the dial plots by indicating the probable density of the torsions via utilizing each rotatable bond present in the ligand, as depicted in Molecular docking and post-MD results revealed that diosgenin penetrated in the active-site gorge region of TcAChE and showed almost similar molecular interactions in the static and dynamic mode. The peripheral anionic site aromatic residues Trp279 and Tyr334 showed hydrophobic or pi-pi stacking contacts with diosgenin in both pre- and post-MD stages. Subsequently, new molecular interactions were also observed and mediated hydrogen bond during 100 ns MD run with the hydroxyl moiety of diosgenin via Asp72 with the distances of 1.68 Å and 2.84 Å and Ser81 with the distances of 2.93 Å. The OH group interacted with Asp72- and Ser81-mediated H-bonding interactions could be the reason for enhancing the binding affinity of diosgenin in TcAChE.These results show that although fenugreek is so popularly used traditionally in GI disorders, it exhibited neither a stimulant nor a relaxant effect on the GI smooth muscles. This is an important finding as it rules out several variables and gives valid information to other researchers to try either a different solvent system for the extraction or different animal species to investigate any potential GI effects of this herb. Isolated smooth muscle preparations not only help to elucidate GI effects but also help look for ACh-like effects, which are also relevant in Alzheimer's disease pharmacology. Although it did not affect the isolated tissues, the extract did exhibit an inhibitory effect on the AChE enzyme assay, providing a rationale for its traditional consumption in memory loss. Diosgenin, a known chemical component of fenugreek, also showed a much more potent inhibitory effect on the AChE enzyme assay, indicating that it must be the responsible compound in fenugreek for this effect. After applying in silico approaches, we concluded that diosgenin permeated the active-site gorge of TcAChE and displayed substantially identical molecular interactions in the static and dynamic states obtained from molecular docking and post-MD findings.