Results obtained from the lower molecular weight series of TBPUs demonstrated potential use in drug delivery and imaging contrast agents due to their high hydrophilicity and degradation rates. On the other hand, the higher molecular weight series of TBPUs exhibited improved hydrophilicity and degradation rates compared to PL-homopolymer. Moreover, they displayed improved tailored mechanical properties suitable for utilization as bone cement, or in regeneration medicinal applications of cartilage, trabecular, and cancellous bone implants. Furthermore, the polymer nanocomposites obtained by reinforcing the TBPU3 matrix with 7% (w/w) bacterial cellulose nanowhiskers (BCNW) displayed a ~16% increase in tensile strength, and 330% in % elongation compared with PL-homo polymer.A novel series of biodegradable polylactide-based triblock polyurethane (TBPU) copolymers covering a wide range of molecular weights and compositions were synthesized for potential use in biomedical applications. This new class of copolymers showed tailored mechanical properties, improved degradation rates, and enhanced cell attachment potential compared to polylactide homopolymer. Triblock copolymers, (TB) PL-PEG-PL, of different compositions were first synthesized from lactide and polyethylene glycol (PEG) via ring-opening polymerization in the presence of tin octoate as the catalyst. After which, polycaprolactone diol (PCL-diol) reacted with TB copolymers using 1,4-butane diisocyanate (BDI) as a nontoxic chain extender to form the final TBPUs. The final composition, molecular weight, thermal properties, hydrophilicity, and biodegradation rates of the obtained TB copolymers, and the corresponding TBPUs were characterized using  Biodegradable polymers are increasingly used in different biomedical applications. This includes drug-releasing implants, bioresorbable surgical sutures, biodegradable vascular grafts, and in sustained drug delivery applications . These pL-lactic acid). These PUs are highly elastic with low degradation rates that makes them suitable for applications as long-term scaffolds , polyethylene glycol as a linker , and Sn(Oct)2 as a catalyst were used to synthesize triblock copolymer. A total 1,4 Butane diisocyanate  and polycaprolactone diol  soft segments were used in chain extending reactions, and the formation of final polyurethane matrix. Diethyl ether, dimethylformamide (DMF), dichloromethane , N,N-dimethylacetamide (DMAc), acetone, and methanol of ACS grade were used in the present work as common solvents during synthesis, purification, and film casting of polyurethane. Deuterated chloroform  was used for NMR analysis. Phosphate buffered saline , sodium azide NaN3, and porcine pancreas lipase type II were obtained from Sigma-Aldrich Canada, (lipase activity: 100–500 units/mg protein (using olive oil (30 min incubation)), 30–90 units/mg protein (using triacetin)). Sulfuric acid (98%) was used for the hydrolysis of bacterial cellulose nanofibers to obtain the nanowhiskers.All chemicals were purchased from Sigma-Aldrich and were used as received unless stated otherwise. Lactide  + [Mn(PEG) − 18]. The obtained Mn values of the triblock PL-PEG-PL were found in the range of . These values were a good match with the calculated value based on GPC measurements; see In addition, the molecular weights, average segmental length of both LA and PEG components, and the composition of the copolymers were also estimated from As presented in 1H-NMR spectra. In this regard, besides the predetermined characteristic signals of the triblock PL-PEG-PL, new chemical shift signals emerged due to the presence of PCL segments; see The structures of the final TBPUs polymer that were produced as a result of the interaction of PCL with triblock PL-PEG-PL were further confirmed using 1H-NMR spectra for all samples revealed neither remarkable change happened in the PL/PEG segment ratio or an increase in the molecular weight of the polymer. This result evidenced that BDI molecules interact only with the terminal OH groups without causing any crosslinking reactions between TB copolymer chains. Therefore, based on the controlled molar ratio of the terminal OH groups of PCL-diol, and the molar ratio of added BDI, , the number average molecular weight of TBPUs can be estimated using the equation:After the coupling reaction between TB copolymer and BDI molecules, n values of TBPUs were found in the range of , see The MThe surface and bulk hydrophilicity of the various TBPU copolymers was determined by water uptake and contact angle measurements. The preliminary lab observations revealed that when the specimens made of homo-PL and TBPU copolymers were immersed in water, the homo-PL specimen floated on the water due to its hydrophobic nature, whereas the TBPU specimens absorbed water rapidly and sank. These observations elucidated that synthesized TBPU copolymers hold more hydrophobic characters than the PL homo polymer. As can be seen in Water contact angle testing reports that better hydrophilicity results in a smaller contact angle . The varIt is worth mentioning that the contact angle measurements of TBPU samples with higher PEG contents  could not be tested. Their higher hydrophilicity and partial solubility in water caused instant spreading of the water drop and sometimes local distortion on the polymer surface.30 increase with increased hydrophobicity, i.e., for the lower PEG content or the longer PL segments in TBPU copolymers. The variations in hydrophilicity as a function of composition and time were further assessed by observing the variation of the water contact angle with time as presented in Many contributing factors are affecting the biodegradation of polyurethane polymers, such as hydrophilicity of polymer segments, the molecular weight of polymers, degree of microphase separation, and enzyme function. In this study, Lipase from the porcine pancreas was employed in the enzymatic degradation testing due to its proven ability to degrade PLA . Enzymatn lead to low degradation rate for polyurethane due to enhanced interaction force among the polymer chains that in turn resists water and enzyme attack [In contrast, weight loss values of 21%, 23%, and 27%, respectively, were achieved after 5 days for the high molecular weight polyurethanes ; see e attack . This exn = 67,500 g/mol; enzymatic degradation rate ~10%), one can conclude that the enzymatic degradation rate of TBPUs was approximately two times faster than homo-PL. This implies that the complete degradation of TBPU sample needs half the degradation time required for a homo-PL sample of comparable molecular weight.Comparing the enzymatic degradation rate of TBPU-3 to and the homo polylactide polymer of comparable molecular weight  needs approximately 1.5 months compared to 6.5 months required for the homo-PL sample. Nevertheless, a slight decrease of 0.5 pH units was observed at the end of depredation due to the release of some acidic byproducts from PL segments upon degradation. The non-significant change in pH associated with the weight loss of TBPUs suggests a reduced inflammatory response in vivo and supports the suitability of these materials for utilization as internal implants [Among the promising TBPU samples, TBPU-3, which had a relatively high Mimplants .− anions that act as strong nucleophiles [Moreover, the effect of changing pH value on hydrolytic degradation of TBPU-3 was also examined by studying the degradation profiles of TBPU-3 in PBS at different pH values; 7.4, 6.0, and 5.0. The degradation rate was found to be faster in PBS solution of pH 7.4 than the other acidic pHs. One probable reason is that pH 7.4 is a mildly basic medium, which promotes the hydrolysis of ester linkage by providing OHeophiles . The othThe surface morphology changes associated with the degradation process of TBPU-3 films were monitored using SEM; see m peaks at 64 °C and 186 °C, respectively. The thermograms of TBPUs copolymers of lower PEG content 10–18%  showed Tm peaks for PL segments only at slightly lower values than the homo-PL. More specifically, Tm values due to PL segments decreased further as PEG content increased. The absence of melting endotherm peaks of the PEG segment in those three TBPUs might be related to the lake of highly ordered and long crystalline domains for PEG soft segments compared to the long enough PL hard segments. However, the thermograms of TBPUs that have higher PEG contents 48–53%  showed broad Tm peaks at temperatures slightly higher than neat PEG, see m values of PEG might be related to the partial obstruction of polymer chain mobility in the crystallization domain of PEG by the long PL segments. Since the Tm of both neat PEG and PL segments had altered one another, it can be used as an indication for a successful copolymerization. Nevertheless, no Tg peaks were observed in DSC curves of TBPUs related to hard or soft segments. A probable explanation is that copolymerization reaction may have caused a decrease in the structure symmetry, which significantly inhibited the chain packing [m peaks corresponding to PEG segments disappeared whereas the Tm peaks for PL segments remained, see The DSC thermograms of TBPU copolymers, neat PEG, and PL homopolymers are shown in  packing . Furtherm of the soft PCL segments was not clearly observed in TBPUs isotherms. This might be related to the low Mn PCL used, which did not allow the formation of enough crystalline domains or because of the overlap of its endotherm in the melting range of PEG segments.Moreover, Tb), tensile strength at break (σb), and tensile modulus (E), see The mechanical properties of TB copolymers and their corresponding TBPUs were tested in this study by evaluating elongation at break  were increased as the chain length of PL increased, and PEG content decreased. Due to the impressive mechanical properties, hydrophilicity, and good degradation behavior, TBPU-3 was chosen among the three highest molecular weight TBPUs as a pilot for comparison and fabrication of nanocomposite.The tensile strength and elongation of PL, TB3, and TBPU-3 were examined at room temperature, and depicted in In this study, a total of six green and biodegradable TBPUs samples composed of PL, PEG, and PCL segments, and BDI as a nontoxic chain linker were successfully obtained and differentiated into two extremes. The low molecular weight polyurethanes with higher PEG content and shorter PL segments showed low mechanical strength and increased hydrophilicity, giving them an advantage for utilization in drug delivery and MRI imaging applications, whereas the other group that had longer PL segments and lower PEG content showed high mechanical strength and improved hydrophilicity, making them a great candidate for utilization in soft bone tissue regeneration. A promising member from the latter group  was chosen as a pilot for developing five different TBPU/BCNW nanocomposites. The maximum enhancement of tensile strength and percent elongation for the nanocomposites was achieved at 7 wt% nanowhiskers loading. Introducing PEG segments in TBPUs chains demonstrated high potential for improving the interaction between the polymer chains and BCNW, keeping the polymer integrity intact and supporting a strong network structure.


Dielectric spectroscopyin frequencies that range from 10 Hz to1 GHz has been used to study the molecular orientational dynamicsof the two types of dimers that form the twist-bend nematic phaseover a wide range of temperatures for both nematic and twist-bendnematic phases. The symmetrical and asymmetrical liquid crystal dimerswith the cyanobiphenyl mesogenic groups were investigated. The resultswere analyzed within the framework of the molecular theory of dielectricpermittivity for nematogens. The two molecular processes can be assignedto the reorientation of the monomeric unit: the high frequency oneto the precessional rotation of the longitudinal components of thecyanobiphenyl groups (CN) and the second (low frequency) to the end-over-endrotation of the CN dipole around the short molecular axis. The precessionmode, which is determined by the local order and is almost unaffectedby the phase transition from the nematic to the twist-bend phase,while the end-over-end rotation clearly slowed down at the transition,as it is affected by the growth of the intermolecular interactions.The latter corresponds well to the fact revealed by IR spectroscopyabout the longitudinal correlation of the molecular dipoles. Bent-coremesogens, for example, exhibit a rich variety of polar phases, whichare not typical for rod-shaped mesogens. The unusual properties ofthe bent-core nematic phases of the resorcinol derivatives, such asthe formation of optically active domains, which enhanced their flexoelectriceffect2 and strong magneto-optical effects,4 have been reported. Conversely, a spontaneous self-assembly canprovide information on the assembly of the components, that is, theirconformation/shape, charge, polarizability, magnetic dipole, mass,and so forth, in a specific phase, as these properties determine theirinteractions. Molecular self-assembly studies provide essential informationabout the influence of intermolecular interactions on the structureof the investigated molecular system. Therefore, such studies arecrucial when anisotropic molecular systems can lead to direction-dependentphysical properties. Helical structures, which have been the subjectof great interest in the field of molecular sciences partly becauseof their potential applications in electro-optical devices, have emergedas one of the most important subgroups of hierarchical structures.8 Even when chiral nematic phase has a pitch of few molecular lengths,the director twists and bends are generally accepted, as has beenconfirmed by several experiments: polarizing optical microscopy (POM),9 freeze fracture transmission electron microscopy,11 atomic force microscopy,11 nuclear magneticresonance (NMR),13 Raman scattering,14 IR spectroscopy (IR),16 and non-resonant hard X-ray scattering,18 as well as C K-edge and Se K-edge resonant X-ray scattering.20 The chirality of the phase is measured directly using the synchrotroncircular dichroism of aligned samples.21 However, the specific structure of the mesophase still remains amatter of debate.24 Although the basic features of this phase are nowestablished, the nature of this phase is still discussed controversially.The existing materials do not behave fully in line with the prevailingmodels.18 Besides, there have been significant theoreticaland simulation efforts toward developing an understating of the interactionsand underlying mechanisms that account for the origin of such nematic–nematic(N–N) transitions, with most recent results indicating stericinteractions and the primary role of the molecular shape as the drivingforce for the phase formation.27Ithas been commonly accepted that the large scale structures anddynamics of soft matter are controlled by weak intermolecular forceson the order of thermal energy. Therefore, thermal fluctuations aresufficient to significantly change the structure of a material. Forentropic reasons, macromolecules often exhibit a spontaneous self-organizationinto complex, ordered structures. These structures in soft mattersystems can occur hierarchically and can range from length scalesthat are comparable to the size of a single molecule to the higherorder, super-molecular scales. Typical examples are the spontaneouslyformed nanometer-sized patterns that are found in block-copolymersystems, or the hierarchical assembly of lipid molecules into membranesin biological materials. A crucial factor for determining the characterof an ordered structure is its mesogenic shape.TB) using dielectric spectroscopy of the bent-shape liquidcrystals. The collective dynamics were studied already in ref (TB) phases, whichare formed by the polar symmetric dimers CBSCnSCB and the similarasymmetric dimers CBSCnOCB. Dielectric relaxation measurements wereconducted for both samples in the entire temperature range up to thepoint of glass transition. The observed relaxation processes wereinterpreted based on the theoretical models. In both nematic phases,the dipolar–dipolar groups that are associated with the terminalcyanobiphenyl (CB) groups led to two relaxation modes that were relatedto the rotational dynamics of the molecules. The low-frequency mode,which is denoted as 1m, was caused by end-to-endmotion of the dipolar groups that are parallel to the mesogen axis.The high-frequency mode, which is denoted as 2m, was the result of the precessional rotation of the dipolar groupsaround the director. These modes contributed to the complex dielectricpermittivity differently depending on the orientation of the directorrelative to the electric field that was being measured. The secondimportant part of the analysis was the study of the glass transition,which was clearly identified below the N–NTB transitionin both longer dimers. The glass transition is characterized by thetemperature Tg and the rate at which variousproperties change with temperature as the liquid phase approachesthe glass transition. For systems that exhibit a certain number ofstructural relaxations, it is possible to identify the dielectricglass transitions that correspond to each of the two types of molecularmotion. The results for the symmetrical and asymmetric liquid crystaldimers presented here are discussed to provide information on howthe changes in the molecular interactions that are observed near theN–NTB transition and below it affect the aggregationprocess and how they are frozen during glass formation.The primary motivation for this work was toanalyze the orientationaldynamics of molecules in the nematic phases: N and twist-bend . In the asymmetric dimers with the acronymCBSCnOCB , the mesogens are linked to analkyl chain with five or seven methylene groups on one side by a thioetherbridge and on the other by an ether bridge. The molecular structureof the investigated compounds is shown in Thesymmetrical and asymmetricalliquid crystal dimers with the CB mesogenic groups were investigated.The symmetric dimers with the acronym CBSCnSCB contain thioether bridge(S–Cn–S) with five/seven methylene groups in the linker.For the higher frequency dielectric measurements, gold-plated cellswere applied. The temperature of the sample was stabilized using aPID temperature controller within ±2 mK. For the high-frequencyrange, we use a parallel plate capacitor made of two circular gold-platedbrass electrodes with 5 mm diameter separated by silica spacers. Thesample was placed at the end of a coaxial line. The planar alignmentin the range of the N and N33.1P to the macroscopicelectric field E.Electric dipoles can be used as effectivemolecular probes to detectchanges in the molecular structure using dielectric measurements.The electric permittivity is by definition an II-order tensor quantity,which relates the induced polarization Generally,a symmetricII-order tensor has six independent components, which can be reducedto three diagonal components if the tensor is defined in the principalaxis system. For axial symmetry, there are only two independent componentsof the permittivity: parallel and perpendicular to a single axis ofsymmetry. Under such assumptions, the dielectric permittivity tensorcan be expressed by the mean value ε and anisotropy Δε.33 In conclusion, we can state that electric dipoles can be used aseffective molecular probes to detect changes in molecular structureby means of dielectric measurements.Dielectric permittivityisa measure of the averaged molecularmean-square dipole moment, which is determined by the conformationallyaveraged sum of the dipole component vectors and is therefore directlyrelated to the distribution of molecular conformations.For anisotropic materials,the direction of the measurement isdefined by the topology of the sample, and where the electrodes aresituated. For a fluid, these directions are determined by externalconsiderations such as the surface area, flow ordering, or the electricor magnetic fields. The ordering of a uniaxial liquid crystal is determinedby a single director and can be arranged parallel or perpendicularto the surface of the electrodes via the appropriate surface preparation.34 The following equations give the dependencies of the dipolarcontribution on the main order parameter, S, andthe frequency of the field, ω.To represent the relationship between the dielectric propertiesof a liquid crystal and the molecular properties of a system of molecules,it is possible to obtain results that are equivalent to Onsager’sequation, but only in the case of an anisotropic fluid. This was firstdone for a uniaxial system by Maier and Meier.These equations assumethat both the cavity and its reaction fieldare isotropic, thus having a value that is the same as for an isotropicmedium. There is some basis for this assumption because the anisotropyof an external field reflects the far-field anisotropy in a radialsystem; for the radial distribution function for molecules, however,many models consider the development of an anisotropic internal fieldin liquid crystals, and these can be used if necessary.S, P, D, and C, which determine the degree of the orderingof molecules due to the phase symmetry. The basic order parameteris the S parameter, and it determines the orderingof the molecular axes with respect to the principal director. If theliquid crystal molecules are biaxial, but the phase is uniaxial, thenit is necessary to introduce the D parameter. Fora biaxial molecule, the two x, y axes that are perpendicular to the long molecular axis behave differently,and therefore, the ordering of these two axes with respect to theuniaxial director of the sample will also be different. The differencebetween them will be precisely the parameter D.Generally,the anisotropy of a material appears through the microscopicorder parameters The Maier–Meier equations presented here for the low-frequencycomponents of permittivity represent the dependencies on all of theorder parameters. These equations predict that the average value ofthe permittivity should be independent of the orientation order. Disregardingany density changes, the development of this value should be continuousthrough all of the liquid crystal phases. These predictions are notalways confirmed by experiments for dipole mesogens, but are oftenobserved for the mean value ε̅ with phase changes, includingin the transition into isotropic liquids.Dielectric measurementsof liquid crystals provide an opportunityto observe the organization of molecules and changes in the componentsin permittivity during phase transitions of a liquid crystal betweenthe nematic phases and from the nematic (N) phase to the various smectic(Sm) phases. Thus, they primarily reflect changes in the orientationalorder and changes in the symmetry.36 The conformationaldistribution is also influenced by the anisotropic nematic interactions,thus favoring the molecular geometries that can better adapt to thenematic ordering and environment. The temperature dependence of thedielectric response of a liquid crystalline phase depends on the rotationaldistribution of the molecular dipoles in the presence of an electricfield and the orientation of the phase. However, the order parameteritself is temperature-dependent and, as indicated earlier, the conformationaldistribution affects via both the shape and the order parameter.In liquid-crystalline dimers,the molecular flexibility that isprovided by the changing distribution of the conformers has a stronginfluence on the energetically favored molecular shapes and thus onthe mesophase properties.i is the subscript for paralleland perpendicular directions.The effect of the macroscopic anisotropy in measuring the dielectricproperties of an LC material was calculated for a model of a polarizablecavity that was immersed in a dielectric continuum. To go furtherbeyond this approach, it was necessary to create a model for the anisotropiclocal field acting on the molecule and for the short-range interactionsbetween the dipole moments inside the cavity. The former depends onthe long-range anisotropy in the radial distribution, while the dipole–dipoleinteraction can be described by the anisotropic Kirkwood coefficientthat is defined for the different directions in the sample. Thesecoefficients are most easily defined as the corresponding dipole correlationfunctions, where g∥  and g⊥ (perpendicular) determine to what extentthe individual dipole moment projections are correlated along themajor axis of anisotropy, assuming the uniaxial case. If the rotationalmotion of the molecules is isotropic, then the correlation coefficientsalong different axes should be the same.Thus, 3.2Ea for the reorientation of a moleculardipole in a dielectric environment.The simplestdielectric absorption model that is applicable to most materials isthe Debye model, which describes the single dipole relaxation process.The temperature dependence of the relaxation time can be representedby the phenomenological Arrhenius equation, which introduces the activationenergy Although theArrheniusequation describes the dielectric relaxation of many simple fluidswell, there are several materials for which the equation fails, andit is necessary to modify equations that describe experimental results.One of the phenomenological equations that are most commonly usedto describe the temperature dependence of relaxation time data (τ)is the Vogel–Fulcher–Tammann (VFT) equation:T0, some theories such asthe Adam–Gibbs entropy model37 andsome more recent theoretical models40 are based on the VFTequation. The basic idea behind these theories is that glass formationis associated with highly cooperative movements in a structurallyfluctuating sample, with cooperativity growing as Tg is approached.Despite some concernsabout the validity ofthe VFT equation, becauseit assumes a dynamic divergence of the relaxation time at some finitetemperature, 3.342 Rotation of the molecule about a short molecular axis in the presenceof the nematic potential gives rise to the low frequency relaxationmode 1m that is detected in the parallel componentof the permittivity. Relaxation of the longitudinal component of themolecular dipole by precession about the director axis contributesto a high frequency mode 2m that is detectedin the perpendicular component of the permittivity. The dielectricrelaxations were related to the rotational diffusion of the electricdipoles: the reorientation of the end-to-end longitudinal dipolesat low frequencies (1m) and the precessionalmotion of the dipolar groups around the director for the high-frequencybranch of a spectrum (2m).This problem is compounded in dielectric studiesof liquid crystals,because of their macroscopic anisotropy and the presence of a nematicpotential. One solution of the rotational diffusion equation for arigid dipolar molecule in the presence of a nematic potential predictsthat the frequency dependence of each component of the electric permittivitycan be characterized by two exponential decays.q/RT, and q is the height of thebarrier separating the two minima along the n direction,while the approximate expressions for the relaxation times were derivedas reported in ref (1m) according to the theoretical model of dielectric relaxationin nematic dimers,33 and the precessionalmotion of the dipolar groups around the director for the high-frequencybranch of a spectrum (2m). The longitudinal,μl, and transverse, μt, componentsof the molecular dipole moment, μ, contribute to the dielectricpermittivity differently, and they relax at different frequenciesof the probe field. In solving the rotational diffusion equation,a uniaxial nematic potential is assumed. The relaxation times of modes 1m and 2m are described,in terms of spherical harmonics, by τ00 and τ10, respectively:D, is the one for rotational diffusion in the isotropic phase.A nematicpotential is assumed to be of the form:d in ref . The die44.133 Therefore,electric dipoles can be used as effective molecular probes to detectchanges in the molecular structure using dielectric measurements.For CB7CB and CB9CB symmetric dimers, the mean square dipole momentis determined by the vector-averaged sum of the dipole moments thatare associated with the nitrile groups that are attached to the terminalmesogenic units. Different molecular conformers will contribute tothe measured components of dielectric permittivity differently.Dielectricpermittivity is a measure of the averaged molecular mean-square dipolemoment, which is determined by the conformationally averaged sum ofthe dipole component vectors and is therefore directly related tothe distribution of the molecular conformations.36 To some extent, the conformationaldistribution is also determined by the nematic interactions, thusapproving geometries that can be better adapted to nematic ordering.The temperature dependence of the dielectric response of a liquidcrystalline phase depends on the rotational distribution of the moleculardipoles in the presence of an electric field as well as the orientationof the phase. However, the order parameter itself is temperature-dependentand, as was indicated earlier, the conformational distribution affectsboth of the molecular dipoles via the shape and the order parameter.The results of calculating the conformational energy as a functionof the bending angle of a dimer indicated that the CB7CB homolog hadtwo peaks: a strong one that was centered around 120° (extendedconformers) and a weaker peak at 30° (hairpin conformers).31 They also showed that as the orientational orderincreased, the population of extended conformers grew; simply, theshape of the molecule adapted to the increased orientational orderin the nematic phase while cooling.43 Aswas expected, the hairpin conformers had a large contribution to theparallel permittivity of the mean-square dipole moment that was measuredalong the director. The extended conformer had a zero mean-squaredipole component, thus contributing to parallel permittivity alongthe dipole. However, for such conformers, there was a non-zero (transverse)contribution of the dipoles to the perpendicular component of permittivity,which was dependent on the angle between the end dipolar groups. Becauseof the large amount of molecular flexibility, each of the dipolargroup had a large contribution to square dipole moment. Another importantphenomenon appeared at the transition to the NTB phase.Because of the specific arrangement of the terminal mesogenic unitsin the local environment, the short-range interactions between dipolemoments increased, which affected both the effective dipole momentthrough the positional correlations, and, consequently the dielectricpermittivity. In addition, the relaxation rates for the transverseand longitudinal dipole polarization were different, and therefore,the changes in the conformational distribution of the interarm anglewere clearly reflected by the dielectric permittivity measurements.We measured the parallel and perpendicular components of the staticdielectric permittivity when cooled from the isotropic phase. Theperpendicular and parallel components of the permittivity  were obtained in the planarand homeotropic orientations, respectively, in the presence of a weakmeasuring field (0.5 Vrms/μm). The opportunity of obtaininga homeotropic orientation in a strong field was rejected, as thisclearly evokes the conformational changes that are induced by thefield. Both components were measured as a function of temperatureat different frequencies.In liquid-crystalline dimers, the molecular flexibility that is deliveredby the linkage and its conformational mobility has a strong influenceon the energetically selected molecular appearances and thus on themesophase properties.|| +2ε⊥)/3 and dielectric anisotropy (Δε= ε|| – ε⊥) are shownin 40 As was expected for the materialswith a positive dielectric anisotropy, the value of the perpendicularcomponent of the permittivity decreased from the isotropic phase aftercooling, while the parallel component increased at the isotropic-nematictransition.The results include the relaxationof the molecular modes and thecollective soft mode but do not include the influence of the ioniceffects and the collective modes below 10 kHz. In addition, the averagedielectric permittivity  according to the theoretical model ofdielectric relaxation in nematic dimers33 and the precessional motion of the dipolar groups around the directorfor the high-frequency branch of a spectrum (2m). In terms of spherical harmonics, their relaxation times are classifiedto be τ00 and τ10, respectively, as defined in The dynamic dielectric responses were measuredin metal cells with 50 μm spacer, in the frequency range of10 Hz to 1 GHz. The dielectric response of CBSCnSCB and CBSCnOCB inthe nematic phase exhibited two relaxation processes whose contributionto the dielectric spectrum depended on the orientation of the directorin the measurement cell. These dielectric relaxations were relatedto the rotational diffusion of the dimer molecules: the reorientationof the end-to-end dipolar groups parallel to the director at low frequencies for the odd dimershad no longitudinal dipole moment if the angle between the terminaldipoles was greater than 90° and their contribution to the orientationaldielectric permittivity was insignificant. However, the hairpin conformerswith small interarm angles, for which both rigid units usually tendedto align along the director, contributed significantly to the parallelcomponent of the permittivity. Thus, the strong decrease of the permittivityafter the temperature was reduced reflected a progressive increaseof the population of the bent (trans) molecular conformers, whichwere accommodated better by the nematic potential.1m and 2m, of the symmetric CBSCnSCB and asymmetric CBSCnOCBdimers, as was also observed by Cestari et al.,44 López et al.,45 and Merkelet al.46 for CBnCB.As was alreadystated, at higher frequencies, the ε″spectra were dominated by two maxima. These corresponded to the twomolecular relaxation modes, S, and the so calledDebye relaxation time, τD, which corresponds to therelative relaxation in the absence of orientational order. This canbe interpreted simply as an extension of the isotropic relaxationtime into the temperature range of the nematic phase. Following 2m, can arise from therotations of a segment of the molecule, that is, the internal rotationof each monomer with the spacer anchored involved the fluctuationsof the CB dipolar moment. Because the length of the spacer in betweenthe two mesogens in a dimer is large enough, such an independent internalrotation of each monomer of a dimer is highly feasible. The temperaturedependencies of δε2 and of the relaxation timeof mode 2 are suggestive of the precessional rotationof the longitudinal component of each thioether CB (SCB) dipole momentaround the director in a planar-aligned cell.We simultaneouslyanalyzed the relaxation times of the two observedprocesses in the N phase and were able to describe them unequivocallyby the orientational order, 1 and δε2 of the 1m and 2m modes  were strongly affected by both an increase in thepopulation density of the bent conformers as well as the increasingstrength of the intermolecular interactions that accompanied the formationof NTB phase. The relaxation times of the modes, however,seemed to be well defined solely by the local orientation order inthe nematic phase. For all of the dimers, the dynamic behavior inthe nematic phase was reproduced quite well by the molecular dynamicmodel, as shown in The results inthe N phase were, however, more complex than forthe monomers since the dielectric amplitude and its relaxation ratewere strongly affected by the reorganization of the conformers interms of their molecular shapes. Two major conformers were consideredto be the bent cores: a stretched conformer with a transversal dipolemoment and a hairpin-shaped conformer with a large longitudinal dipolemoment. This was clearly reflected in the results of the temperaturedependencies of the average dielectric permittivity (ε̅),which are presented odes see S1 were s1 and τ2, for mode 1 and 2, respectively,were well fitted using S, and τD were unknownvalues. The results are shown in D and in S. The temperature dependencies of the orientational parametersquite well correspond to the results obtained by infrared spectroscopy,15 but their values are slightly smaller than referenced(IR) because dielectric ones are related to the S-CB or (O-CB) dipolemoments, but the IR ones correspond to the molecular (bow) axis.The experimental relaxation times, τTB phase, the temperature dependenciesof theτ10 kept their trends without any step at the transitiontemperature, despite the fact that the director became tilted withrespect to the symmetry axis. It seems that this process is determinedexclusively by the local orientational order , which was growing continuously with a decreasing temperature.If the orientational order is calculated using dielectric strengthsor birefringence, then we have S in the other referencesystem: where the z-axis is along the helical axis.In such a case, the molecular tilt at the N to NTB transitiontemperature affects the S parameter. The resultscan be converted from one unit system to another by considering thetilt of the director.In the N1m). First of all, therelaxation time dependenceclearly exhibited a kink at the transition temperature, which indicatedan increased relaxation rate at the transition temperature. This wasmore likely due to critical fluctuations in the director in the vicinityof the transition.47 Then, in NTB phase, the temperature trend of the relaxation time suddenly increasedwith respect to the trend in the nematic phase. We simply interpretedthis fact as an apparent growth of the potential barrier, q, which separated the two minima along the n direction after entering the NTB phase. This findingcorresponded well with the orientational correlations effect of thelongitudinal dipoles, which was revealed by IR spectroscopy.16This was not the case for the end-over-endrelaxation time  up to about 18 kJ/mol in the NTB phase;, however,the temperature trends for the shorter dimers (n =5) are clearly different than for the longer ones (n = 7).TTB, regardless of the cooling rate. Both of the longerlinker dimers, CBSC7SCB and CBSC7OCB, exhibited a smaller/slower increaseof the potential barriers that extended in the range of 20–30K, after which they reached their saturation, while the samples underwentvitrification. It is worth mentioning that the barriers increasedin two steps: the first step corresponded well to an orientationalcorrelations effect of the longitudinal dipoles (g∥ < 1) along the major axis of anisotropy, andthe second step appeared at the temperature at which the interactionsalong the perpendicular axis became important (g⊥ > 1) that was revealed by IR spectroscopy.16For the shorter linker in dimers, the potentialbarrier increasedsteeply, and finally, both samples (CBSC5SCB and CBSC5OCB) soon crystallizedat a few degrees below the transition temperature, TB, during which the typical glass-formingbehavior was observed on the cooling for the two longer dimers thathad seven carbons in the link . The phenomenologicalequations, the VFT formula 0 for the 2m mode was of the orderof 10–11 s.We will now focus on the temperature rangethat is well below (∼20K) the transition N-N–9 to 10–10 sin the isotropic, nematic, and disordered smectic phases. Thus, interpretingthe dielectric relaxation processes at the MHz or even kHz frequenciesin terms of the rotation of a single molecule is unlikely to be correct.The low frequency relaxations that were observed in liquid crystalsare the result of a collective molecular motion, although the modelsoutlined above are useful for analyzing the results and comparingmaterials.Themodels that were used to interpret the low frequency relaxationin liquid crystals are often based on a single particle relaxationprocess; however, spectroscopic probes of molecular motion such asmagnetic resonance, neutron scattering, and time-resolved fluorescencedepolarization suggest that the reorientation times for mesogens areof the order of 1049 and the resultsfor CBSC7SCB and CBSC7OCB are presented.50A more detailed method for studying the dynamicsof glass formingbehavior is to analyze the derivatives of the relaxation time withrespect to the inverted temperature,5The dielectric relaxationspectra for two symmetric CBSCnSCB andthe asymmetric CBSCnOCB dimers were analyzed in terms of their relaxationprocesses as predicted by the rotational diffusion model. For theodd dimers when considering a nematic environment, the continuoustorsional potential model predicted a broad conformational distributionthat was dominated by the bent conformers, but with an appreciablecontribution of the hairpin molecular configurations at high temperatures.S, and therefore, their isotropic relaxation time, τD, which corresponded the relaxation of the system in the absenceof an orientational order. The high frequency relaxation process originatedfrom the precession rotation of the longitudinal component of thedipole moment of the monomers of each SCB dipole moment around thedirector. The relaxation rate was accelerated with respect to theisotropic relaxation because τ10 was shorter comparedto τD. The low frequency relaxation originates fromthe “end-over-end” rotation of the dipole moment ofthe monomers. The relaxation rate of that mode was lower with respectto the isotropic mode. It was found that the model reproduced boththe relaxation times dependence very well in the nematic phase forboth modes. The temperature dependencies of the orientational parameters, S, that were obtained, corresponded quite well with theresults that were obtained with infrared spectroscopy, although theirvalues were slightly smaller because they were related to the S-CBor (O-CB) dipole moments unlike in the case of the IR ones, whichdescribed the molecular axis. It is interesting to note that the relaxationtime maintained its temperature trend during the transition from theN to NTB phase, as was predicted by the power law approximationof the S parameter. It seems that it was determinedby the local orientational order, which grew continuously regardlessof the tilting of the director that appeared at the transition. Thelow-frequency relaxation time, τ00, was significantlyaffected by the transition from the N to the NTB phase.Initially, it exhibited a kink at the transition temperature, whichwas likely due to critical fluctuations of the director in the vicinityof the transition at which the τ00 relaxation timebegan to grow much more steeply. We interpreted this fact as an apparentgrowth of the potential barrier, q, that separatedthe two minima along the n direction after enteringthe NTB phase. This corresponded well with the effect ofthe orientational correlations of the longitudinal dipoles, whichwas revealed using IR spectroscopy.The experimental spectra were dominated by the two relaxation processesfrom the rotations of a segment of a molecule, that is, the internalrotation of each monomer with the spacer anchored that involved thefluctuations of the CB dipolar moment. Because the length of the spacerbetween the two mesogens in the dimer was large enough, such an independentinternal rotation of each monomer of the dimer was highly feasible.By simultaneously analyzing the two processes in the N phase, theywere described unequivocally in terms of their orientational order,


Bovine tuberculosis (bTB) is a severe zoonotic disease that has major impacts on both health and the economy, and which has been subjected to specific eradication programmes in many countries for decades. This manuscript highlights the relevance of this disease in the context of the European Union (EU) and summarizes the epidemiological situation and the main tools  used to control and eradicate bTB in the various EU countries with a focus on the situation in Spain. A comprehensive description of the specific bTB epidemiological situation in Spain is provided, together with an assessment of the evolution of different epidemiological indicators throughout the last decades. Moreover, the main features of the Spanish bTB eradication programme and its control tools are described, along with the studies carried out in Spain that have allowed the updating of and improvement to the programme over the years with the aim of eradication, which has been established for 2030. Mycobacterium tuberculosis complex (MTBC), mainly Mycobacterium bovis, from animal origin), especially in Africa and Southeast Asia. As an example, in 2016 there were 147,000 new cases of zTB in the world, and 12,500 people died from it [Mycobacterium bovis from M. tuberculosis is limited or even absent. An accurate estimate of the importance of zTB is impossible in those mostly low and middle income countries, thus making it a clearly underestimated disease [According to data published by the WHO, it is estimated that more than 140,000 people become ill and more than 12,000 people die each year as a result of zoonotic TB  in endemic areas, animals can also be affected by reverse zoonosis, including multi-drug resistant strains [The control and eradication of TB infection in cattle through the use of diagnosis and cull strategies continues to be the mainstay as regards reducing the risk of zTB. Given the high burden of human TB (caused by  strains .M. bovis or M. caprae were reported in the EU in 2021 (32 of them in Spain). The EU notification rate in 2021 was 0.03 cases per 100,000 population members, which was identical to that reported in 2020. This EU notification rate decreased by 28.3% and 25.3%, respectively (i.e. with or without taking the data concerning the UK into account), when compared with the mean rate observed during the pre-COVID pandemic years from 2017 to 2019. Interestingly, M. bovis and M. caprae notification rates did not differ significantly between EU countries with a disease-free status as regards cattle and those with a non-disease-free status [According to the European Food Safety Authority (EFSA), 111 confirmed cases of human TB resulting from e status .M. tuberculosis complex  in bovine animals and other mammalian species are included in the list of notifiable animal diseases (Commission Implementing Regulation (EU) 2018/1629) of Regulation (EU) 2016/429 on transmisible animal diseases  that was adopted in March 2016. Commission Delegated Regulations (EU) 2020/688  and 2020/689  have suplemented Regulation (EU) 2016/429 and replaced Council Directive 64/432/ECC. The eradication of bTB has been an important issue since the beginnings of the European Economic Community (EEC) in 1957, and current EU policies on the eradication of the disease are best understood after considering the progressive development of relevant legislation.In the EU, the situation regarding bTB is heterogeneous and has improved in recent years, although not significantly. The strategy employed to eradicate bTB includes a variety of measures. At the EU level, the Subgroup of the Bovine TB Task Force (Task Force Subgroup DG SANTE) has provided reports prepared by groups of experts in the field. Of these, the document “Working Document on Eradication of bovine TB in the EU” is worth highlighting, since it establishes specific guidelines that Member States should follow in order to accelerate the eradication process . Infection with The scientific report published annually by EFSA and the European Centre for Disease Prevention and Control (ECDC) on the situation of zoonotic diseases in Europe  providesIn 2021, the overall proportion of cattle herds testing positive for bTB was very low , but slightly higher than that reported in 2020 (0.4%). Several MSs did not report cases of bTB , whereas other MSs  reported the presence of bTB in their territory. The overall bTB herd prevalence was 1.3%. When comparing the data for 2021 with those for 2020, the overall number of cattle herds infected annually, the prevalence and the total number of cattle herds increased in these non-disease-free zones. This increase was mainly attributed to the withdrawal of the United Kingdom from the EU in 2020 and to the subsequent (2021) inclusion of data concerning Northern Ireland (UK), a region with a bTB prevalence that is relatively higher than in the UK as whole. The prevalence of bTB in 2021 varied widely among those MSs with a non-disease-free status: Ireland (4.6%) and Spain (1.3%) were the only MSs to report a prevalence higher than 1%. Northern Ireland, which has no disease-free zones, reported a prevalence of 11.3% [M. bovis were reported in Bulgaria, France, Germany, Hungary, Ireland, Italy, Poland, Romania and the United Kingdom (Northern Ireland), whereas infections owing to M. caprae were reported by Austria, Germany and Romania. No cases of M. tuberculosis infection in cattle were reported [Most of the MSs reported MTBC infections in 2021 without specifying the species involved. Infections with reported .In Spain, herd prevalence had undergone a moderate decrease in the 15 years prior to 2013, after which this indicator increased, especially in 2015 and 2016, reaching a level similar to that reported in 2001 , but the disease has been reported in a wide range of other domestic and wildlife species on the Iberian Peninsula that can act as reservoirs , 7. In SThe highest prevalence of bTB in Spain is in the west-central and southern regions Fig. , where tIn order to monitor and control the distribution of MTBC members in animals in Spain, it is necessary to collect all the information of epidemiological interest, such as the geographical location, the animal species from which the microorganism was isolated or the genotype . It is, therefore, essential to develop databases containing this information that users can consult and use in their epidemiological studies. The Spanish Ministry of Agriculture, Food and Fisheries (MAPA) has several databases. The REGA database (register of livestock establishments) is a multi-species register containing all the data concerning the establishments located in Spain. The REGA, REMO (register of movements) and RIIA  are essential parts of SITRAN, which is an integral traceability system. Moreover, in 2009, the BRUTUB database was created so as to include all the epidemiological surveys conducted on positive farms in order to allow their analysis and identify risk factors related to the occurrence of bTB outbreaks.M. bovis and M. caprae) obtained in Spain. These data include the animal species of origin, the geographical location, the year of isolation, the spoligotyping profile and the MIRU-VNTR profile (if available). The mycoDB.es database [Additionally, databases containing epidemiological and molecular data regarding the MTBC isolates recovered are also available. From the point of view of Animal Health, Mbovis.org  is one of the most important international databases on animal TB . Its maidatabase  containsTransmission models have also been used in order to study TB infection in more specific conditions in Spain. Alvarez and collaborators estimated the coefficient of intra-herd transmission using data obtained from herds in the Autonomous Region of Madrid that included the main farming industries in Spain  . In thisThe identification of both the origin of bTB outbreaks and the main factors that contribute to an increased risk of infection can provide important indications for the design of effective prevention and control strategies. According to the Spanish bTB eradication programme, for each newly infected herd confirmed by culture (i.e. a bTB breakdown), the official veterinary officers (VOs) identify the possible source of the breakdown by conducting a questionnaire-based epidemiological investigation. At the end of each questionnaire, the VOs should also indicate what, in their opinion, is the most likely cause of the breakdown. Since 2009, these data have been recorded in the BRUTUB database, which is held by MAPA. Furthermore, since 2012, epidemiological surveys have also been conducted on skin-test negative farms (i.e. control farms) selected by the VOs through pairing by herd size and location in order to identify potential risk factors, and the data obtained have been recorded in the BRUTUB database.Guta and collaborators investigated the origin of the bTB breakdowns reported in Spain between 2009 and 2011 by analysing the data recorded in this database . The investigation showed that the most frequent cause was residual infection, followed by interaction with wildlife reservoirs , resultsIn addition to these studies, great efforts have been made at national and regional levels to discern which other risk factors may shape the risk of occurrence and the duration of bTB outbreaks. In this respect, studies have been carried out in order to both analyse the movement of animals as a source of bTB  and idenAll the information obtained from epidemiological studies is valuable for stakeholders and its implementation in future strategies in eradication and control programmes.The first actions taken against bTB began in Spain in the early 1950s. In 1965, a national plan to control and eradicate bTB and brucellosis was established, focused mainly on the dairy cattle population located in the north and centre of Spain. After the inclusion of Spain in the EEC, Spain presented an Accelerated Eradication Programme in 1987, in accordance with Directives 77/391/EEC and 78/52/EEC and Decision 87/58/EEC. Efforts have since been made to apply the eradication campaign in a homogeneous manner throughout Spain and in all production types .The National Programmes for the Eradication of Bovine Tuberculosis 2006–2010 represented a qualitative change in the approach employed to attain the objectives, since they laid down the principles required to guarantee continuous actions over time using a multiannual approach, established over a 5–year period. One of the main objectives of these programmes was the gradually increase in diagnostic sensitivity (Se) at both herd and individual levels. Moreover, specific measures based on previous studies were gradually implemented, such as the control of wildlife reservoirs or the effective implementation of a post-mortem surveillance system in slaughterhouses. In fact, up to 8–9% of all bTB cases in Spain are detected as the result of the passive surveillance at the slaughterhouse, whereas the remaining cases are detected as the result of the active component of the eradication programme . However, according to a recent study carried out in a region of Spain, the probability of detecting animals with bTB lesions is highly influenced by the slaughterhouse at which an animal is slaughtered, ranging from 603 to 3,070 per 10,000 animals for the reactors and 0.2–16.1 per 10,000 animals for the non-reactors . In addiThe official diagnostic tests used are those established by Royal Decree 2611/1996, Sect. 2 of Annex III (diagnostic methods for the granting and maintenance of disease-free status) and article 9  of Regulation (EU) 2020/689 and the National Eradication Programme. Ante-mortem diagnostic tests are carried out in animals older than 6 weeks in the case of the single and comparative intradermal tuberculin (SIT and CIT) tests and 6 months in the case of the gamma-interferon release assay (IGRA), which are typically used in parallel with the intradermal test in confirmed bTB-infected herds. The frequency of tests is established according to the prevalence in the different areas and is specified in the National Eradication Programme. The IGRA is carried out by laboratories authorised in accordance with the aforementioned Royal Decree 2611/1996, which periodically participate in inter-laboratorial tests carried out by the Spanish NRL. Although the use of the IGRA can help to overcome some of the disadvantages associated with the use of SIT/CIT tests, its use as a stand-alone test is limited owing to its comparatively lower diagnostic specificity (Sp). The fact that different commercial kits are available signifies that a careful assessment of the performance of these kits help to adjust the cut-off values on the basis of the strategy of use and the objectives [The routine technique employed in order to obtain and maintain the officially bTB free status is mainly the SIT test, although the CIT test can also be used under specific epidemiological circumstances and at the discretion of the competent authority. Since Regulation (EU) 2016/429 came into force, the IGRA test can be used as an alternative to the SIT/CIT tests in animals older than 6 months, always at the discretion of the competent authority, as a routine test by which to grant or maintain the OTF status and as a pre-movement test, in accordance with Sect. 2 of Annex III of Regulation (EU) 2020/689 and the protocol published for this purpose on the European Union Reference Laboratory (EU-RL) website, in accordance with Article 6 of Regulation (EU) 2020/689. In this respect, the scientific opinion published by the EFSA regarding the suitability of the IGRA for inclusion as an official primary or stand-alone test and as an equivalent to the intradermal test used to define the infectious status  has beenNevertheless, the EFSA employed previous studies as the basis on which to conclude that the Sp of the IGRA might not, in certain conditions, be as high as that of the SIT test, which is the skin test with the lowest Sp approved in the EU. Therefore, and taking into consideration ongoing research on improved antigens, the EFSA recommended further evaluations of the performance of newly available specific antigens for blood stimulation in the IGRA with the aim of improving its Sp . In thisSeveral studies have been carried out in order to evaluate the performance of the official diagnostic tests (ante-mortem and post-mortem) and the overall effectiveness of the bTB diagnostic strategy in Spain , 30, andThe eradication programme in Spain allows the use of strategies depending on bTB prevalence: in OTF regions (A) or regions with 0 prevalence , the competent authority may extend the interval between routine herd-tests from one to two years. In addition, in regions that have maintained a herd prevalence of below 1%  for two consecutive years (B) the interval between the routine herd-tests may be extended to two years (24 months) in historically TB-free (hOTF) establishments . This occurs in provinces in which 100% of the target herds are included in the national programme and specific herds such as bullfighting herds or herds using shared pastures are excluded. In (non-bullfighting) hOTF establishments, SIT or CIT tests can be used as a routine test on the basis of a previous risk assessment. If positive animals are detected by using the CIT test, the results should be re-evaluated on the basis of SIT. The IGRA protocol published by the EU-RL can also be used as a routine test with the same considerations as those of the SIT test. Similarly, if the competent authority decides to apply the CIT test, those animals that attain a positive result in the single interpretation  but a negative result in the CIT test (owing to a larger increase in the skin fold thickness on the avian PPD inoculation site) are considered “follow-up cattle”. Their movements are subsequently restricted and they are subjected to post-mortem sampling when sent to the slaughterhouse or in the by-product processing plant in order to carry out bacteriology. SIT or IGRA tests are used for the routine testing of the remaining OTF herds. Non-OTF herds must be tested at least twice a year. Finally, in regions with a bTB prevalence higher than 1% , the control measures are more severe and the SIT and IGRA tests are used for routine testing even in OTF herds .In Spain, the measures adopted in order to deal with positive cases are described in Chapter II of Royal Decree 2611/1996 and in articles 24 to 31 of Regulation (EU) 2020/689. Particular emphasis is placed on those establishments in which a positive animal has been detected following the prophylactic measures established in article 24 of Royal Decree 2611/1996. Specifically, the OVs will certify that the cleaning and disinfection measures have been correctly carried out, that a minimum period of 60 days of sanitary vacuum for the reuse of pastures has been applied, and that manure has been correctly managed. These certificates may be issued on the basis of others that have been completed by accredited and duly approved companies, mainly with regard to the management of manure and the disinfection of the facilities.As described previously, the actions related to the control of TB in wildlife included in the National Eradication Programme were complemented with PATUBES, agreed at the central level with the other official authorities with competences in matters related to the environment and hunting and with the competent authority in matters regarding public health and food safety. This is in accordance with the recommendations made by the European Commission and anticipates the future European Legislation on animal health by incorporating wild species into the control of diseases that affect domestic livestock. The materialisation of PATUBES took place in Royal Decree 138/2020, as a basic legislative instrument that aims to control the risk to public health, animal health and the environment by reducing the spread of the disease among the different susceptible species of domestic and wild animals or those that are hunted.M. bovis, bovine PPD tuberculin) developed at the end of the 19th century by Koch and initially conceived in order to cure the disease [Finally, it is important to highlight that the cornerstone of all bTB eradication programmes comprises the tuberculin tests, which are based on the intradermal injection of a reagent , which in the case of Spain and has been established for 2030. The objective is not easy, but the great advances made in recent years in aspects related to the epidemiology, diagnosis and control of the disease foster our optimism. In this respect, it is noteworthy that several regions of Spain have already attained this objective and that bTB surveillance is improving in the rest of Spain. Despite this, tuberculosis research remains essential, particularly as regards antemortem diagnosis, which is still a great challenge. Moreover, the consideration of sociological aspects in the eradication programme has gained relevance in recent years, especially with regard to the motivation of the veterinarians involved and to the improvements made to communication with the farmers in order to help them to realise they are an essential part of the eradication process and for them to appreciate the potential benefits in the medium and long term. There is currently a consensus among researchers and official veterinary services as to the feasibility of the objective established for 2030 if the positive evolution in the indicators of the programmes is maintained and the numerous advances in research continue to be applied.


CHMP4C is one of the charged multivesicular protein (CHMP), and is involved in the composition of the endosomal sorting complex required for transport III (ESCRT-III), facilitating the necessary separation of daughter cells. CHMP4C has been proposed to be involved in the progression of different carcinomas. However, the value of CHMP4C in prostate cancer has not yet been explored. Prostate cancer is the most frequently occurring malignancy among male and remains a leading cause of deaths in cancers. So far, clinical therapy of prostate cancer is more inclined to molecular classification and specific clinical treatment and research. Our study investigated the expression and clinical prognosis of CHMP4C and explored its potential regulatory mechanism in prostate cancer. The immune status of CHMP4C in prostate cancer and relative immunotherapy were then analyzed in our study. Based on CHMP4C expression, a new subtype of prostate cancer was established for precision treatment.We studied the expression of CHMP4C and relative clinical outcome using the online databases TIMER, GEPIA2, UALCAN, and multiple R packages. Meanwhile, the biological function, immune microenvironment and immunotherapy value of CHMP4C in prostate cancer were further explored on the R software platform with different R packages. Then we performed qRT-PCR, Western Blotting, transwell, CCK8, wound healing assay, colony formation assay and immunohistochemistry to verify the expression of CHMP4C, carcinogenesis and potential regulatory mechanisms in prostate cancer.We found that the expression of CHMP4C is significant in prostate cancer and the high expression of CHMP4C represents a poor clinical prognosis and malignant progression of prostate cancer. In subsequent vitro validation, CHMP4C promoted the malignant biological behavior of prostate cancer cell lines by adjusting the cell cycle. Based on CHMP4C expression, we established two new subtypes of prostate cancer and found that low CHMP4C expression has a better immune response while high CHMP4C expression was more sensitive to paclitaxel and 5-fluorouracil. Above findings revealed a new diagnostic marker for prostate cancer and facilitated the subsequent precise treatment of prostate cancer. CHM. CHM7). 22.1https://cancergenome.nih.gov/) and used for validation of the difference analysis and paired difference analysis with “ggpubr” and “limma” packages (The timer database (TIMER2.0 (cistrome.org)) was applied to compare the differences in the expression of CHMP4C between prostate tumor samples and normal tissue samples . Prostatpackages .2.2http://gepia2.cancer-pku.cn/#analysis), “survival” package and “survminer” package were applied to analyze the prognosis of CHMP4C in prostate cancer (The GEPIA2 database (e cancer . UALCAN e cancer  20)..http://g2.3To further investigate the regulatory role of CHMP4C in cell proliferation, we performed co-expression gene analysis and GSEA analysis using the “limma” package and “enrichplot” package and identified important regulatory roles of CHMP4C in the cell cycle. To deepen our understanding of the biological functions of CHMP4C, we conducted grouping differences analysis of CHMP4C using the “limma” package and performed KEGG and GO analysis based on the grouping results. KEGG and GO gene sets were got from the Gene Set Enrichment Analysis (GSEA) website (GSEA (gsea-msigdb.org) . Then th2.4limma package, estimate package and reshape2 package were used to analyze the immune and mesenchymal scores of CHMP4C in prostate cancer. CIBERSORT is a deconvolution algorithm that can be used to transform the normalized gene expression array into the composition of infiltrating immune cells. Based on the result of CIBERSORT, we obtained the relationship of CHMP4C with the infiltration level of 22 immune cells using “limma” packages. We then explored the relationship of CHMP4C expression with 49 immune checkpoint genes with “ggplot2” and “reshape2” R packages and set the p-value filter to 0.001.2.5https://tcia.at) and utilized to predict the response to ICI in patients with low and high expression of CHMP4C. External independent immune validation queue data from GEO database . The “pRRophetic” package was applied to predict the drug sensitivity of CHMP4C.IPS-CTLA4 blocker and IPS-PD1/PD-L1 blocker data for prostate cancer from TCGA were got in TCIA  with 10% added fetal bovine serum  and 1% penicillin and streptomycin . The environmental conditions for incubation were 37 degrees, 5% CO2 humidified incubator.2.7Total RNA Kit  was used to isolate and purify total RNA from cell lines, and BIOG cDNA Synthesis Kit  was used for reverse transcription.In qRT-PCR, GAPDH primers were designed as follows  and CHMP4C primers were designed as follows . Relative expression differences were calculated using the 2-ΔΔCt method with the GAPDH gene as a control. si-NC and si-RNA of CHMP4C were designed as follows .2.8RIPA buffer with PMSF was used to get the total protein, and the total protein concentration was determined with the BCA method . The protein was separated on 10% SDS/PAGE gels and transferred to PVDF membranes. After transferring, the membranes were blocked with 5% skim milk and incubated overnight in primary antibody at 4°C. Bound antibodies were detected by horseradish peroxidase-labeled secondary antibody. Western blot analysis was performed with ECL luminescent reagent . The antibodies were displayed as follows .2.9Pathology slides from patients with prostate cancer and benign prostate hyperplasia (BPH), and we cut paraffin sections of the corresponding tissues. Immunohistochemical antibodies were obtained from Abcam, Cambridge, UK, ab272638. DAB reagent was used for staining .2.10In CCK8 assay, si-NC and si-CHMP4C cells were inoculated into 96-well plates at a density of 2 × 103 cells/well, incubated at 37 degrees for 3 hours, and absorbance was measured at 450nm for 3 consecutive days. In the colony formation assay, 2 groups of cells were inoculated in 6-well plates at a density of 1000 cells/well and cultured until visible colonies were formed. Cell colonies were fixed in 4% paraformaldehyde  for 20 min and then stained with 0.1% crystal violet solution  for 20 min. In the wound healing assay, we inoculated cells on 6-well plates and cultured cells until fusion reached 80%-90%, using pipette tips for cell scoring and PBS for cell rinsing. Photographs were taken under the microscope at 15h, 30h, and 45h, respectively. In the transwell assay, the matrigel was melted and spread in a 24-well transwell chamber. The lower chamber was added with 10% fetal bovine serum. After 12 h of starvation, cells were transferred to transwell chambers and incubated at 37° C for 36 h. The remaining cells in the upper chamber were erased with cotton swabs and fixed with paraformaldehyde for 20 min. The cells were stained with 0.1% crystal violet solution for 20min.2.11Statistical analysis of bioinformatics was conducted on the R software platform and experimental data were counted using GraphPad Prism version 9.0. We adopted a t-test to compare differences between the two groups and all statistical tests were set as two-sided .2.12All studies involving human tissues in this study have been reviewed and approved by the Medical Ethics Committee of the Second Hospital of Tianjin Medical University (KY2023K038), and all experiments were conducted in accordance with relevant requirements and guidelines.33.1We first performed immunohistochemical staining on prostate cancer tissues and benign prostatic hyperplasia tissues, revealing that CHMP4C was higher in prostate cancer tissues   has achieved success in tumor immunotherapy. To forecast the response to ICI, we computed scores for four subtypes based on machine learning. The results suggested that the group with lower expression of CHMP4C was more likely to respond to anti-PD1, anti-CTLA-4, and comprehensive therapy . The pre4Prostate cancer is the second most prevalent tumor in men, with a 5-year survival rate of 60% in Asia. In the United States, the 10-year survival rate for localized prostate cancer is nearly 100%. However, when distant metastasis occurs, the 5-year survival rate is only 32.3%  24). Gl. Gl24). in vitro prostate cancer cell growth, invasion, and metastasis were all considerably reduced when CHMP4C was knocked down. The above results fully illustrated that CHMP4C could be regarded as a novel diagnostic and prognostic marker in prostate cancer. CHMP4C has been shown to be up-regulated in a variety of tumors and is related to malignant behavior. CHMP4C as a model gene for pyroptosis was more closely associated with prostate cancer prognosis implying prognostic value of CHMP4C in prostate cancer  can be found in the article/The studies involving human participants were reviewed and approved by The Medical Ethics Committee of the Second Hospital of Tianjin Medical University. The patients/participants provided their written informed consent to participate in this study.ZH researched and designed all bioinformatics analyses. DL, BY, LZ did the basic experimental part, and SX, DL, ZQ, SY and KW critically reviewed the manuscript. YX, HZ, KY and RL provided administrative, technical, and material support. All authors contributed to the article and approved the submitted version.


The challenge of antimicrobial resistance (AMR) continues to receive significant global attention as common infections become increasingly resistant to the drugs used to treat them. Once an infectious microbe has developed a mechanism of resistance, it can cause longer, more damaging infections which are more costly, time‐consuming, and sometimes impossible to treat. Such impacts occur across the health of humans, animals, plants, and the environment. Thus, AMR is considered a One Health issue. However, current narratives on AMR focus on humans, food‐producing animals, crops, and their immediate environments. Very little attention is given to wildlife in terms of the impact of AMR on their health, nor their role in the evolution and spread of AMR. This article (1) discusses an absence of wildlife in current AMR guidance, (2) suggests how this absence of wildlife could limit understanding of, and action on, AMR, (3) proposes that considering AMR as a form of human–wildlife conflict could enable AMR guidance to better incorporate wildlife into action planning and create a truly One Health approach to tackle AMR. AMR natHowever, the majority of AMR research, practice, and guidance focuses on humans, domestic animals such as livestock and pets, crops, and the environments in which these species live use, which drives AMR; heavy metal and agro‐chemical/pesticide (mis)use, temperature, humidity, and radiation changes are all recognized to challenge the same mobile genetic elements, which drive resistance in microbes .Struggles that emerge when the presence or behaviour of wildlife poses actual or perceived, direct and recurring threats to human interests or needs, leading to disagreements between groups of people and negative impacts on people and/or wildlife As discussed, the spread and evolution of AMR can be rooted in HWC, including habitat degradation, intensive farming, pollution, and biodiversity loss. Thus, could the evolution and spread of AMR be considered a form of HWC in its own right? The International Union for the Conservation of Nature (IUCN) hosts a Human–Wildlife Conflict and Coexistence Specialist Group who consider HWC to be:The consideration of wildlife as a vehicle for the spread of AMR would certainly appear a “struggle” that could frame AMR as an example of HWC. Accepting this narrative would present critical questions to engage with on AMR , which discuss a need for awareness raising, cross‐disciplinary collaboration, data‐informed planning, and the development of long‐term solutions with political backing WHO, . As suchIn combination, the framing of AMR as a form of human–wildlife conflict and engaging with stakeholders and data in the HWC area will significantly enrich our understanding of AMR by accelerating transdisciplinary action to tackle AMR via a holistic One Health approach. It is becoming impossible to disregard the health implications of interactions between humans, wildlife and domesticated animals and environments ; data curation ; formal analysis ; funding acquisition ; investigation ; methodology ; project administration .This work was funded by the University of Leeds via an internal Research Cultures Award to Dr Jessica Mitchell in January 2022.None to declare.Table S1Click here for additional data file.


For the ground-state properties of gas-phase nanomolecules with multi-reference character, thermally assisted occupation (TAO) density functional theory (DFT) has recently been found to outperform the widely used Kohn–Sham DFT when traditional exchange-correlation energy functionals are employed. Aiming to explore solvation effects on the ground-state properties of nanomolecules with multi-reference character at a minimal computational cost, we combined TAO-DFT with the PCM (polarizable continuum model). In order to show its usefulness, TAO-DFT-based PCM (TAO-PCM) was used to predict the electronic properties of linear acenes in three different solvents . According to TAO-PCM, in the presence of these solvents, the smaller acenes should have nonradical character, and the larger ones should have increasing polyradical character, revealing striking similarities to the past findings in the gas phase. Electronic structure methods are commonly used to compute the properties of gas-phase molecules. Nonetheless, the properties of molecules in solutions can be very different from those of gas-phase molecules. Therefore, it is important to realize the effects of solvents on the properties of solute molecules, especially when polar solvents are involved. While it is possible to adopt explicit solvent models (wherein solvation effects are taken into consideration by explicitly including the molecular details of each solvent molecule) ,2,3, theIn order to circumvent this difficulty, several implicit solvent models have been devised to study the properties of molecules in polarizable solvents over the past few decades. Among them, the Kirkwood–Onsager model ,5,6 is pIn order to resolve the shortcomings of the KO model, recently developed implicit solvent models, such as the PCM (polarizable continuum model) ,20,21,22On the other hand, among the presently available electronic structure methods, Kohn–Sham (KS) density functional theory (DFT)  has beenFor instance, KS-DFT with the commonly used local, semilocal, and hybrid XC energy functionals may lead to unreliable results for the GS properties of gas-phase molecules with multi-reference (MR) character. Therefore, it can be anticipated that the corresponding KS-PCM can also lead to incorrect solvation effects on the ground-state properties of these molecules. Typically, accurate MR electronic structure methods ,33,34,35In order to predict the GS properties of gas-phase nanomolecules with MR character, thermally assisted occupation (TAO) DFT , i.e., aal local , semilocal local , and hybal local  XC energal local ,42,43,44al local ,54,55,56al local , and speal local ,58 propeIt is worth mentioning that some interesting electronic structure methods ,61,62,63nctional  ) ,68 at soFT-DFT)) ,44,53 fon-acene . Furthermore, some shortcomings of KS-PCM related to the issues of MR character are found to be greatly resolved by TAO-PCM.Accordingly, here, we combined TAO-DFT with the PCM to model solvation effects on the GS properties of nanomolecules with MR character. The resulting TAO-DFT-based PCM, denoted as TAO-PCM, is expected to improve on the widely used KS-PCM for the properties of solute molecules with MR character when solvents are involved. In large part, this is because the MR character of solute molecules is inherently quantum-mechanical, which should be better described by TAO-DFT than by KS-DFT when traditional XC energy functionals are employed. In order to show its usefulness, we also employed TAO-PCM to explore the effects of solvents on the electronic properties  of d states ,43,44,45N electrons) surrounded by solvent molecules. In the PCM [Consider a solute molecule  is the  of Ref. ) is the In order to obtain the solute electron density i-th TAO-orbital i-th TAO-orbital Here,  of Ref. ) is the  of Ref. ) is the For the  density , has then-acenes) [We performed all calculations using the 6-31G(d) basis set with the software package of Q-Chem 4.4 . As mentrization  of the Lnctional  obtainednctional  of the Lperties) ,40,44, T-acenes)  when com-acenes) . In addiharacter ,51,52,53On the other hand, for the PCM part, we adopted C-PCM , which hFor comparison with the results of TAO-PCM , we also reveal some results obtained with the corresponding KS-PCM , wherein KS-LDA  in the continuum solvent, we fully optimized the geometries of the lowest singlet and triplet states of n-acene in the continuum solvent using spin-unrestricted TAO-PCM , and computed the ST gap of n-acene in the continuum solvent usingn-acene in the continuum solvent. For comparison purposes, we additionally report the results of the corresponding KS-PCM . As presented in n-acenes in three different solvents  are essentially the same as those of n-acenes in the gas phase, indicating that the solvation effects play insignificant roles here. In addition, for all the cases investigated, n-acenes in different media possess singlet ground states. As molecules with MR character are commonly characterized by small ST gaps, similar to the gas-phase situations [n-acenes, which have smaller ST gaps, are expected to have more significant MR character than the smaller n-acenes.A singlet–triplet energy gap (ST gap) can provide many insights on chemical processes. In order to obtain the ST gap of Equation ) associatuations ,43,44,45n-acene in a monotonic manner, but the ST gap of KS-PCM exhibits an unexpected increase beyond 10-acene. Therefore, we also explored the potential causes of such discrepancies by examining the expectation values of n-acene in the continuum solvent, calculated by spin-unrestricted KS-PCM , the n-acenes in different media, obtained from spin-unrestricted KS-PCM, are heavily spin-contaminated. Consequently, the larger n-acenes can have MR character in the lowest singlet states, and the unexpected increase in the ST gap of KS-PCM beyond 10-acene could simply be an artifact related to spin contamination.On the other hand, the ST gap of TAO-PCM decreases with the size of -PCM see . For thet state) ,53,77. An-acenes in different media, obtained from spin-unrestricted TAO-PCM and KS-PCM. Because of the spin-symmetry constraint [n-acene in the continuum solvent, calculated by the spin-restricted formalism, has to be identical to that calculated by the spin-unrestricted formalism. In order to assess whether this constraint can be obeyed in TAO-PCM, we fully optimized the geometries of the lowest singlet state of n-acene in the continuum solvent using spin-restricted and spin-unrestricted TAO-PCM, and subsequently computed the n-acene in the continuum solvent usingn-acene in the continuum solvent, computed using the spin-restricted and spin-unrestricted formalisms, respectively. For comparison, we also report the corresponding KS-PCM results. As shown in n-acenes in the continuum solvent, calculated by KS-PCM, are rather large for some larger n-acenes . Consequently, for the lowest singlet state of larger n-acene in the continuum solvent, an electronic property (which depends on the spin densities) obtained with spin-unrestricted KS-PCM can be rather different from that obtained with spin-restricted KS-PCM (even when these electronic properties are required to be the same due to the spin-symmetry constraint). Clearly, such an unphysical spin-symmetry-breaking feature of spin-unrestricted KS-PCM is very undesirable. By contrast, the n-acenes in the continuum solvent, calculated by TAO-PCM, are essentially zero for all the cases investigated, leading to essentially no unphysical spin-symmetry-breaking results in the corresponding spin-unrestricted TAO-PCM calculations.Furthermore, we can also assess the effects of spin contamination in the lowest singlet free energies of nstraint ,38,39, fEquation ) associan-acenes in the continuum solvent, computed using KS-PCM, can be severely influenced by spin contamination, seriously degrading the accuracy of KS-PCM in predicting the ST gaps and possibly other electronic properties . As will be shown later, the lowest singlet states  of the larger n-acenes in different media exhibit an increasing polyradical nature, wherein KS-PCM could lead to unreliable electronic properties. Consequently, we merely present the TAO-PCM results hereafter.In summary, the ST gaps of the larger N electrons) in a continuum solvent, the free energy gained when an electron is added to the neutral solute molecule  is defined as the vertical electron affinityFor a neutral solute molecule Egn-acene  in the continuum solvent.In this study, n-acene in three different solvents  and in the gas phase, obtained with spin-unrestricted TAO-PCM . As shown, for each solvent studied, the n-acene increases. In addition, for each n-acene studied, the n-acene in the continuum solvent of larger n-acene is the largest in the gas phase . Note that the Similar to the TOONs in TAO-DFT ,38,39,44entropy) :(10)SvN=n-acene in the continuum solvent increases with n, implying that the respective strength of MR character should also increase with n. In addition, the n-acenes in three different solvents  are essentially identical to those of ground-state n-acenes in the gas phase [n-acenes.As shown in as phase ,39,43,45n, we reveal the active TOONs of ground-state n-acene (with N electrons) in the continuum solvent, obtained with spin-restricted TAO-PCM . The In order to see why the aforementioned n-acene in the continuum solvent possess interesting patterns. First, the smaller n-acenes  are essentially the same as those of ground-state n-acenes in the gas phase  [37,39,behavior ,44,45, in-acenes  in the continuum solvent, obtained with spin-restricted TAO-PCM . As n increases, the active TAO-orbitals exhibit an increasing trend to localize near the edges of n-acene. In addition, the active TAO-orbitals of ground-state n-acenes in three solvents  and in the gas phase [In real space, we reveal the active TAO-orbitals see , such asas phase  look vern-acene  with n = 2–20 in three different solvents  and in the gas phase. For comparison, our TAO-PCM results  were also compared with the corresponding KS-PCM  results.In conclusion, we developed TAO-PCM, combining TAO-DFT with the PCM, to study solvation effects on the GS properties of nanomolecules with MR character at a minimal computational cost. In addition, we adopted TAO-PCM  to explore the electronic properties  of n-acenes have been found to reveal MR character in the lowest singlet states , KS-PCM can yield incorrect electronic properties . For example, the ground states of some larger n-acenes in different media, obtained from spin-unrestricted KS-PCM, have been shown to be highly spin-contaminated, yielding unphysical spin-symmetry-breaking effects. While accurate MR methods may remedy this issue, these MR methods and related PCMs could be computationally impossible for the larger n-acenes in the gas phase and solution phase, respectively. Hence, it is well justified to use TAO-PCM to explore the electronic properties of n-acenes in different media because of its decent compromise between accuracy and efficiency.In the three solvents and in the gas phase, since the larger n-acenes , and has a significant impact when the net charge is varied in the process .According to TAO-PCM, in the three solvents, the smaller tuations ,43,44,45While TAO-PCM seems to be promising for the study of solvation effects on the GS properties of nanomolecules with MR character, TAO-PCM may not work well in situations where the underlying PCM (or other implicit solvation models) could fail badly. Therefore, in the near future, we plan to explore the possibility of combining TAO-DFT with an efficient explicit solvent model to resolve this issue.


Parkinsonism is a rare manifestation of brain tumors that has most commonly been reported in association with gliomas and meningiomas. In this paper, we describe a unique case of secondary Parkinsonism that was precipitated by a craniopharyngioma. A 42-year-old female presented with resting tremors, rigidity, and bradykinesia. Her past medical history was significant for a craniopharyngioma resection four months prior. The postoperative course was complicated by severe delirium, panhypopituitarism, and diabetes insipidus. Notably, she was taking haloperidol and aripiprazole daily for four months to manage her delirium and psychotic episodes. Her preoperative brain MRI showed a compressive effect of the craniopharyngioma on the midbrain and nigrostriatum. Drug-induced Parkinsonism was initially suspected given extended treatment with antipsychotics. Haloperidol and aripiprazole were stopped, and benztropine was started with no improvement. Consequently, the patient was treated with carbidopa/levodopa with symptomatic improvement. A dopamine transporter (DaT) scan was done after starting carbidopa/levodopa and showed asymmetric decreased uptake in dopamine transporter in the striatum. Only one other case of Parkinsonism following craniopharyngioma resection was found in the literature review. Unlike our example, the symptoms resolved following surgical intervention and did not require a long-term treatment with carbidopa/levodopa. The purpose of our case report is to highlight brain tumors as a potential cause of secondary Parkinsonism in younger patients for an early surgical intervention can be curative. Parkinsonism is a motor syndrome manifested by rigidity, tremors, and bradykinesia . In overCraniopharyngioma is a rare central nervous system (CNS) tumor with a prevalence of 0.5 to 2 cases per million persons per year. It usually arises in the suprasellar region and can extend to involve multiple brain segments, producing a variety of symptoms. The most common manifestations of craniopharyngioma are elevated intracranial pressure, visual impairments, and endocrine deficiencies . In thatA 42-year-old woman presented to the Emergency Department with a two-month history of progressive resting tremors in her hands. Her medical history was relevant for cutaneous lupus erythematosus and a papillary craniopharyngioma resected four months prior to the presentation. The postoperative course was complicated by diabetes insipidus, panhypopituitarism, and severe delirium, which were managed with DDVAP, levothyroxine, haloperidol, and aripiprazole. Her original preoperative presentation was significant for two weeks of fatigue, bilateral blurry vision, bilateral lower extremity weakness, and urinary incontinence. Family history was noncontributory.The neurological examination showed hypophonic speech and an intermittent resting tremor. The tremor was more noticeable in her left upper extremity. Provoked mental math induced tremor spread to the head and lower extremities. There was cogwheel rigidity with more prominence in the right upper extremity compared to the left. She also exhibited bradykinesia on hand grips, finger taps, and toe taps Video .The initial diagnosis was drug-induced Parkinsonism given that the patient had been taking haloperidol 10 mg and aripiprazole 10 mg for about four months for delirium. She had originally been recommended to take those medications as needed, but further questioning revealed that she was taking them daily. Knowing that she had a craniopharyngioma measuring 3.8 x 2.3 x 2.0 cm with midbrain compression on preoperative MRI, we decided to obtain a brain MRI to assess the integrity of the anterior midbrain and nigrostriatal system. The new MRI was significant for the loss of normal swallow tail in T2/SWI with expected surgical changes  scan on an outpatient basis after starting treatment with carbidopa/levodopa. It demonstrated an asymmetric bilateral decrease uptake of radiotracer more predominant in the right caudate and putamen Figure , which c.Craniopharyngioma is a rare primary CNS tumor. It can extend to multiple brain structures, with most symptoms due to the mass effect . SecondaSecondary Parkinsonism due to craniopharyngiomas is rare, and only a single case was reported by Parvaresh in 2015 . In thatIn this case, the craniopharyngioma extended to compress substantia nigra and striatum bilaterally, precipitating irreversible secondary Parkinsonism. A DaT scan was used to confirm nigrostriatal dopaminergic neurodegeneration, which is seen in Parkinsonian syndromes ,7. AlthoThis case report demonstrates a rare case of papillary craniopharyngioma causing secondary Parkinsonism due to likely compression on the midbrain and substantia nigra. Diagnosis and management of such cases can be challenging, especially if there are overlapping factors like the use of antipsychotics. DaT scan is used to confirm the loss of neurons in the substantia nigra and to differentiate from purely vascular causes of Parkinsonism, where dopamine uptake remains normal. We hope to encourage the early consideration of brain tumors as a cause of Parkinsonism in young patients as an early surgical intervention might be curative.