Cortical Bone Mechanics Technology™ (CBMT) comprises certain improvements over a previous method known as Mechanical Response Tissue Analysis (MRTA). Both methods are dynamic 3-point bending tests intended for measuring the mechanical properties of cortical bone in living people. MRTA presented a theoretical potential for direct measurement of skeletal fragility, but it had acquired a reputation for error and fallen into disuse. We found sources of error in both MRTA data collection and data analysis. We describe here the fundamentals of MRTA, the major sources of error we found in MRTA, and our innovations for avoiding them.•Data collection at many sites across the mid-shaft of the ulna bone in the forearm.•Parameter estimation by fitting analytical complex compliance and stiffness transfer functions to empirical complex compliance and stiffness frequency response functions.•Optimization by selecting results from frequency response functions with the smallest deviations between fits to compliance and stiffness frequency response functions. Specification TableRecently, we reported the excellent accuracy of a novel non-invasive, radiation-free method for measuring the bending stiffness and estimating the bending strength of cortical bone in ulnas of living people . This meB = F/x is defined as the slope in the linear  range of the graphical relationship between force and displacement. To measure bending strength, the displacement is increased until the beam finally breaks. The peak moment Mpeak = FpeakL/4 prior to fracture is then defined as bending strength. QMT cannot be used clinically because the first step in QMT measurements of bone stiffness and strength is to remove the bone from the body.Our interest in MRTA derived from the long-known observation that quasistatic measurements of bone bending stiffness accurately predict quasistatic measurements of bone bending strength . In QMT QMT is quasistatic, because bone is a visco-elastic material. That is, the relationship between force and motion in bone depends strongly on the speed as well as the amount of displacement. Due to inertial effects, the relationship also depends strongly on the acceleration of displacement. By proceeding very slowly, QMT reduces the influences of these effects to negligible levels so that the bone’s elastic behavior  can be measured in isolation.MRTA was expected to overcome the clinical limitations of QMT by applying small, rapidly oscillating forces spanning a range of audio frequencies to the skin overlying a long bone. Such dynamic mechanical testing induces viscous and inertial as well as elastic effects in both the skin and bone, and enables the mechanical properties causing those effects  to be quantified in the resulting frequency-dependent vibration response. Then, the reasoning went, the bone’s strength could be accurately predicted from its stiffness.S, KS and DS) while the other is comprised of the mass stiffness and damping of the underlying bone .B is calculated by QMT is typically more than 100 N. In MRTA, the applied static force is typically less than 20 N, and the applied oscillatory force is typically 1 N. This static force is similar to the force on one’s fingertip when pressing an elevator button, and the oscillating force is similar to the force one feels holding an electric toothbrush or electric razor.The amount of force required to bend an ulna bone into the linear portion of its range of motion where KNote, too, that the accelerance FRF in The off-putting terms “real” and “imaginary” are nothing more an artifact of the history of mathematics when authorities in mathematics, including Rene Descartes, could not imagine how the square root of minus 1 Fsxs=Substituting the complex frequency s = jω (where ω = 2πf and j = (1)The complex accelerance FRF (a/F) is integrated twice to obtain the complex compliance FRF (x/F);(2)The complex compliance FRF is inverted to obtain the complex stiffness FRF (F/x);(3)the polynomical coefficients are obtained by fitting Eq. (4)the parameter values are then obtained by solving the simultaneous equations in Eq. (3).These relationships enable the values of the parameters of the mechanical model in Alternatively, the parameter values can be obtained in three steps by skipping Step 2 and fitting the inverse of Eq. and it is impossible for either the subject or the operator to know where to put it. Third, the bone being tested in B L3/48, where E = the elastic modulus of bone material, I = the cross-sectional moment of inertia of the bone’s geometry at the mid-span, and L = length of the bone). B ranged from 143 to 653 Nm2 with a mean of 320 Nm2 and a standard deviation of 199 Nm2 for a coefficient of variation CV of 62%.The property of a long bone that accurately predicts it’s bending strength is flexural rigidity  assumed in the derivation of Eq. 2. In our experience, such differences from averages of the paired estimates have ranged up to almost ±200%.The first source of error we encountered in MRTA data analysis related to fits of compliance and stiffness TFs to corresponding compliance and stiffness FRFs. Theoretically, the information in a rational function (y = numerator/denominator) and its inverse (1/y = denominator/numerator) should be the same. However, contrary to theoretical expectation, we observed that estimates of parameter values derived from fits of the complex compliance TF and its inverse (the corresponding complex stiffness TF) to a complex compliance FRF and its inverse (the corresponding complex stiffness FRF) often differed widely from each other. The values of EI calculated from fitting the complex compliance FRF with an extraneous peak in The second source of error we encountered was that estimates of parameter values varied greatly with the frequency range over which the TF was fitted. 2) to judge the goodness of fit of a TF to a FRF. MRTA practitioners commonly rejected fits to FRFs with R2 < 0.90 [2 > 0.90 accepted all the FRFs shown in 2 for the fits of TFs to the complex compliance FRFs shown in 2 were 0.9988 (left) and 0.9996 (right). In our experience testing forearms, we have rarely seen R2 as low as 0.95.The third source of error we encountered lay in the selection by previous practitioners of the coefficient of determination  to calculate the coefficients that would normally be obtained by fitting Eq. (2) to an empirical FRF. We then substituted those coefficients and values of frequency at 1 Hz intervals from 40 to 1200 Hz into Eq. (2) to obtain a synthetic complex stiffness FRF of the same size as an empirical FRF. We then inverted this perfect complex stiffness FRF to obtain the corresponding perfect complex compliance FRF. Then we fitted compliance and stiffness TFs to these perfect FRFs just as we would fit empirical compliance and stiffness FRFs. We found the estimated parameter values from the two fits to be identical and equal to the initial values substituted into Eq. (3). After confirming this with other sets of initial values, we concluded that the discrepancies are a measure of the extent to which an FRF deviates from the 7 parameter model shown in B but hardly at all in values of R2 crowded near 1. For example, values of RMS7 for the FRFs shown on the left and right of B estimated from the complex stiffness FRF  as a candidate goodness-of-fit statistic. We found that RMS7 varied widely (from 0.2% to 90.5%) for FRFs that varied widely in measures of KK_B,S in  and fromB at particular sites such as those plotted in correct values of KB remained to be determined, however.Two clarifications should be made at this point. First, the values of KB in 35 cadaveric human arms to measurements by QMT in the ulnas excised from those arms. Male and female arm donors ranged widely in age (17 to 99 years) and body mass index (13–40 kg/m2). Because QMT force-displacement curves were typically nonlinear, QMT measurements of bending stiffness were recorded as the peak tangent stiffness. Discrepancies between CBMT and QMT measurements over all 35 arms were minimized by requiring RMS7 ≤ 9% in fits of TFs to FRFs. After correcting for a fixed bias thought to be due to ligamentous binding of the ulna to the radius in the intact arm, the linear relationship between CBMT and QMT measurements of ulna EI approximated the identity line . Linear relationships (y = b0 + b1x) between ulna bending strength measured by QMT and ulna EI measured by CBMT (adjusted) and QMT (both R2 = 0.99) were indistinguishable from one another  vs 1.28 ± 0.03 (CBMT), p ≥ 0.80).As reported  in the c


Recently, it was also shown to be involved in horizontal transfer of a few known Xer-dependent mobile elements. Here, we show that plasmids of various Acinetobacter species, including clinically important strains, often contain multiple pdif sites that are mainly located within their accessory regions. Chromosomes of Acinetobacter strains may also contain additional dif sites, and their similarity with plasmid pdif sites is higher than with the main chromosomal site dif1. We further identify putative mobile genetic elements containing pdif sites on both flanks of adaptive genes and analyze their distribution in Acinetobacter species. In total, we describe seven mobile elements containing genes with various adaptive functions from permafrost strains of A. lwoffii group. All of them are also spread in modern plasmids of different Acinetobacter species including A. baumannii. We could not detect pdif sites and corresponding mobile elements in closely related bacterial genera, including Psychrobacter and Moraxella. Thus, the widespread distribution of dif modules is a characteristic feature of Acinetobacter species and may contribute to their high adaptability both in the environment and in the clinic.The  Acinetobacter strains are common everywhere due to their ability to adapt to various environments , was revealed in an A. baumannii plasmid pABVA01a. It was shown that this element (later designated dif module) is also present in a different context in another Acinetobacter plasmid, suggesting its horizontal transfer  are similar to the chromosomal dif1 site recognized by the dif/Xer system of site-specific recombination, which plays an essential role in the resolution of chromosomal and plasmid dimers . Initially discovered in plasmids from ancient (permafrost) strains of A. lwoffii it was also found in plasmids of modern Acinetobacter strains . These were (i) 38 strains of A. baumannii described by Acinetobacter species obtained from public databases before January 2018. Bacteria were grown in lysogeny broth (LB) medium or solidified LB medium (LA) at 30°C.analysis . In thisrpoB gene sequences with the reference Acinetobacter genomes1. The rpoB gene nucleotide identity level of 98–100% was regarded as evidence that the compared genomes belong to the same species.Species identification was confirmed by comparison of partial The genomes were sequenced with a Roche GS FLX genome sequencer  using the Titanium protocol to prepare shotgun genomic libraries. The GS FLX reads were assembled into contigs using the GS De Novo Assembler (Roche); protein coding genes were identified and annotated using the RAST web server. To identify potential plasmid contigs we used read coverage depth and annotation information, such as existence of genes for mobilization and/or replication of plasmids. Using 454 assembly graph file (output of the GS De Novo Assembler) we extended and merge our potential plasmid contigs with each other. Some additional merging of contigs was done using PCR. All manually assembled regions were checked using PCR. A plasmid was considered as finished if it was circular according to the 454 assembly graph and PCR check. The 454 reads were mapped back to the finished sequences using 454 GS Reference Mapper, and read mapping was visualized in IGV browser and manually inspected for potential missassemblies.Acinetobacter species (dif sites by BLAST software. The XerC/XerD (3916–3943 bp) and XerD/XerC (6899–6926) pdif sites of plasmid pM131-6 [NC_025120] were used as a reference. Only sites that were at least 75–80% identical to the reference were taken into account. The same algorithm was used in the search for the main and additional dif sites in Acinetobacter chromosomes.A collection of plasmids isolated from different  species  was scre2 was used. Similarity searches were performed using BLAST  in this region candidates for mobile modules were revealed. At the final stage, using the Blast software, the distribution of the identified modules among the ancient (isolated from permafrost) and modern strains of Acinetobacter’species was investigated. The presence of the same module with an identity level of 98–99% in different regions of different plasmids (contigs) isolated from Acinetobacter strains belonging to different species was regarded as evidence of its mobility.Identification of 6 cells per ml) were plated onto LA supplemented with Na2TeO3 (Tel) or tBHP. The concentrations tested were as follows: Tel – 0.01; 0.02; 0.04 and 0.1 mg/ml and tBHP – 0.05; 0.1; 0.2; 0.3 and 0.4 mM. The plates were incubated at 30°C for 24 h and visually inspected. In each case, three independent experiments were performed.The level of resistance was determined by the agar dilution method . OvernigThe accession numbers for nucleotide sequences of the plasmids deposited in this work in the GenBank database are as follows: CP032112.1 (pALWED1.2); CP032113-CP032116.1 (pALWED1.4-1.7); CP032117.1-CP032124.1 (pALWED2.2-2.9); CP032287.1-CP032289.1 (pALWED3.2-3.4); CP032290.1 (pALWED3.6); CP032102.1 (pALWEK1.1); CP032105.1-CP032107.1 (pALWEK1.2-1.4); CP032108.1- CP032111.1 (pALWEK1.6-1.9); CP032103.1 (pALWEK1.10); CP032104.1 (pALWEK1.11).pdif sites in Acinetobacter plasmids, we studied plasmids found in strains of various Acinetobacter species, divided into three groups : (1) plasmids of permafrost strains of A. lwoffii and A. pseudolwoffii isolated and studied in our group  together with plasmids from A. lwoffii ZS207 , while very small plasmids (<6 kb) as a rule did not contain them.The presence of pe groups . In most or more . Most pdpdif sites were found both in very large and in middle-size plasmids (22–400 kbp)  .dif sites relative to the backbone and accessory plasmid regions, we analyzed their distribution in four plasmids, two middle size (pALWED2.3 and pOXA58-AP_882) and two large (pALWED2.1 and pXBB1-9), each containing more than 10 sites  and 8 plasmids from 6 strains of Moraxella (1313–44215 bp) described by dif sites were detected in any of these plasmids. Therefore, the presence of a large number of pdif sites may be a unique feature of Acinetobacter plasmids.To our knowledge, there is currently no data on the distribution of typical pdif modules was performed on the set of plasmids from five permafrost strains from our collection . In total, putative dif modules were detected in 12 plasmids; 6 of them contained one module; 4 contained two modules, and 2 contained three different modules   , we reveuspA dif .Acinetobacter strains belonging to different Acinetobacter species  in four permafrost plasmids, pALWED1.4 from A. lwoffii ED23-35, pALWED2.1 from A. lwoffii ED45-23, pALWED3.6 from A. pseudolwoffii ED9-5a and pALWEK1.1 from A. lwoffii EK30A (terC (978 bp), encoding resistance to tellurium. We also found the terC dif module in two modern plasmids and several contigs from modern strains belonging to different species of Acinetobacter , and the chromosomal terC gene was not flanked by dif sites.However, the plasmidic and chromosomal terC dif module, we compared the level of tellurium resistance of the permafrost strains of A. lwoffii and A. pseudolwoffii containing the terC dif module with the strain A. lwoffii VS15 that lacked this module in its plasmids. The results of three independent experiments showed that strains ED23-35, ED9-5a and EK30A have a significantly higher resistance level (MIC from 0.04 to 0.1 mg/ml) compared to strain VS15 (MIC < 0.01 mg/ml); whereas strain ED45-23, characterized by very slow growth, does not differ from VS15. Thus, additional studies are needed to confirm the contribution of the terC dif module into the tellurium resistance of corresponding host strains.To determine the functional activity of the dif sites (both 768 bp long). Both modules contained two genes, one (297 bp) encoding a killer protein and another (291 bp) – an antidote protein. The first module was located in large plasmid pALWED2.1; the second was detected in medium-sized (22771 bp) plasmid pALWED2.3 and large plasmid pALWEK1.1. The difference in nucleotide sequences of the modules was 4%.Most prokaryotic chromosomes as well as many plasmids contain toxin-antitoxin (TA) systems consisting of a pair of genes that encode 2 components, a stable toxin and its cognate unstable antitoxin. TA systems are also known as addiction modules . In partadd dif modules in plasmids and contigs of modern strains belonging to different Acinetobacter species   . In mostohr (429 bp) and a second gene ohrR (432 bp) encoding transcription regulator from the MarR family of transcription factors . However, the size and the boundaries of this module differed between the plasmids. We therefore analyzed its structure and distribution in more detail.The ohr/ohrR genes with plasmid localization were first found by A. baumannii ATCC 19606. The authors showed that A. baumannii ATCC 19606 is resistant to the organic peroxide-generating compounds cumene hydroperoxide (CHP) and tert-butyl hydroperoxide (t-BHP). We analyzed the genome of pMAC and detected typical pdif sites on both flanks of the ohr genes suggesting that pMAC contains an ohr dif module. The permafrost and clinical versions of the ohr dif module are overall closely related (96% nucleotide identity) but reveal some specific differences: (i) the ohr module from the modern strain is 5 bp longer than the ancient one (1068 bp vs.1063 bp); (ii) both modules contain identical ohrR genes but their ohrA genes differ by 3%; (iii) region adjacent to the XerD/XerC site contains multiple substitutions. We therefore designated the ancient variant ohr1 dif and the modern variant ohr2 dif and studied the distribution of each of them separately.The ohr1 dif module in the plasmids of modern strains, it was revealed among unassembled genomic sequences of various Acinetobacter species, in most cases in small-size contigs  contained numerous copies of the ohr 2 module, 14 in the first plasmid and 8 in the second.Interestingly, two plasmids  or lacking (group 2: ED45-23 and ED9-5a) this module, to tert-butyl hydroperoxide (t-BHP) . The results of three independent experiments demonstrated a higher level of resistance of group 1 strains (MIC = 0.2–0.4 mM) in comparison with group 2 strains (MIC = 0.1 mM).To test the functional activity of the sulP encodes sulfate permease (484 aa) that perform transport of inorganic anions into cells (The gene to cells . It is ato cells .sulP gene. The first module sulP dif (2159 bp), first found in our previous work , detected in this work in the plasmid pALWEK1.10 from A. lwoffii EK30A, contained the uspA gene encoding a universal stress protein in addition to sulP. The sulP genes from the two modules are related (the identity level of 75%). The distribution of both dif modules in modern plasmids (contigs) is presented in sulP-uspA dif module in different plasmids.We revealed two different mobile elements containing the ous work , containdif module containing the kup gene (1878 bp) encoding a potassium uptake transporter , we revealed additional dif sites in most completely sequenced circular chromosomes of different species of the Acinetobacter genus. In total, we analyzed 20 sequenced chromosomes of 13 Acinetobacter species, and only one of them did not contain additional dif sites.To reveal whether the presence of multiple terminus . Surprisdif sites varied from one to five and they were located far from dif1 . Interestingly, we found two Acinetobacter strains that contained multiple additional dif sites in their chromosomes, Acinetobacter indicus SGAir0564 with 14 additional chromosomal dif sites [CP024620.1] and Acinetobacter sp. SWBY1 with 15 additional dif sites [CP026616.1].As a rule, the number of discovered dif sites were found occasionally when studying the distribution of different dif modules. In particular, the terC dif module, almost identical to that from plasmid pALWED1.4 (99% identity), was revealed in the chromosome of A. indicus SGAir0564 (CP024620.1). It was flanked by typical pdif sites (XerC/D and XerD/C) and was located at 2363397–2364617 bp, far from the main chromosomal dif1 site at position 1526563. Similarly, we found a variant of the add dif module in the chromosome of the Acinetobacter sp strain SWBY1 [CP026616.1].It should be mentioned that strains containing multiple chromosomal dif sites, we compared their sequences with the main dif1 sites from Acinetobacter chromosomes and with pdif sites from A. baumannii and A. lwoffii . In total 109 pdif sites from 23 plasmids of A. lwoffii, 115 pdif sites from 33 plasmids of A. baumannii, 20 main dif1 and 40 additional chromosomal Acinetobacter dif sites were analyzed.To get insight into the origin and specific structure of the additional chromosomal dif sites were revealed in all studied groups  are very close to each other in their consensus structure and form two clusters with different consensus sequences of five central nucleotides; (ii) additional chromosomal dif sites differ significantly in their structure from the main chromosomal dif1 site, especially in their central part, while (iii) they are more similar to the plasmid pdif sites from both clusters. It should be mentioned that the structure of the main chromosomal dif1 sites of Acinetobacter corresponds well to the consensus of dif sites from Proteobacteria chromosomes  pdiomosomes . Thus, idif sites and associated dif modules in plasmids and chromosomes of various Acinetobacter species including pathogens, commensals, and free-living bacteria belonging to different phylogenetic branches of the genus. Neither the additional copies of dif sites nor the mobile elements containing them are commonly found in other bacteria, even closely related to Acinetobacter, suggesting that their presence is a characteristic feature of the Acinetobacter genus. It should be noted that previous studies were mainly limited to the description of dif modules with antibiotic resistance genes  strains revealed novel dif modules encoding different adaptive functions, including the resistance to chromium  and organic peroxides (ohr1 dif), potassium (kup dif) and sulfate (sulP dif) transporters, toxin-antitoxin systems (add dif). All the detected genetic elements also have almost identical copies in the plasmids of modern Acinetobacter strains belonging to different species. It should be emphasized that the dif modules described in the present work were revealed in plasmids from only five studied permafrost strains of A. lwoffii group, and many more dif modules containing other adaptive genes are likely to be found in the future.The results obtained in this study suggest that multiple dif modules can be distinguished: (i) mobile elements that encode metabolic and regulatory functions  and ensure survival under natural conditions, and mainly found in plasmids of environmental strains of Acinetobacter (described in this study); (ii) mobile elements found in both clinical and natural strains ; (iii) mobile elements containing genes of antibiotic resistance and found almost without exception in plasmids of clinical strains of Acinetobacter, mainly A. baumanni (Acinetobacter module encoding a putative virulence factor (Sel1-repeat protein) described by A. baumannii plasmids, similarly to classical dif modules.Based on their distribution, three groups of known baumanni . It is adif modules described in this work and those containing antibiotic resistance genes described previously . Thus, it can be proposed that the additional chromosomal dif sites may participate in the movement of dif modules encoding antibiotic resistance and various adaptive functions from plasmids to the chromosome and backward. Indeed, a dif module with the Acinetobacter-specific carbapenemase gene OXA-58 was recently found inserted into the XerC/XerD site of a prophage in the Proteus mirabilis chromosome (Acinetobacter antibiotic resistance genes within a bacterial chromosome.We demonstrated that multiple hia coli . Thus, aromosome . As far dif modules is to increase the fitness of their respective bacterial hosts in their habitats. Our results suggest that the dif modules possess the properties of mobile genetic elements capable to translocate between plasmids and chromosomes in Acinetobacter strains belonging to different species. Additional research is highly needed to assess the vast diversity of dif modules in this clinically important genus and reveal their contribution to horizontal gene transfer and plasmid variability.It is natural to assume that the common role of all types of AM, AB, and MP conducted the sequencing and assembly of plasmids. All authors performed the bioinformatic analysis. MP performed the experiments. SM, MP, and AM wrote the manuscript. All authors contributed to the discussion and provided comments on the manuscript.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.


International Journal of Molecular Sciences is a member of the Committee on Publication Ethics (COPE) and takes very seriously the responsibility to enforce strict ethical policies and standards.Experimental results from  were fou


Chestnut is a popular food in many countries and is also an important starch source. In previous studies, physicochemical properties of starches have been compared among different Chinese chestnut varieties growing under different conditions. In this study, nine Chinese chestnut varieties from the same farm were investigated for starch physicochemical properties to exclude the effects of growing conditions. The dry kernels had starch contents from 42.7 to 49.3%. Starches from different varieties had similar morphologies and exhibited round, oval, ellipsoidal, and polygonal shapes with a central hilum and smooth surface. Starch had bimodal size distribution and the volume-weighted mean diameter ranged from 7.2 to 8.2 μm among nine varieties. The starches had apparent amylose contents from 23.8 to 27.3% but exhibited the same C-type crystalline structure and similar relative crystallinity, ordered degree, and lamellar structure. The gelatinization onset, peak, and conclusion temperatures ranged from 60.4 to 63.9 °C, from 64.8 to 68.3 °C, and from 70.5 to 74.5 °C, respectively, among nine starches; and the peak, hot, breakdown, final, and setback viscosities ranged from 5524 to 6505 mPa s, from 3042 to 3616 mPa s, from 2205 to 2954 mPa s, from 4378 to 4942 mPa s, and from 1326 to 1788 mPa s, respectively. The rapidly digestible starch, slowly digestible starch, and resistant starch ranged from 2.6 to 3.7%, from 5.7 to 12.7%, and from 84.4 to 90.7%, respectively, for native starch, and from 79.6 to 89.5%, from 1.3 to 3.8%, and from 7.1 to 17.4%, respectively, for gelatinized starch. Starch is synthesized and stored as semi-crystalline granules in seeds, fruits, and some metamorphic roots and stems ,2,3. StaCastanea spp. which belong to the beech family Fagaceae [Castanea mollissima) in China and Korea, Castanea sativa in Europe and South America, and Castanea crenata in Japan [Chestnuts are the fruits of Fagaceae . In 2016Fagaceae . The cheFagaceae . The majin Japan . The ediin Japan ,14. TherC. sativa [The dry kernel of a chestnut contains about 50% starch and is an important starch source ,16,17,18. sativa ,20,21 an. sativa ,26,27,28. sativa ,24,25,27In this study, nine Chinese chestnut varieties were planted in the same farm and grew under the same conditions. Starches were isolated from fresh kernels of mature nuts, and their physicochemical properties were systematically investigated using multiple physicochemical techniques, including X-ray powder diffraction (XRD), attenuated total reflectance Fourier transform infrared (ATR-FTIR), small-angle X-ray scattering (SAXS), thermogravimetry analysis (TGA), differential scanning calorimeter (DSC), rapid visco analyzer (RVA), and some routine physicochemical measuring methods. Our objective was to investigate and compare the physicochemical properties of starches from different varieties under the same growing conditions. This study may provide some information for the quality breeding of Chinese chestnut varieties and their utilization in food and non-food industries.C. sativa varieties [The fresh nuts of nine Chinese chestnut varieties are presented in arieties ,17. The The C. sativa starches [The iodine absorption spectra of nine starches were determined, their derived parameters including maximum absorption wavelength, iodine blue value, and absorbance ratio of OD 620 to OD 550 (OD620/550) had no significant differences among nine starches (data not shown), but the apparent amylose contents ranged from 23.8 to 27.3% . Some listarches ,20,21. TB-type starches, and the other starches had typical C-type starches  and 1100–900 cm−1 (C-O-H bending). The bands in the region 1100–900 cm−1 have been shown to be sensitive to changes in starch structure, especially the bands at 1045 and 1022 cm−1. The band at 1045 cm−1 is linked with order/crystalline region, and that at 1022 cm−1 arises as a result of absorption by stretching modes in amorphous structure [−1 can reflect the ordered degree in the starch [−1 (−1 ranged from 0.61 to 0.65 (The ATR-FTIR spectra of nine Chinese chestnut starches are shown in tructure . The abse starch . The ninarch [−1  and thei to 0.65 .−1) to account for variations in sample concentration, leading to the spectra being at the same relative scale and directly comparable. The main scattering peak at scattering vector of about 0.066 Å−1 is thought to arise from the periodic arrangement of alternating crystalline and amorphous lamellae of amylopectin and corresponds to lamellar repeat distance or Bragg spacing [The semi-crystalline growth rings formed by alternating amorphous and crystalline regions can be detected by SAXS. The SAXS patterns of nine Chinese chestnut starches are shown in  spacing . The pea spacing ,38. The C. sativa starch [The thermal properties of nine Chinese chestnut starches were measured with a DSC. The DSC thermographs are presented in a starch  are lowea starch  reporteda starch , the staa starch .The pasting properties of starch are important functional properties and determine the quality and utilization of starch in food industry. The pasting properties of starch have been analyzed in some papers ,42. The The TGA curves are usually used to evaluate the differences in thermal stability of starch caused by structural distinctions. The TGA curves of nine starches are presented in Aspergillus niger amyloglucosidase (AAG) (The native and gelatinized starches were in vitro digested by both porcine pancreatic amylase (PPA) and se (AAG) . Starch −1 in FTIR spectrum (IR) and the absorbance ratio of OD620 to OD550 in iodine absorbance spectrum (OD620/550). The gelatinization onset, peak, and conclusion temperatures  were significantly positively correlated to the PI and negatively correlated to the IR and OD620/550. The pasting peak and breakdown viscosities (PV and BV) were negatively correlated to the gelatinization temperatures. The digestion of gelatinized starch was positively correlated to the starch iodine blue value (IBV), apparent amylose content (AAC), PV, and BV, and negatively correlated to the PI and gelatinization temperatures  were obtained from the Institute of Forest and Pomology, Beijing Academy of Agricultural and Forestry Sciences, China. They grew in the Shachang Village, Miyun, Beijing, China, in 2017. Their mature nuts were harvested and stored at 4 °C before use.Nine popular varieties  of Chinese chestnut . Twenty nuts were randomly chosen and weighed. Their seed coats were removed, and the obtained kernels were weighted and sliced into small pieces. The kernel pieces were dried at 110 °C for 2 h and 80 °C for 2 days, and then weighted and ground extensively. The flour was passed through 100-mesh sieve, and its starch was determined using the colorimetric method of anthrone-Ho et al. .w/v) sodium metabisulfite aqueous solution in a home blender . The homogenate was squeezed through four layers of cheesecloth, and filtered with 100-, 200- and 300-mesh sieves. After the starch suspension was settled overnight at 4 °C, the starch precipitate was dispersed in 0.2% (w/v) NaOH and centrifuged  . The starch precipitate was repeatedly treated three times using 0.2% NaOH, and then washed three times with distilled water and two times with anhydrous ethanol. Finally, the starch precipitate was dried at 40 °C, ground into powders, and passed through a 100-mesh sieve.The kernels were homogenized with 0.5% (w/v) starch suspension in 50% (v/v) glycerol was observed and photographed using a polarized light microscope  under normal and polarized light. The dry starch powder was photographed using an environmental scanning electron microscope  following the method of Cai et al. [The 1%  following the method of Cai et al. . The tes−1, and the deconvolution was carried out using a half-width of 19 cm−1 and an enhancement factor of 1.9.The starch was analyzed using a FTIR spectrometer  with a DTGS detector equipped with an ATR cell following the method of Cai et al. . The speThe lamellar structure of starch was analyzed using a SAXS instrument  equipped with Vantec 2000 detector and pin-hole collimation for point focus geometry. The test settings were described previously by Cai et al. .Five milligrams of starch and 15 μL of deionized water were mixed and sealed in an aluminum pan for 2 h at room temperature. The sample was scanned using a differential scanning calorimeter  from 25 to 130 °C at 10 °C/min.Three grams of starch was dispersed in 25 mL deionized water. The sample was analyzed using an RVA . The starch-water slurry was dispersed by rotating the paddle at 960 rpm for the first 10 s and then a constant speed of 160 rpm. The temperature program was set as follows: holding at 50 °C for 1 min, heating to 95 °C at 12 °C/min, maintaining at 95 °C for 2.5 min, cooling to 50 °C at 12 °C/min, and holding at 50 °C for 1.4 min.The TGA of starch was carried out using a TGA system  following the method of Zhang et al. . The sam3, 2.5 mM CaCl2, PPA (4 U/10 mg), and AAG (4 U/10 mg)) at 37 °C using a ThermoMixer C  with shaking at 1000 rpm. The released glucose was determined using a glucose assay kit .The native and gelatinized starches were digested by both PPA  and AAG  following the method of Lin et al. . The gelThe one-way analysis of variance with post hoc contrasts by Tukey’s test and the normal distribution of all measuring data by Shapiro-Wild test were carried out using the SPSS 19.0 Statistical Software Program . The physicochemical properties with the significance of normal distribution over 0.05 were used for principle component analysis using Minitab V. 16.0 software .Chinese chestnut kernels have high starch content and are an important starch source. Starches from different varieties growing under the same conditions showed some differences in their physicochemical properties. The lamellar peak intensity was negatively correlated to the ordered structure. The gelatinization temperatures were significantly positively correlated to the lamellar peak intensity and negatively correlated to the ordered structure. The pasting peak and breakdown viscosities were negatively correlated to the gelatinization temperatures. The digestion of gelatinized starch was positively correlated to the apparent amylose content and the pasting peak and breakdown viscosity, and negatively correlated to the lamellar peak intensity and the gelatinization temperatures. This study could provide some information for the quality breeding of Chinese chestnut varieties and their applications.


The surgical procedure for orthotopic liver transplantation (OLT) is well standardized, and most groups use the retrohepatic caval preservation or piggyback technique to improve hemodynamic tolerance. However, when a discrepancy between the site in the right upper quadrant of the liver recipient and a small graft is present, this technique can provoke a rotation on the axis of the vena cava and cause an occlusion of the suprahepatic vein drainage. This problem can be detected intraoperatively, and several methods have been described to resolve it by placing different devices to correct the position. Early withdrawal may cause the development of clinical hepatic congestion with ascites unresponsive to medical treatment. We present three cases of OLT who developed obstruction of the venous drainage solved intraoperatively with the placement of a Sengstaken-Blakemore tube. As a novelty, prior to the withdrawal of the device, a transjugular hemodynamic study was performed to ensure the correct position of the liver with adequate venous drainage. Orthotopic liver transplantation (OLT) represents the best definitive treatment for patients with cirrhosis and some variants of tumors such as hepatocellular carcinoma. The surgical procedure is well standardized, and most groups have adopted the piggyback technique which consists in suturing the proximal vena cava stump of the graft to the common suprahepatic vein ostium in the recipient , 2. WithHowever, if there is a discrepancy between the size of a small graft and a liver recipient with an oversized site in the right upper quadrant , using the piggyback technique can cause a liver malposition due to rotation on the axis of the vena cava which produces an occlusion of the suprahepatic veins resulting in an acute Budd-Chiari syndrome, with ascites, impaired liver function, and risk of graft loss .Usually, this problem is detected intraoperatively, and several methods have been described in the literature to resolve this malposition/rotation by placing different devices such as Foley probes , breast Removal of these devices such as the Sengstaken-Blakemore tube or the Foley catheter is usually performed after a few days without any control, assuming that the liver graft has already formed adhesions to the parietal peritoneum and is permanently well positioned.Early withdrawal may cause the development of clinical hepatic congestion with ascites unresponsive to medical treatment, which may sometimes require the placement of prostheses into the vena cava to rectify the anastomosis increasing the risk of further complications .The transjugular hemodynamic study is a safe method to quantify portal hypertension. It can be performed in an ambulatory setting with local anesthesia and with a very low complication rate. The procedure has been previously described by our group , 10. A vWe present three cases of OLT who developed obstruction of the venous drainage of the liver caused by excessive rotation and were solved intraoperatively with the placement of a Sengstaken-Blakemore tube. As a novelty, prior to withdrawing the device, a transjugular hemodynamic study was performed to verify the absence of a transanastomotic gradient and the correct venous drainage of the liver. The report is according to the CARE guidelines .The first case is a 51-year-old male with HCV+genotype 3a chronic liver disease. OLT is indicated because of the presence of a hepatocellular carcinoma (HCC) complying with the Milan criteria. The physical measures of the patient were as follows: height, 176 cm; weight, 90 kg; abdominal perimeter, 109 cm; and thoracic perimeter, 111 cm. A first OLT was performed with a graft from an 81-year-old donor. In the first 48 hours, the patient developed severe primary malfunction, for which a second OLT was indicated. He received a graft from a second donor with the following measures: height, 160 cm; weight, 68 kg; abdominal perimeter, 93 cm; and thoracic perimeter, 95 cm. The recipient/donor weight ratio was 1.3. The surgery was performed following our habitual technique with vena cava preservation (piggyback) with an anastomosis between a common stump from the recipient suprahepatic veins and the donor's cava after completely resecting the cuff of the previous graft's vena cava.In the course of the intervention, a rotation of the liver was observed due to a discrepancy between the graft's size and the space in the hepatic fossa that impaired the venous drainage causing hepatic congestion. To solve this problem, a Sengstaken-Blakemore tube was placed under the liver in the right hypochondrium with the gastric and esophageal balloons filled, thus correcting its rotation and achieving an immediate improvement in the venous drainage .In the 7th postoperative day, a liver hemodynamic study was performed through the jugular vein. The THCG was measured with the balloons full and empty, observing that it did not increase after deflating them, so subsequent removal of the catheter was deemed safe. The patient was discharged 4 weeks after the surgery with optimal liver function, without ascites or other associated complications.The second case is a 39-year-old patient with familial amyloid polyneuropathy with associated gastrointestinal involvement. OLT was performed with a “domino” strategy (the explanted liver was used as a graft for a compatible liver recipient with decompensated cirrhosis). The physical parameters were as follows: height, 1.90 cm; weight, 90 kg; abdominal perimeter, 100 cm; and thoracic perimeter, 107 cm. An OLT was performed using a graft from a donor with an abdominal perimeter of 99 cm and thoracic perimeter of 95, with a weight of 68 kg, and a size of 163 cm. The recipient/donor weight ratio was 1.3.th postoperative day, a direct hepatic hemodynamic study was performed through the jugular vein checking the absence of a THCG after deflating the Sengstaken-Blakemore tube's balloons proceeding to its withdrawal. The patient was discharged with a correct hepatic function, without ascites or other associated complications.A piggyback technique was performed as described. After reperfusion of the graft, hepatic congestion was observed due to poor venous drainage in relation to excessive rotation of the anastomosis of the suprahepatic vena cava. This was corrected by placing a Sengstaken-Blakemore tube in the right hypochondrium with both balloons inflated raising the liver, thus modifying the position of the liver and achieving adequate venous drainage. The postoperative course was uneventful. In the 7The third case is a 51-year-old male patient with a 3 cm HCC. In the period on the waiting list, 2 treatments were performed with transarterial chemoembolization (TACE). Arterial spasm during one of them occurred as a complication. The physical parameters were as follows: size, 179 cm; weight, 82 kg; abdominal perimeter, 102 cm; and thoracic perimeter, 106 cm.OLT was performed with the piggyback technique. He required acute retransplantation in the first 48 hours due to arterial thrombosis.The second graft came from a donor with the following parameters: size, 165 cm; weight, 63 kg; abdominal perimeter, 95 cm; and thoracic perimeter, 90 cm. The recipient/donor weight ratio was 1.3. The second procedure was performed following the same piggyback technique, and the arterial intake necessitated the interposition of an aortohepatic graft. The disproportion of size conditioned a rotation on the axis of the caval anastomosis causing venous congestion. A Sengstaken-Blakemore tube with both balloons inflated was used to correct the position. A first liver hemodynamic measure was carried out in the 6th postoperative day, which showed an augmented THCG (6 mmHg) after deflating the gastric balloon of the Sengstaken-Blakemore tube. The balloon was again inflated, correcting the hepatic venous hypertension, so it was maintained in place for 72 hours more. A second measure was then performed showing a favorable result after emptying the balloon, so the tube was withdrawn without incident. The patient was discharged two days later in good clinical condition.The surgical procedure for liver transplantation has become highly standardized, and most groups perform vena cava preservation with the piggyback technique, using an end-to-side anastomosis from the donor's caval stump to a cuff tailored with the recipient's suprahepatic veins. One of the reported complications is acute obstruction of the hepatic venous drainage, which is usually associated with technical problems related to anastomotic stenosis or liver malrotation. This event is more frequent when using the piggyback technique (3-4%)  than witTwo of the three cases presented here occurred after acute retransplantation. In these occasions, a longer venous cuff is usually obtained by leaving in place the previous anastomosis and using the failed graft's proximal cava, thus facilitating the second anastomosis and reducing the necessity of partial clamping of the recipient's cava . HoweverBy the same causal mechanism, this regional Budd-Chiari syndrome can occur after a right hepatectomy due to a rotation of the left hemiliver, impairing the venous drainage through the trunk of the left suprahepatic vein .In the field of transplantation, this complication occurs more frequently in pediatric and living donor settings. It can also happen when using cadaveric adult donors if there is a discrepancy between the graft size and the recipient volume and the piggyback technique is used, and the probability is even higher in retransplantation where the urgency of the case does not allow to adapt the perimeters and sizes between the donor and the recipient. A recipient/donor matching is usually attempted in our institution according to the weight and the thoracic and abdominal perimeters. Unfortunately, sometimes there is no choice but to use grafts from smaller donors in larger recipients where rotating problems such as those described by the size discrepancy may occur.Diagnosis is usually done intraoperatively, becoming evident after reperfusion when the graft acquires a congestive, bloated aspect due to poor venous drainage, and is corrected by rectifying its position in the right hypochondrium, elevating it to avoid rotation of the suprahepatic veins.Various solutions have been reported to avoid this rotation and maintain a correct position of the liver, such as the placement of a breast prosthesis behind the right liver. This procedure has the disadvantage that the prosthesis cannot be removed without a new surgical procedure . Other mIn the reported cases, the removal of these devices is performed “blindly” assuming that the adhesions of the liver to the peritoneum and its adaptation to the new space can maintain the correct position. However, if this is not achieved, malfunctioning of the graft may occur after removing the device and the patient develops a Budd-Chiari-like syndrome, with ascites refractory to conventional medical treatment and impaired liver function.In order to avoid this, we propose to do a direct hemodynamic study via right internal jugular vein to measure THCG before and after deflating the balloon, so that we can be sure that once the device is removed, the liver will drain correctly. Maybe with a Doppler study, we can obtain similar information on the inversion of the flow at the level of the suprahepatic veins, but the acute local inflammatory changes together with the interference produced by the Sengstaken-Blakemore's tube makes the assessment more difficult using an ultrasound approach. We preferred to make a hemodynamic study, and despite being an invasive technique, we considered that this approach is a safe procedure and provides us relevant information before removal of the device in real time, monitoring the correct drainage through the suprahepatic veins.The optimal moment for the tube removal is in the early postoperative course but no sooner than 5 days after the surgery, to permit the formation of natural adhesions that will keep the organ in place. In our institution, the study is performed by the hepatology team with accumulated extensive clinical experience. This study can be performed easily with a few complications.In conclusion, the placement of a removable device such as a Sengstaken-Blakemore balloon intraoperatively helps to correct the position of the liver avoiding its malrotation and obstruction of the venous drainage. Performing a hemodynamic study before its withdrawal allows to objectively verify adequate venous drainage.


In relation to the article of the Journal of Clinical and Experimental Dentistry “Calvo-Guirado JL, Aguilar-Salvatierra A, Guardia J, Delgado-Ruiz R, Ramírez-Fernández MP, Pérez-Sánchez C, Gómez-Moreno G. Evaluation of periimplant bone neoformation using different scanning electron microscope methods for measuring BIC. A dog study. J Clin Exp Dent. 2012 Feb 1;4(1):e8-e13”, the authors have used three figures that are the same as those published in three different publications . The copyright of the mentioned publications was consequently not respected. Retraction of the article is therefore decided.


One of the many ways ofdefining globalization is as the integration of information, communication, and economyon a worldwide scale, with a direct influence on all levels of higher education. In thismanner, the internationalization of postgraduation may be seen as a response toglobalization, taking shape in the form of programs and policies put in place byacademic institutions and governments in order to increase student and faculty exchangesand to stimulate and strengthen partnerships in research, among other actions.Universities and research centers have, in fact, been practicing these actions for along time, but they have expanded significantly, particularly during this century.Globalization has left its mark on the 211-3 have repeatedly shown that collaborativeresearch that involves authors from multiple institutions and/or countries have anidentifiably greater impact than research involving only one group or institution. InBrazil, the internationalization of postgraduation has been highly valued by theCoordination for the Improvement of Higher Education Personnel (CAPES), generatingimmense efforts on the part of postgraduate programs (PGPs) to achieve the goalsdefined. In a study conducted with PGPs ranked 6 or 7 by CAPES, Ramos1 observed that internationalization inthese PGPs encompasses everything from international mobility, international cooperationnetworks, academic output , toaccess to resources through the sharing of research facilities and internationalfunding. In Brazilian PGPs, the most popular internationalization strategies wereinternational mobility of faculty, researchers, and students and international researchcollaboration, implemented mainly through international cooperation agreements. Thisstudy, nevertheless, detected inequalities between institutions in the provision ofadequate conditions for internationalization. The availability of financial resources,the existence of regulatory frameworks, and organizational support were consideredimportant requisites for achieving this goal.1Various studies2 may or maynot be considered positive, given that more well known institutions often have a headstart in the competition for funds, to the detriment of other academic centers. Theexistence of national policies that support internationalization, including thepublication of national journals, is extremely important, as can be seen in the exampleof countries that have successfully invested in this form of support.3Thus, due to the demands of CAPES, the need to internationalize has led to aninstitutional “race” between PGPs in search of partners, with or without governmentsupport, and often in a competitive manner. The strategies adopted by each institutionvary in accordance with their “scope” (based on professors and researchers’ contacts orpreviously established partnerships) and their level of resources and complexity capableof influencing international visibility and competitiveness. This “academicentrepreneurship”In order to meet the demands imposed by CAPES, PGPs and medical societies are faced withthe challenge of making a joint effort to provide all academic institutions with accessto opportunities and to allow the scientific output that originates from these PGPs tobe disseminated by their national and international peers. In this context, theBrazilian Society of Cardiology (SBC) has held “Postgraduate Meetings in CardiovascularSciences.” The theme of the fourth meeting, in 2018, was “The Internationalization ofBrazilian Postgraduate Programs.”During this meeting, the international guest speaker Professor Fausto J. Pinto, theDirector of the Faculty of Medicine of the University of Lisbon, spoke aboutpartnerships between European and Brazilian universities and announced the signing of arecent agreement between the Portuguese institution and the Federal University of Rio deJaneiro’s School of Medicine for the bilateral recognition of diplomas and dual degrees.He emphasized the importance of reinforcing contacts between universities in Brazil andEurope, especially in Portugal, in order to make the dissemination and adaptation ofexisting models possible by establishing a network of Portuguese-language medicalschools as an element to facilitate exchange, in addition to the creation of an“Erasmus-like” program for the Community of Portuguese Language Countries (CPLP).Arquivos Brasileiros de Cardiologia (ABC Cardiol) andthe International Journal of Cardiovascular Sciences. The ABCCardiol, which is indexed in the main databases, including ISI Web ofScience, Index Medicus, MEDLINE, PubMed Central, EMBASE, Scopus, SciELO and LILACS,obtained an Impact Factor (IF) of 1.318 from Journal Citation Reports (JCR), as well asa B2 rating from the CAPES Qualis System in its most recent evaluation.4The visibility of national research is another important aspect of internationalization.It is of the utmost importance to disseminate research carried out by the Brazilianscientific community in the area of cardiovascular disease (CVD), which is recognized asthe leading cause of mortality in Brazil and worldwide. The SBC has two journals indexedin SciELO: the ABC Cardiol is Latin America’s main journal for the publication ofresearch in the area of Cardiology and Cardiovascular Sciences and it has an importantand increasing degree of internationalization, as more than 20% of its articles are ofinternational origin. It is worth emphasizing that Portuguese language countries (PLCs)have access to the bilingual version of the ABC Cardiol, which ispublished in all Lusophone countries by the Portal of the Federation of CardiologySocieties of Portuguese Language Countries (http://www.fsclp.org), representingapproximately 245 million people. In PLCs, the large differences, related mainly tosocioeconomic conditions, in the relative impact of CVD burden are noteworthy.5 The highest quality of scientific outputpublished in the ABC Cardiol continues to originate from Brazilianpostgraduate programs, which are increasingly exposed, in the meanwhile, tointernational competition to find publishing space in the ABC Cardiol,added to the lower incentive to publish in this journal as a direct result of itscurrent CAPES ranking. It is also very important to valorize the publication of scienceand knowledge, which are public goods, in open science journals such as the ABC Cardioland other journals in the SciELO network.The CAPES values the social involvement of PGPs, with the objective of promoting improvementsin the population’s living conditions. However, Brazilian studies focusing onpopulations with peculiar socioeconomic characteristics rarely receive the interest ofthe international community; their dissemination would need to be driven by CAPES’evaluation system in order to strengthen a national exchange network that included PLCs.The innovative scientific contributions that result from PGPs and their mission forcivil society are also worth highlighting. In this sense, it would be desirable for thisregulatory agency to create a system, mediated by Qualis, for valuing the leadingjournal in the fight against the CVD epidemic in a manner that makes it possible toshare successful experiences in fighting CVD with these countries.6 as well as topromote the development of a more just and equitable society through improvements in theliving conditions of the population; this, upon final analysis, is the main objective ofresearch undertaken by PGPs. The recognition of national research must also occurethically and meritocratically through the valuing of the means responsible for itsdissemination. The growth of Brazilian journals will have to be the fruit ofintellectual investment on the part of researchers who produce quality science andinternationalization through international partnerships, as well as stimulation andvalorization through better rankings in CAPES’ evaluation system.An unprecedented academic revolution is taking place in higher education, leading to areal need for the internationalization of PGPs. Internationalization offers newopportunities for study and research that are limited neither by national boundaries norby boundaries to knowledge. However, “internationalization is not a goal in itself, butrather a means to accomplish improvements in teaching, research, andinnovation,”


Ipomoea batatas) noodles are a traditional Chinese food with a high nutritional value; however, starch adulteration is a big concern. The objective of this study was to develop a reliable method for the rapid detection of cassava (Manihot esculenta) components in sweet potato noodles to protect consumers from commercial adulteration. Five specific Loop-mediated Isothermal Amplification (LAMP) primers targeting the internal transcribed spacer (ITS) of cassava were designed, genomic DNA was extracted, the LAMP reaction system was optimized, and the specificity of the primers was verified with genomic DNA of cassava, Ipomoea batatas, Zea mays, and Solanum tuberosum; the detection limit was determined with a serial dilution of adulterated sweet potato starch with cassava starch, and the real-time LAMP method for the detection of the cassava-derived ingredient in sweet potato noodles was established. The results showed that the real-time LAMP method can accurately and specifically detect the cassava component in sweet potato noodles with a detection limit of 1%. Furthermore, the LAMP assay was validated using commercial sweet potato noodle samples, and results showed that 57.7% of sweet potato noodle products (30/52) from retail markets were adulterated with cassava starch in China. This study provides a promising solution for facilitating the surveillance of the commercial adulteration of sweet potato noodles from retail markets.Sweet potato ( Ipomoea batatas) is a dicotyledonous plant that belongs to the family of Convolvulaceae. The roots of sweet potato are starchy, sweet-tasting, and tuberous vegetables. Sweet potato noodles are favored by many consumers due to this food’s ease of cooking, good nutritional value, and easy digestion. There is a huge market for this food product in Asia, including China, Japan, and Korea. The food labels of noodle products are required to specify the starch source specifically to prevent adulteration and to protect consumers. However, the mixture of low-priced starch into high-priced starch is still very common. For example, cassava starch is often incorporated into sweet potato starch during the production of sweet potato noodles; therefore, a reliable method for the identification of starch species is critical for the surveillance of commercial adulteration [Sweet potato  and sensory evaluation , which aManihot esculenta). In addition, the starch from Zea mays or Solanum tuberosum is sometimes used as the counterfeit material. The specificity of a newly developed real-time LAMP method for the rapid detection of cassava-derived ingredients was tested with starch from cassava (Manihot esculenta), Ipomoea batatas, Zea mays and Solanum tuberosum.Loop-mediated isothermal amplification (LAMP) has been developed to amplify nucleic acids under isothermal conditions, and it is a very specific, sensitive, and rapid technology . The metManihot esculenta was 193 bp. The identity of the sequences with the corresponding sequences of Ipomoea batatas (GenBank: MH792118.1), Zea mays (GenBank: KU182536.1), and Solanum tuberosum (GenBank: AY875827.1) was 79.91%, blasted with DNAMAN ; thus, the real-time LAMP assay using the cassava primers had a high specificity.As shown in Manihot esculenta) along with negative results for all negative control (water only) reactions. Given the amplification efficiency and specificity, 58 °C was chosen as the most suitable reaction temperature.The LAMP reaction with the newly designed primers was carried out at 56 °C and 58 °C for 60 min, and as shown in Manihot esculenta, Ipomoea batatas, Zea mays, and Solanum tuberosum. The reason for this type of sample selection was that, alongside the most common material for sweet potato noodle fraud, starch from Zea mays or Solanum tuberosum is sometimes used as the counterfeit material. As shown in The specificity of the LAMP assay was tested with starch samples of The sensitivity of the LAMP assay was determined with genomic DNA extracted from sweet potato starch mixed with different proportions of cassava starch at 58 °C for 60 min. The detection limit was found to be the sweet potato starch mixed with 1% cassava starch, as shown in Out of 52 sweet potato noodle samples collected from Chinese retail markets that were tested, 30 were found to be positive for cassava starch using the newly established real-time LAMP assay, indicating that fraud of sweet potato noodles with cassava starch may be very common in China.Since the LAMP technique was first reported in 2000 , researcSince starch contains a high carbohydrate content, and sweet potato starch was subjected to a heat treatment, it was difficult to extract enough high-quality genomic DNA from the noodles for the use of sweet potato noodle fraud testing with the DNA-based method. The improved CTAB method was used as the DNA extraction protocol in this study, and the internal transcribed spacer (ITS), with high copy number, was selected as the target sequence, significantly increasing the sensitivity and practicability of the newly established real-time LAMP assay.There is no national standard or industry standard for sweet potato noodles in China, and the current national standard (GB/T 23587-2009 Vermicelli) does not have a method to distinguish noodles made of sweet potato starch from those made with low-cost cassava starch; consequently, sweet potato noodle fraud with cassava starch is very common in China. The adulteration of sweet potato noodles economically impacts sweet potato plantations and seriously impacts consumers. The advantages of the real-time LAMP assay are its cost-effectiveness, simplicity, high specificity and sensitivity, which make it suitable for the surveillance of commercial sweet potato noodle products that are adulterated with cassava starch.Manihot esculenta (GenBank accession No. MK114629.1) were designed using PrimerExplorer V5 (http://primerexplorer.jp/e/) and Oligo 7 . The primer sequences are listed in A set of LAMP primers targeting the internal transcribed spacer (ITS) of Manihot esculenta, Ipomoea batatas, Zea mays, and Solanum tuberosum was extracted according to the method of Rajoo et al. [g for 15 min at 4 °C. The aqueous phase was mixed with 7 mL of chloroform: isoamylalcohol (24:1) by inversion for 15 min and centrifuged at 8000× g for 15 min at 4 °C. The aqueous phase was transferred to a fresh tube, and 0.6 volume of ice-cold isopropanol was added and incubated at −20 °C for 2 h to precipitate the DNA. After centrifugation at 4000× g for 5 min, the pellets were washed with 70% ethanol, dried in the air and dissolved in sterile double distilled water. RNase was added to the DNA solution at a concentration of 10 mg/mL, followed by incubation at 37 °C for 1 h. An equal volume of phenol: chloroform: isoamylalchohol (25:24:1) was added to the DNA solution, mixed for 15 min, and centrifuged at 10,000× g for 15 min at 4 °C. The aqueous phase was re-extracted for phenol. An equal volume of 100% ice-cold ethanol was added to the aqueous phase and incubated at −20 °C for 2 h to precipitate the DNA. After centrifugation at 4000× g for 5 min, the pellets were washed with 70% ethanol to remove salts. The pellets were air dried and dissolved in 500 µL double distilled water. Two hundred microliters of 30% PEG 8000 were added to 500 µL of the DNA sample, mixed, incubated at room temperature for 30 min, and centrifuged at 14,000× g for 15 min. The pellets were washed with 80% ethanol, air-dried, and dissolved in nuclease free water or Tris-EDTA (10:1) buffer. The samples were frozen at −20 °C until further use. The noodle samples were homogenized using mortars and pestles before the DNA extraction.Genomic DNA from the starch samples of o et al. . Briefly4)2SO4, 6 mM MgSO4, 0.1% Triton X-100, 7.5% DMSO [Manihot esculenta, and 3.2 U Bst 2.0 WarmStart DNA polymerase  [TM System .The real-time LAMP assay was performed in a 10-μL reaction mixture containing 0.8 mM each of forward inner primer (FIP) and backward inner primer (BIP), 0.2 mM each of forward outer primer (F3) and backward outer primer (B3), 0.4 mM of forward loop primer (LF), 1.0 mM dNTPs, 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM  . The reaManihot esculenta, Ipomoea batatas, Zea mays, and Solanum tuberosum was used for determining the specificity of our developed LAMP assay, and the amount of genomic DNA template used was 1 ng per reaction.Genomic DNA of The sweet potato starch was mixed proportionally with the cassava starch; the mass percent of cassava starch was 10%, 5%, 1%, and 0.1%, and genomic DNA was extracted according to the method of Rajoo et al. . The detA total of 52 samples labeled sweet potato noodles were collected from Chinese markets; the samples were homogenized, their DNA was extracted according to the method of Rajoo et al. , and the


To evaluate the interactive influence of these variables, a statistical learning model based on gradient boosting is applied. The results showed that the effect of the sintering temperature has an optimal (maximum transmittance) point. Increasing the temperature beyond this point reduces the transmittance of the material for all types of zirconia. Moreover, the optimal transmittance point is affected by the chemical composition of each type of zirconia. In addition, the results showed that the transmittance of all types of zirconia had an inverse relationship with the density, zirconia Zpex-Smile being the most sensitive to this parameter. Furthermore, the transmittance of 3Y-SBE, Zpex, and Zpex-4 decreases approximately linearly with the specimen thickness, whereas zirconia Zpex-Smile has a sublinear decrease, which is expected due to the optical isotropy of the cubic phase.Research has increasingly been conducted to improve the toughness and aesthetics of zirconium oxide (zirconia) used in prosthetic dentistry. However, the balance between better mechanical properties and greater translucency, to ensure aesthetic requirements, is still a challenge in the development of a novel zirconia for dentistry applications. This study evaluated the transmittance of visible light for four types of pre-sintered zirconia blocks used in dentistry . The objective is to analyze the simultaneous influence of sintering temperature, in the range of 1450–1560 °C, as well as the chemical composition (%Y By adding a certain percentage of dopant oxide, such as cerium oxide (CeO2), magnesium oxide (MgO), and most commonly yttrium oxide or yttria (Y2O3), it is possible to induce metastability in the tetragonal phase at room temperature, obtaining the tetragonal zirconia [2O3, known as nY-TZP, where n is the yttria percentage.Pure zirconia, (ZrOzirconia . The polt→m) induced by stress. This phase transformation is accompanied by a volumetric expansion by 5%, which generates compression stress at the crack tip and impairs its propagation, thereby increasing the material’s toughness [The Y-TZP has good toughness, which is associated with the transformation of the metastable tetragonal phase to monoclinic  were used. Zirconia powders with commercial designations TZ-3YSBE, Zpex, Zpex-4, and Zpex-Smile, supplied by TOSOH-Japan , were used to produce the blocks. The powders were uniaxially pressed at 80 to 90 MPa, according to TOSOH instructions.2O3 content which is 3 mol % (5.2 wt %) for TZ-3YSBE and Zpex, 4 mol % (7.0 wt %) for Zpex 4, and 5.2 mol % (9.3 wt %) for Zpex-Smile.2O3 content), sintering temperature, density, wavelength, and thickness on the material transmittance, 160 specimens of each type of zirconia were prepared. The specimens were sintered at 1700 °C in the NBC model furnace, from Nobody Materials Science and Technology Co. . All specimens were sintered at a heating and cooling rate of 5 °C/min. Final plateau sintering temperatures were 1450, 1500, 1530, and 1560 °C. The sintering time was 120 min at plateau). After sintering, samples had thicknesses of 1.00, 1.25, 1.50, and 1.75 mm. The thickness measurements were verified with micrometer, and a maximum variation of 0.01 mm was observed.To analyze the influence of the chemical composition  Empyrean diffractometer, with Cu-Kα radiation, 2θ range of 20–80°, a step width of 0.02°, and an exposure time of 5 s. The crystalline peaks were identified by comparison with the JCPDS data files using X’Pert HighScore Plus software and quantification was estimated according Scott, Matsui et al. and Chen et al. [The crystalline phases present in the sintered ceramics were performed by X-ray diffraction (XRD) using a Panalyticaln et al. ,11,12.To obtain the percentage of transmittance, the experiment was performed on a spectrophotometer that operates in the wavelength range of visible light . The light source of the equipment is a xenon arc lamp, and the generated data were computed in the wavelength range of 360–740 nm, at intervals of 10 nm. The transmittance test was performed using a standard two-blot black-and-white card . The results obtained from the test correspond to transmittance values at every 10 nm, between 370 and 740 nm. According to the Technical Standard ISO 4007: 2018, there are no precise limits for the spectral range of visible radiation since they depend upon the amount of radiant power reaching the observer’s retina. The lower limit is generally taken between 360 and 400 nm and the upper limit between 760 and 830 nm. The limits of the visible spectrum are usually taken to be 380 nm (blue violet) to 780 nm (red).For characterizing and quantifying the surface roughness, step heights, critical dimensions, and other topographical features, the NewView™ 7100  light interferometer (profilometer) was used. The NewView™ 7100 provides affordable versatility in non-contact optical 3D surface profiling . The parThe analysis of the influence of the various parameters and the zirconia processing in the transmittance is difficult due to the interaction among several variables. The attempt to employ parametric models that isolate one effect can generate a bias of omitted variables due to simplifications in the model. In addition, the nonlinear influence of each variable requires complex mathematical models to represent the joint dependence of all variables studied.Recent parametric approaches, such as the use of Rayleigh scattering model, require strong assumptions with respect to the shape of grains, pores, and particles sizes. Alternatives, such as the use of the Mie scattering model, makes use of similar assumptions and involves simulations and numerical solutions to solve electromagnetic waves equations.In the present work, we used a statistical learning model based on gradient boosting ,15. The The model work as follows: we assume that the transmittance is described by a set of measured variables that affects directly or indirectly the transmittance of zirconia. In this case, the variables are density, sintering temperature, composition, and thickness. It is important that these variables also define other variables that were not measured, such as grain size. Therefore, since these variables are all defined implicitly, the model will be able to capture the interaction among them and the effect in the transmittance of zirconia.To estimate the function, we assume that it can be approximated by a combination of simpler functions that are easier to estimate, called base learners . The mety,x) and we are using the squared error as our loss function. In other words, we are seeking the best function that predicts average transmittance given the chemical composition, sintering temperature, density, and thickness of the sample.More formally, we want to find a function F is restricted to an additive expansion of the form:Since we have finite data, the risk function defined in Equation (1) must be replaced by the empirical risk function given by Equation (2):In Equation (3), each function posed by  will be m−1 iteration, the gradient in the m-th iteration is calculated as Equation (4):Assuming that the model was calculated up to the nditions  we have Once we have the estimation of the gradient Then, we calculate the total step in the direction of the new base learner m-th step are given by Equation (9):Finally, the model and forecast in the M iterations the model will be given by Equation (10):M is the total base of learners and After For the quantitative analysis of the transmittance results, the modeled variable Y was chosen as the percentage transmittance of each of the zirconia samples tested. The set of analyzed variables were the thickness of the sample, the composition of the zirconia , the sample density, and the sintering temperature.In this work, the BooST algorithm was used to estimate the model . The maiEach STR-tree (base learner) has 4 terminal nodes (parameter). The number of trees used in the model was selected using 5-fold cross validation. To estimate the model, the software R and the algorithm proposed in  was used2 cubic phase increases with increasing percentage of Y2O3.The results from XRD analysis identified two distinct phases present in all samples . The phaWhen comparing the tetragonal phase percentage of sintered 3Y-SBEat different temperatures, it can be observed that the sintered samples at 1450 °C showed the highest percentage of tetragonal phase. The samples sintered at 1560 °C showed the lowest percentage of tetragonal phase. The gap between the highest and the lowest percentage was 9%. For the other groups of zirconia, the differences between the gaps of tetragonal phase after sintering were from 6–10%, as a function of sintering temperature. This result indicates that only the sintering temperature is not statistically significant to modify the relationship between the percentages of tetragonal and cubic phases.2O3 content (from 3–5.2 mol %) in the composition of the different zirconia, the variations of the percentage of phases were between 40% and 43% among the sintered samples at the same temperature. This result indicates that the influence of yttria content in the percentages of the phases is significant.On the other hand, comparing the influence of the variation in the YBefore presenting the results obtained with the statistical model, one can see in x-axis, one can see the thickness of the sample. Each facet corresponds to a specific type of zirconia , each color represents a different sintering temperature, and the x-axis is the mean thickness of the specimen. The y-axis is the value of the transmittance. The wavelength was fixed at 710 nm since changing the wavelength does not affect the relationship of transmittance with the explanatory variables.In Naturally, the thickness variable influences the translucency of the material, since the larger the thickness, the greater the interaction with the factors responsible for scattering the incident electromagnetic wave. It is also expected that the sintering temperature influences the transmittance of the zirconia because it directly affects the grain size and the level of porosity of the sintered zirconia.p < 0.01). The translucency of enamel and dentin increased in direct proportion to wavelength and in inverse proportion to thickness (r = 0.87–0.91).Yu et al.  determinx-axis is the thickness of each specimen. The y-axis is the value of the transmittance.In addition, the final density is also correlated with the level of porosity, and the difference in chemical composition between different types of zirconia affects the type of metastable phase at room temperature, which also affects the transmittance of the specimens. In With the adjusted model, it was possible to analyze the influence of the sintering temperature, the thickness, density, and composition in the transmittance. By fixing a specific density value—say, the average—the other parameters can be varied to understand the impact of these variables on the transmittance. In The model was able to identify the influence of the density for each zirconia group. The difference among groups suggests that the zirconia microstructure plays an important role in how significant the impact of density (and consequently porosity) is on the scattering of the incident light.For the Zpex-Smile group, the higher sintering temperature and high density generates a zirconia with higher transmittance. For the other types of zirconia, the density also affects the transmittance. However, the partial effect is clearly lower. In addition, it is possible to verify that for the Zpex-Smile zirconia, there is less influence of the sample thickness regardless of density level. However, for the other types of zirconia, the influence of sintering temperature is still approximately linear.Based on the results obtained by the proposed methodology, it is observed that at lower sintering temperatures than that for maximum densification, the positive effect of grain size decreases. Above a certain sintering temperature, grain growth induces microcrack nucleation and decreases the novel zirconia for dentistry (NZD) transmittance with a negative effect.The results of the mathematical models showed a higher transmittance sensitivity of the sintered NZD-4 at higher temperature. For this group, if the zirconia reached the expected maximum densification around 1550 °C, where the highest transmittance is obtained. If the Zpex-Smile has not yet reached the maximum densification, the sensitivity in relation to the sintering temperature is similar to that for the other groups.It also worth noting that for the zirconia Zpex-Smile, the partial effect of the sintering temperature is surprisingly high. Specifically, for this type of zirconia, the model points out that high density and high sintering temperature are both fundamental for the best transmittance results of the material.Based on the data of the roughness parameters shown in Even though the grain size is known to be an important parameter in the transmittance of zirconia, the dependence of the grain size with the sintering temperature and the powder microstructure is well established  and suffOne analysis of interest is to understand the partial effect of the grain size in the transmittance of zirconia. However, for practical use, the control over the sintering temperature and type of zirconia is easier, especially for practical applications, since it avoids the use of techniques such as SEM and indirectly measuring the same effect.Based on experimental results and in the statistical learning method proposed, it was possible to analyze the effect of the sintering temperature in different types of zirconia commercially available. The proposed statistical model was fundamental to identify the nonlinear dependencies among the sintering temperature, chemical composition, density, and thickness of the samples in the zirconia transmittance.The sensibility analysis presented no linear dependence between transmittance and specimen thickness for 3YSBE, Zpex, and Zpex-4, and a sublinear dependence for zirconia Zpex-Smile. Increasing the sintering temperature has a positive impact in the transmittance of the zirconia up to a certain level, which varies with density and chemical composition. After this point, increasing the sintering temperature decreases the transmittance of the material. The group deriving the greatest benefit from high sintering temperature was zirconia Zpex-Smile.


The unconventional nature of Holy See diplomats rests in the composite character of their ecclesiastical role as the Pope’s representatives and their legal diplomatic status and commencement to ordinary diplomatic practice. Holy See diplomacy is a form of conduct created by a set of mixed secular and religious standards in which agents are guided by practices. I locate this argument within a classical English School and a conventional understanding of practice, diplomacy, and agency while incorporating understandings of the diplomat as a stranger. The article situates a Holy See diplomat’s mode of agency as a hybrid one by nature, located at the intersections of political and religious modes of agency and substantial and relational conceptions of international politics. I probe this conceptual framework of hybrid agency by analysing episodes involving papal diplomats in turmoil-ridden historical episodes, and correspondence with informed agents. From many points of view, the HS is unique to the world in its ability to pursue its own agenda. The Vatican, with its diplomatic relations […] is second only to the United States. Diplomats are aware of the scope and influence of the HS diplomatic trajectory, but International Relations tend to set aside the HS diplomatic trajectory and its societal practices as historical footnotes or proxy variables.By placing the diplomatic entanglement of the HS in international politics to the historical backburner, international studies disdain insights with grave consequences. Today, the Church’s members and officials are increasingly from all over the world and inter-religious dialogue, for example, becomes important as part of the engagement in political and social policy issues under the aegis of diplomacy. Diplomacy itself is a key part of this transformation which offers expanded space for actors engaging in diplomacy . HS diplWhereas ordinary diplomats are serving the goals of a territorial unit, the unconventional nature of HS diplomats is nested in the hybrid character of their ecclesiastical role as the Pope’s representatives and their legal diplomatic status and commencement to ordinary diplomatic practice. Their papal patron’s global perspective renders them unusually dedicated to influencing the conditions beyond their principal’s immediate possessions (such as a territory). This standing places them at an advantage in an international society in which such contextual factors are centrally important. This advantage is reinforced by the degrees to which HS diplomats constitute a combination of transnational identity and national interest. This difference is obvious when looking at instances of how papal diplomats mediate between a conventional approach and the approach of the HS. Between ‘its supernatural mission of salvation and the mundane reality of world politics’, the institution of the papacy ‘remains a singular and surprisingly vital factor in the international scene’ : 474. ThGiven the religious nature of its principal, its ordained agents, and international society’s requirements for diplomatic standards and practice, papal diplomacy cannot be like ordinary diplomacy. Papal diplomacy is the result of a religious and political entanglement that generates a hybrid mode of agency of which this article proposes the constituents of its diplomatic practice. This argument is located within a classical English School understanding of practice, diplomacy, and agency while incorporating constructivist understandings of the diplomat as a stranger. The article situates HS diplomat’s mode of agency as hybrid, located at the intersections of political and religious modes of agency and substantial and relational conceptions of international politics, an angle that has not been applied to HS diplomacy by scholars of International Relations and diplomacy.After a review of the literature on the HS in international society, the next section illustrates the hybrid mode of diplomatic agency. This hybrid diplomatic agency resolves questions at the intersection of substantialist and relational conceptions of international relations and political and religious modes of agency. HS diplomats act on behalf of a religious and a political entity and logic in religious and secular environments. The next step unpacks the historical trajectory of the HS diplomatic practice. The article draws on a hybrid mode of agency by illustrating how the different dimensions and conceptions matter in blueprinting a comprehensive picture of HS diplomacy. The remainder of the paper demonstrates how the hybrid mode of agency varies by degree. The framework of hybrid agency is then probed by analysing incidents involving papal diplomats in turmoil-ridden historical episodes and unstructured interviews and correspondence with informed agents. This section teases out points of broader interest and illustrates the complex entanglement of the religious and political. The conclusion locates the findings in the context of the theoretical framework, the general political interest in the Church, its entanglement in international society, and points out future avenues of research.1 international law  which is an international personality in international law. In terms of international law, the ‘State of the Vatican City’ and the HS are different entities. They are linked by the personal-union with the pope, the supreme head of the Catholic Church and the Vatican City. The foreign relations of Vatican City are managed by the HS, which is subject to international law. In operational terms, the management comprises the HS Secretary of State and its subordinate, the Secretary of Relations with States.10 In 2017, Pope Francis created a third section, the Secretary of States (‘Section for the Holy See diplomatic staff’), which is exclusively dedicated to the work of the nuncios .12 Today, the HS has diplomatic relations with almost all countries around the globe and newly consolidated nations keep seeking recognition from the HS  of the diplomatic Corps has been recurrently the nuncio, at least in Roman Catholic countries.15 In terms of international law, nunciatures hold the same status as embassies. Canon law offers a range of functions for papal legates, most of them dealing with internal matters of the Church such as assisting local bishops. These internal matters include traditional clerical duties, such as the appointments of bishops, which influence domestic and international politics as the contested cases in the People’s Republic of China illustrate  is intended to underscore the particular nature of their mission. : 302HS Secretary of State Pietro Parolin characterises HS diplomacy as ‘human diplomacy’ because ‘real people must be at the centre of all diplomatic action’.An eye ever open to serene observation of the moral, religious and social state of France, in order to render a true and objective account to the Supreme Pastor. A heart watchful and sensitive to the needs of the people of Christ, as hungry for spiritual foods as they are for bodily sustenance. The Pope’s own heart indeed, for his mission is not to busy himself with material conquests and earthly interests, and not to follow in the ways of intrigue and hatred, but to observe the law of brotherhood and love. Finally, a hand to point to the right road, a hand which, in the Lord’s name, succours, encourages and blesses. This is still and will always be the Pope’s own hand outstretched, reproduced in the person and office of his Nuncio, in France and in every other nation. : 106The ‘Apostolic Nuncio is one of the most characteristic signs of the Pope’s presence in a nation’, as Pope John XXIII, former nuncio to France, summarised it. Moreover, pontifical ‘and national diplomacy have a common basis in frankness and in the constant effort to improve relations. But they differ in their essential characteristics’ : 105. ThThis instruction is similar to conceptualizations of ordinary diplomatic modes of agency and conventional notions of practice, which is obvious in both the first and the last sentence of the quote. It stresses the importance of the diplomat’s task of gathering information and communicating interests. It combines the view of diplomats as representing a principal in behaviour (to act for others) and in status (to stand for others) : 100. ItThis instruction is also an expression of the religious mode of diplomatic agency and as such it is illustrative of the HS hybrid character with regard to diplomacy. Pope Francis, addressing the diplomatic academy, reinforced the main elements of the religious mode of agency in this instruction. In particular, he stressed the need for HS diplomats to keep their spirituality amid their worldly tasks and pointed out that their future life will be inserted in different societal contexts. He also pointed out that HS diplomats need to be examples in their lives, characterised by closure, dialogue, listening but also tangible verbal action. What is more, he stressed that HS diplomats need to serve the evangelization without proselytising as the Church grows by attraction .The remainder of this section turns to three cases of nuncios in turmoil ridden historical episodes. The cases illustrate that although there are outlines of an ideal-type nuncio such as illustrated above, their practical mode of agency remains a hybrid one as it comes into existence through the interaction of different origins. Although any hybrid mode has its own outstanding characteristics and remains hybrid in nature, it varies in degree. This variance is no different in the case of HS diplomats. The three episodes of the nuncios Joseph Hurley, Jose Laboa, and Fernando Filoni illustrate that the mix of principal steered possession and milieu goals, diplomatic practice, and the self-understanding of the agents add up to a contextualization of the HS diplomatic service and the practices of its members as a hybrid mode of agency.Joseph P. Hurley from the United States was perhaps one of the most influential yet largely unknown papal diplomats in the 20th century. Hurley originally served in the Japan mission in the 1930s and was a warning voice of National Socialism and Fascism. He served again as nuncio in the 1940s in Yugoslavia and became known for his fierce stand against Communism. Hurley is an outstanding example of a papal diplomat as he resembles many of the aforementioned ideal type features of ordinary diplomats. He was sympathetic towards an approach of diplomacy understood as ‘high politics’ such as secretively operating behind the scenes and keeping a low public profile. Yet he also believed his role to be the ‘Pope’s own hand outstretched’. Throughout his life, he was torn between serving his two masters: the United States and the Church, and rival interpretations over the mission of them : 1–3. InThe episode of Hurley illustrates how difficult it is to grasp the practice of HS diplomats along the religious model of diplomatic agency. During several stages of his career Hurley, in many respects, might have come close to a conventional diplomat but he remained so in ordinary diplomatic terms. Although he assumed his role as serving church and country, he sometimes placed the mission of the latter over the former. Arguably, in this regard Hurley was a classic example of a power politic driven agent, mediating between different political entities. As Hurley tried to serve two principals, he encountered difficulties in doing justice to both as well as becoming even more disappointed when the two principals parted ways in their goals in the 1950s. The story of Hurley illustrates that HS diplomacy is not always conducted along standardised diplomatic practices. In Hurley’s case, this divergence meant that he drifted from representing to governing, mediated by personal beliefs regarding the political and the religious realm. Yet still, he complied with the art of diplomacy as a form of high politics and certainly was seen as an outstanding diplomat.Another episode illustrates how a papal agent got caught between the principal’s foreign policy goals and ended up improvising. It is the episode of how Panamanian leader Manuel Noriega was eventually handed over into the hands of US authorities. In 1989, rather unexpectedly, Jose Sebastian Laboa from Spain and nuncio to Panama was faced with the difficulties that serving possession goals bring with them when Noriega took refuge in the nunciature. Without being able to consult the HS at first, the nuncio extended the diplomatic immunity to another house to separate Noriega from his aids. Eventually, Laboa, who had been encouraging anti-Noriega forces before, was able to sway Noriega out of the embassy and into the hands of US forces. He did so through a carefully orchestrated psychological campaign, warning Noriega of the danger of being lynched or by talking about loyalty at the Catholic Mass. All of this practice aimed to convince Noriega that his only realist option was to give up . In his The episodes of Hurley and Laboa illustrate that, on the surface, HS diplomats operate like ordinary diplomats, face the same challenges, and rely on the same instruments. However, they also know how to use their authority as ordained diplomats. Without an in-depth study into the agents motives, the episodes only hint towards a genuine hybrid mode of agency. We can find this mode in operation in the more recent case of nuncio Fernando Filoni, originally from Italy.28 Filoni’s actions constituted a modality of practice and discourse of a Church of the margins as it is today attributed to Pope Francis’ conception of the Church (Recalling the mediation of difference and estrangement, Filoni’s case illustrates how HS diplomats are an inherent part of the host society, and not only the Catholic part of it. Filoni was the representative of the Pope in Iraq in 2014 but first became famous for remaining on his post as nuncio in Iraq during the 2003 US bombing of Bagdad . Explaine Church  and its Seen in chronological order of the historical episodes described above, HS diplomatic practice seems to move from a realist system, over the rationalist society, towards the revolutionist world society approach . The epiDespite the weakening influence of the Eurocentric Catholic Church, HS diplomacy continues to play a vital role for the Church itself and in international society. In conflicts around the world either a Catholic majority or a significant Catholic minority is at home. Moreover, in a global Church its officials are from all over the world rather than only from the Roman ecosystem. Globally, religion wields an arguably growing influence in international politics , and theA renewed interest in diplomacy is not least propelled by the current Pope and his intermingling in conflicts around the world , 2017. HHS diplomats, as the three episodes of nuncios illustrate, exercise authority under legal, moral, and expert modalities, weaving together secular and religious discourses and practices. They are essentially and functionally recognised agents in the international society that grounds its dialogue on diplomacy. All three trained as diplomats and experts in their tasks and missions. Based on their religious affiliation, they are nominally also moral experts. The three episodes illustrate how modalities and discourses interact and sometimes trump each other. In doing so, they also illustrate that international society is not a one-way street to a justly governed world society : 266.29 The theoretical conceptualizations of power, diplomacy, and international society demonstrate insights into the HS diplomatic conduct while not only framing diplomacy as a category of analysis but also as one of explanation. Diplomacy is a practice because political units need to interact and want to stay apart. In the conduct of international society, this gap is filled by the practice of diplomats, no less so by the ones of the HS. They are even more likely to bridge this gap because estrangement on the cultural and religious level tends to be low but remains intact when it comes to secular duties where social distance remains a condition for pursuing foreign policy goals. The findings of this article suggest a renewed research interest looking into the interplay between the hybrid agency of diplomats and structural issues of international society.Early English School scholars put forward the claim that religious institutions are constitutive for international society. Those claims, however, have been picked up only reluctantly in subsequent studies. The entanglement between religious institutions and international society, after all, remains an understudied topic.