This classification scheme has been included into clinical recommendation for post-operative risk-based management, although there have been few Asian studies on this topic. The present study aimed to evaluate the prevalence and clinical outcomes of endometrial carcinoma using this classification in Northern Thailand and the feasibility of implementation in resource-limited settings.Endometrial carcinoma is molecularly categorized into four subgroups: POLE mutation testing. Clinicopathological variables and outcomes were analyzed. The costs of the molecular information-based approach were compared to those incurred by the conventional approach (without molecular classification).Endometrial carcinomas from hysterectomy specimens were classified using immunohistochemistry for MMR proteins and p53, as well as POLE testing was used to identify patients for treatment omission.Of 138 patients, 52.9% in the NSMP subgroup, 28.2% were in the MMR-d, 13.8% in the p53-abn, and 5.1% in the POLE-mut. After adjusting for other variables, patients with POLE-mut showed the most favorable outcomes, while those with p53-abn had the poorest survival. When estimating the costs for post-operative management, the use of molecular classification resulted in a 10% increase over the conventional approach. However, the cost increased only by 1% if only POLE mutation testing in early-stage patients should be considered.In Northern Thailand, endometrial carcinoma had comparable subgroup distribution and prognostic implications to previous reports, supporting the implementation of management guidelines that incorporate molecular information. In resource-limited settings, at least  Endometrial carcinoma is the sixth most common cancer in females worldwide and the third most common cancer in the female genital tract in Southeast Asia, with an annual incidence of 32,000 new cases , 5.POLE ultramutated (polymerase-E exonuclease domain-mutant: POLE-mut), microsatellite instability hypermutated (mismatch repair-deficient: MMR-d), copy number high , and copy number low (no specific molecular profile: NSMP) ,–27,21–27POLE mutation testing, patients with grade 3 tumors would typically be classified as having at least an intermediate risk, which warrants adjuvant therapy [All seven POLE-mut tumors in our cohort had endometrioid histotype, and 42.9% of them were FIGO grade 3, which aligns with the findings in a recent systematic review (43.4%) . Without therapy . The fav therapy , 26.p53abn [The p53-abn subgroup, on the other hand, is the most aggressive, necessitating adjuvant therapy. Serous histology is seen in a significant proportion of tumors in this subgroup. It should be noted that serous adenocarcinoma has a broad histological spectrum and is sometimes confused with low-grade endometrioid carcinoma . In casep53abn , and, asPOLE mutations cause multiple subsequent mutations, including MMR and p53 genes [POLE mutation testing, some POLE-mut tumors in our study could have been misclassified as MMR-d. This is significant because POLE mutations have been shown to be a prognostic driver even when MMR deficiency or p53 abnormalities are present [MMR-d and p53-abn may be found in 9.8% and 15.7% of POLE-mut tumors, respectively, since 53 genes . Without present .POLE mutation testing into early-stage patient management may not result in a significant increase in overall costs when compared to the conventional approach. This was primarily due to the fact that omitting adjuvant therapy in POLE-mut patients saved money. Interestingly, by carefully selecting patients with at least intermediate risk, doing POLE mutation testing alone could result in a 1.6% cost reduction, suggesting that testing for POLE mutations could be useful in early-stage patients, even in resource-constrained settings. In such cases, a single molecular testing center could serve as a reference laboratory. As technology advances and the volume of testing increases, the cost tends to decrease.One of the challenges of implementing molecular-based classification is the cost of molecular testing , which iPOLE mutation status can be determined using DNA sequencing techniques, such as next-generation sequencing, Sanger sequencing, or hotspot mutation analysis [POLE mutations have been developed [analysis . In our eveloped . Table 5In this new era of endometrial carcinoma treatment, molecular-based classification is becoming increasingly important in determining the best therapeutic approach. The treatment is rapidly evolving, driven by the development of novel strategies –38. For This is the first study to look into the molecular data of endometrial carcinoma in Southeast Asia, and it provides information on the prevalence and outcomes of each molecular subgroup in this population. Because molecular-based classification has become a current topic of interest, this study focuses on its application in routine clinical practice in resource-limited settings. Limitations of our study include the study’s retrospective nature, small sample size, study design, and proportion of cases with suboptimal DNA preservation (25.8% of recruited cases), which may all affect the distribution of the subgroups. The DNA preservation factor could be attributed, at least in part, to our tropical environment and/or the duration of FFPE block storage. Given that FFPE blocks are typically transferred for testing immediately after diagnosis in clinical practice, DNA degradation is expected to be minimized. Because our classification algorithm begins with MMR IHC, cases with MMR-d and POLE-mut coexistence would be classified as MMR-d subgroup. These tumors are uncommon, accounting for only 5% of endometrial carcinomas, and data are limited . AnotherPOLE mutation testing in early-stage patients should be considered in resource-limited settings.Our study of molecular-based classification of endometrial carcinoma in Northern Thailand discovered that subgroup distribution and prognostic implications were comparable to the previous reports. These findings support the implementation of management guidelines that include molecular information in this region. At least


VDP–Au junction followexceptionally well the single-level modeling with strong gate couplingethene). Ethaline is also an idealsolvent for the measurement of very short molecular junctions, asit grants a greatly reduced snapback distance of the metallic electrodesupon point-contact rupture. Our work demonstrates that DESs are viablealternatives to often relatively expensive ionic liquids, offeringgood versatility for single-molecule electrical measurements.The environment surrounding a molecular junction affectsits charge-transportproperties and, therefore, must be chosen with care. In the case ofmeasurements in liquid media, the solvent must provide good solvation,grant junction stability, and, in the case of electrolyte gating experiments,allow efficient electrical coupling to the gate electrodes throughcontrol of the electrical double layer. We evaluated in this studythe deep eutectic solvent mixture (DES) ethaline, which is a mixtureof choline chloride and ethylene glycol (1:2), for single-moleculejunction fabrication with break-junction techniques. In ethaline,we were able to (i) measure challenging and poorly soluble molecularwires, exploiting the improved solvation capabilities offered by DESs,and (ii) efficiently apply an electrostatic gate able to modulatethe conductance of the junction by approximately an order of magnitudewithin a ∼1 V potential window. The electrochemical gatingresults on a Au– There are a range of scanning-probe-basedmethods, which differ in how the junctions are formed but have thecommonality of tethering a molecular target between a pair of electrodesto form a metal | molecule | metal junction. The most widely usedmethod for recording single-molecule electrical characteristics isthe scanning tunneling microscopy break junction (STM-BJ) technique.8 Such measurements are typically performed either in a solvent containingthe molecular target or with the target formed as a self-assembledmonolayer. The former “in situ” approachis often preferable over the “ex situ”self-assembly of molecular monolayers on substrates, in which casethe solvent properties are very important. Advantages of the in situ approach over the ex situ approachcan be that often the optimal conditions for the latter are unknownand might have to be optimized, while the in situ measurement directly from solutions of the target molecule removesthis surface assembly preparation step. In addition, the insitu method provides a fresh supply of the molecular targetfrom solution, as the junctions are continuously made, broken, andremade during the cyclic STM-BJ process.It is now possible toreliably form and measure the electricalcharacteristics of single-molecule junctions in a wide variety ofsolvents and electrolytes under a wide variety of experimental conditions.The electrical behavior of the junction can be recorded by using oneof many techniques, such as scanning probe microscopy (SPM)–2 to 10–6 M, depending on experimental requirements. The solvent may alsobe required to show a low vapor pressure to avoid evaporation andpotential damage to instrumentation from solvent vapors in some scenarios.It is also desirable for the solvent system to have a low toxicity.Typical solvent systems used in the literature include mesitylene,1,2,4-trichlorobenzene, propylene carbonate, 1,3,5-trimethylbenzene,and aqueous solution.14 Further requirements are added when experiments require electrochemicalcontrol. Here, the solvent requires sufficient ionic (electrolytic)conductivity, and in the case of the STM-based techniques, all butthe very end of the STM tip needs to be covered with a wax or otherinsulating coating. This is to reduce tip leakage currents to lowlevels, which typically need to be much lower than setpoint tunnelingcurrents. Aqueous electrolytes have been used, but these can be unsuitablewhere a wide electrode potential window is required. Ionic liquidshave been used to greatly extend the electrode potential window.14 Using these, multiple redox switching events have been observedwhich are not accessible with aqueous electrolytes.16 Although ionic liquid deployment can be very effective, it doeshave some potential drawbacks. These include the fact that they canbe relatively expensive or require specialist synthesis and/or purification.With this in mind, we evaluate here deep eutectic solvents as electrolyticmedia for single-molecule electronics studies.An important considerationfor the measurement liquid for two-terminalSTM-BJ experiments is that it is a poor ionic (electrolytic) conductorso that leakage current is not an issue and the target molecule hassufficient solubility. Typical solution concentrations for such measurementsare in the range of 1019 This promotedour interest in extending the choices of electrolyte medium for EC-STMbreak-junction studies. There are several requirements for such candidates;they should (1) form an ionically conducting electrolytic solution;(2) have a wide electrochemical window for measurements; (3) be easilysynthesized or purchased; (4) be cheap; (5) ideally be nontoxic andenvironmentally friendly. Based on the above criteria, water or aqueouselectrolytes may seem to be the most obvious choice. However, in previousstudies, it has been shown that aqueous electrolytes are often notthe best suited medium for EC-STM break-junction studies, since thetarget molecule may be water insoluble. In some instances, this canbe circumvented by preadsorption of the molecule on the substratefrom a suitable solvent. This can be successfully implemented whenthe target molecules form self-assembled monolayers (SAMs), but thisis not always straightforwardly the case. Perhaps more importantly,aqueous electrolytes provide a relatively narrow potential window,which may mean that the required potentials cannot be reached to observeeither the redox transition or multiple redox states. For example,only one redox transition was observed for a pyrrolo-tetrathiafulvalenecontaining molecular bridge (pTTF) in aqueous electrolytes,15 while “soft gating” with a verybroad peak was recorded for viologen-based junctions.20 By contrast, ionic liquids have been shown to perform wellas solvent/electrolytes in both of these situations. Two separatedtransitions of a pyrrolo-tetrathiafulvalene containing molecular bridgewere reported by the in situ break-junction methodin ionic liquid, which contrasted with the single observed redox transitionin aqueous electrolytes.15 In addition,the electrochemical conductance gating of a viologen-based junctionin the ionic liquid 1-butyl-3-methylimidazolium triflate (BMIM-OTf)fitted the two-step (hopping) mechanism, exhibiting a “hardgating”, which contrasts with the broad conductance featurein aqueous electrolytes.20Although thebreak-junction method can work in air, in liquid,and in vacuum, limited liquid media can be chosen for electrochemicalSTM (EC-STM) break-junction studies as highlighted above.24 In addition, DESs can be relatively cheap and readily synthesized.DESs are eutectic mixtures composed of Lewis or Brønsted acidsand bases with inherent ionic conductivity and good solubilizing properties,making them attractive electrolytes for electrochemistry. Despitethe viscosity of the liquid resulting in a lower ionic conductivity,some low-viscosity DESs still have a conductivity on the order oftens of mS cm–1.23 Dueto the large electrochemical window and acceptable ionic conductivity,DESs can display advantages over aqueous electrolytes and severalnonaqueous solvents for electrochemical studies. There are now manyexamples in which DESs have been exploited for metal electrodeposition,electropolishing, and extraction.25 There are also a growing number of publications in which fundamentalelectrochemical behavior is examined in deep eutectic solvents, examplesof which include diffusion coefficients of redox species,28 electron transfer kinetics,29 interfacial single-crystalelectrochemistry,30 metal underpotentialdeposition,32 and the electrochemistry of graphenein DESs.33 Moreover, in a recent study,fast electron transfer (ET) has been observed in a typical DES, ethaline(1:2 choline chloride:ethylene glycol). The ET rate constants measuredin ethaline are just slightly lower than that in acetonitrile forferrocene and comparable with those in water for ferrocyanide.29 This provides an additional incentive for investigatingDESs as alternatives to ionic liquids for single-molecular junctionstudies.Deep eutecticsolvents (DESs) are a relatively new class of ionicliquid analogue that share many physical properties with ionic liquids.VDP) in ethaline was examined within a bipotentiostaticallycontrolled four-electrode cell. The conductance of VDP junctions varies as a function of the sample potential applied tothe gold substrate electrode. This is consistent with previous findingsemploying aqueous HClO4 electrolytes.12In this study, ethaline has been used with both theSTM-BJ andEC-STM techniques as the solvent or electrolyte environment and hasbeen shown to be an effective alternative to an ionic liquid for suchmeasurements. Several two-terminal single-molecular junctions havebeen successfully measured by STM-BJ, including some molecules thatshow low solubility in common solvents and had not been successfullymeasured before. Conductance data from the molecular junctions havealso been compared with those measured in a common nonaqueous solvent,mesitylene. Following this, the electrostatic (or electrolyte) gatingof the molecular conductance of 1,2-di(pyridine-4-yl)ethene . With further tip retraction,the ultrathin gold metallic structure snaps back, and then, moleculescan self-assemble in the nanogap to form a metal | molecule | metaljunction. This results in an additional plateau in the current–distanceplot. Otherwise, a rapid exponential decay is observed if no singlemolecular junction is formed. The STM-BJ system can be further adaptedby adding extra electrodes for the electrochemical implementation(EC-STM). Here, a four-electrode bipotentiostat configuration appliesindependent electrode potentials to both the STM tip and the substrate.18 Data were acquired and processed using bespokePython scripts and plotted using commercial software (Origin 2020b).In this study,a scanning tunneling microscope (STM) is used toform and break metal-to-metal contacts repeatedly between the STMtip and the substrate. A fresh Au–Au single-atom junction isformed when the STM tip is pushed into the substrate, followed bytip retraction to thin the Au–Au constriction down to a singleatom   was recrystallizedfrom absolute ethanol, followed by filtration and drying under vacuum.ChCl was mixed with 2 equiv of ethylene glycol  underN2.215 The STM-BJ technique as described in the I–s) traces and withelectrochemical control of single-molecule conductance versus electrodepotential responses. In each cycle, the gold STM tip is crashed intothe gold substrate and then retracted to form a single atomic pointcontact. Further retraction results in the metallic contact breakingand snapping back. Following this, a single molecule can then bridgeinto the freshly opened gap. Following further retraction, this molecularbridge can break, itself resulting in a current jump in the current–distance(I–s) trace. . In the electrochemical STM (EC-STM) implementation, a four-electrodebipotentiostat configuration is added to the STM-BJ setup. This facilitatesindependent electrode potential control of both the tip and the substrate.To minimize the faradaic leakage currents, an additional requirementfor this technique is the application of an insulating coating onall parts of the STM tip, except the apex, which is left uncovered.We used Apiezon for the tip coating, which was stable for the electrochemicalSTM-BJ data collected for VDP in ethaline (see later). A homemadeAg/AgCl electrode has been used with the EC-STM setup as the referenceelectrode, together with a Pt counter electrode and the insulatedAu tip. To prepare a Ag/AgCl reference electrode, two silver wireswere dipped into a 0.1 M HCl solution. One wire was connected to thepositive terminal of a 1.5 V AA battery and the other to the negativeend. After a few minutes, the positively polarized wire was coatedwith a uniform AgCl layer, while H2 gas bubbles evolvedat the other electrode. Details can be found in the Supporting Information.34The detailed experimental procedure is similar to that of our previousmolecular conductance studies employing ionic liquids.3.03.138 In this study, we find a snapback distance of 0.16nm in ethaline in the absence of any target molecules, which is considerablyshorter than for other media. Details of the calibration method canbe found in the Supporting Information . The surprisingly short snapback distance holds great promisefor small-molecule junction measurement.TheSTM-BJ method relies on the single-atom Au–Au junction beingbroken immediately prior to any molecular junction being formed. Whenthe Au–Au contact breaks, the tip of the two gold electrodesrelaxes, and a small gap  opens. Thetip retraction continues, and the target molecule can bridge withinthis expanding gap until the resulting metal | molecule | metal junctioncleaves. Therefore, the actual molecular junction length consistsof two parts: the snapback distance and subsequent electrode separationof the retraction. The average snapback distance depends on the differentsolvent or electrolytic environments and ideally needs to be estimatedfor each one .3.2G/G0)) for each sample . The experimental molecular junction length and thecalculated junction geometry (indicated by tilt angle α) foreach junction are also listed. Despite the different solvent environments,values for Au–pyridyl compound–Au junctions .46 When the HC and LC values of VDP in these respectiveliquids are compared (Table S2 in the Supporting Information), the HC value measured in ethaline is slightly lower in conductancethan that in mesitylene, while the LC values are similar in both solvents.Moreover, the slope (tilt) of the conductance plateau of the two-dimensional(2D) histogram , showing that the solvent type has a marked influence onthe junction evolution during the stretching process. Tilted junctionplateaus have been observed for conjugated molecular wires, and thesehave been related to a decreasing electrode–molecule couplingas the orientation of the molecular wire changes during junction stretching.47 Direct through-space tunneling between the electrode and the molecularwire π orbitals would be expected to be more pronounced formore tilted junction geometries. The differing solvation properties,molecular size, and viscosity might affect the junction formationand orientation in subtle ways, thereby impacting the tilt of theplateau in the 2D histograms. The solvating ability and viscosityof the DES may help to fix the molecule junction geometry as the tipretracts and may promote either more upright geometries or perturbthe direct coupling between the molecule π-orbitals and theelectrode. Evaluation of such a hypothesis would however require ahigh level of computational simulations to model the noncovalent interactions,the complex solvation, and the junction evolution and dynamics duringthe stretching, which are beyond the scope of this study.In addition,to highlight the advantages of measuring single-moleculejunctions in ethaline compared to other commonly deployed solvents,both BP and VDP, ethaline shows good solubility forseveral metal complexesand also target compounds for molecular conductance studies, whichwere previously found to be insoluble in solvents commonly used inSTM-BJ studies. This includes TA-1, which has never beensuccessfully measured before in other solvents, despite several attemptsdue to poor solubility. Although the compound was still not fullysoluble in ethaline during preparation, reasonable conductance signaturescould be clearly observed in the 2D histograms. This shows that ethalineprovides additional opportunities for electrical conductance measurementsfor molecular wires, which have proven to be insoluble in organicsolvents commonly used in STM-BJ studies.Besides the model pyridyl molecular wires, 3.3VDP–Au junctions withdifferent electrode potentials applied to the gold substrate. Thesemeasurements were made with a Ag/AgCl reference electrode, which consistedof a Ag wire with an insoluble layer of AgCl, suitable for the EC-STMcell. The electrochemical gating voltage ranged from −0.2 to+0.7 V. Below −0.2 V, no junctions were observed, while theEC-STM system become very unstable when the sample electrode potentialwas set above +0.7 V. For each effective individual sample electrodepotential, ∼4800 unselected traces were collected. Then, eachset of data was plotted as a 1D histogram . The data presentedin the main paper were measured using a Ag/AgCl reference electrode,while complementary data with a Pt quasireference electrode are presentedin the Supporting Information. These datasets are consistent, although the Ag/AgCl reference system is favorable,since it possesses greater stability . Detailed information concerning each sample potential with thetwo reference electrodes is summarized in Table S3.istogram 4a. A cor12 or in aqueous phase.14 The advantage is that the electrolyte conductivity of ethaline isintrinsic without any requirement for the addition of a supportingelectrolyte. The molecular conductance values shift higher when thesample electrode potential is adjusted to more negative values (S3). The rate of change of conductance with electrodepotential is different for the HC and LC peaks, and this could arisefrom different anchoring configurations yielding different couplingvalues in the single-level model .As discussed in Quek et al.,37 the low-conductancefeature of 4-pyridyl junctions has both weaker coupling and betteralignment to the electrode Fermi level, which would yield the higher“gating rate” we observed. Furthermore, a similar behaviorfor VDP has also been found by Brooke et al. in Ni–molecule–Nijunctions.12 In the absence of experimentalvalues for gamma and epsilon for the low-G feature,we focus our later discussion on the high-G feature,where indeed we observe excellent agreement between our data and thesingle-level model.In F) position relative to the molecular orbital caused by thepotential applied between the electrode and the electrolyte solution.48 When the potential is moved to more negative values, εF is raised. Thus, the energy barrier between the Fermi leveland the lowest unoccupied molecular orbital (LUMO) decreases, leadingto an increase in conductance as pyridyl anchoring groups generallyimpose LUMO mediated tunneling. This is consistent with previous experimentalresults employing aqueous electrolyte gating.12As explained in previous works, the electrochemicalgating effectcan be attributed to the change in the electrode Fermi level . When the geometry of the junction becomes more tilted, the nitrogen–goldbond tilts away from the plane of the pyridine ring, leading to anenhancement of the electronic coupling between the gold s-states andthe LUMO π* orbital. This could then contribute to a conductanceincrease through the molecular junction.The increase in conductance may also be, in part, due to changesin junction geometry between the different gate voltages. 52ELevel is the alignment of the molecular orbital relative to themetal Fermi level and Γ is the broadening of the molecular orbitaldue to hybridization with the metal electrode. The junctions withAu electrodes have been well studied experimentally, and the transmissionfunction has been well approximated by a single-Lorentzian form. Ithas been reported from experimental data that on average ELevel and Γ are, respectively,1.1 eV and 40 meV for Au–VDP–Au junctions in the high-conductancestate.49 It can be seen in 20 This good correspondence between the experimentalgating data for the Au–VDP–Au junction in ethaline electrolyteswith the two-level phase coherent model verifies well the proposedmechanism of the electrostatic gating of VDP in ethaline.A fundamental classicalmodel for phase coherent charge transportthrough a metal–molecule–metal junction involves a coherenttransmission of electrons and holes from one electrode to the otherthrough the molecular orbital that dominates transport. The transmissioncoefficient can be described using a Lorentzian form using the Breit–Wignerformula:4.0VDP) system. Future work will focus on the evaluationof this solvent for the electrochemical gating of redox-active single-moleculejunctions.In summary, we have employeda novel solvent medium for our STM-BJ/EC-STMsystem, and we have demonstrated that the deep eutectic solvent, ethaline,is a good alternative to ionic liquids for single molecular junctionelectrical measurements. This liquid is able to solvate two metalcomplexes and other previously insoluble molecular wire targets. Moreover,the gold snapback distance when the Au–Au junction breaks inDESs is shorter than that in other commonly used media for STM-BJstudies. 2D conductance histograms in DESs show flatter, better definedconductance plateaus, which are likely to result from the solvationof the molecular wire within the junction. We hypothesize that thismight be a result of a reduction in through-space tunneling betweenthe tilted molecular junctions and the contacting electrodes. Thisstudy also shows that ethaline is an exceptional medium for the measurementof single-molecule conductance under electrochemical conditions, withSTM systems equipped with a four-electrode bipotentiostat control(EC-STM). Effective electrostatic gating with gate coupling closeto unity can be achieved with this kind of solvent for the 1,2-di(pyridin-4-yl)ethene(


A heterostructured nanocomposite MCM-41<SFA> was formed using the encapsulation method, where a silicon dioxide matrix—MCM-41 was the host matrix and synthetic fulvic acid was the organic guest. Using the method of nitrogen sorption/desorption, a high degree of monoporosity in the studied matrix was established, with a maximum for the distribution of its pores with radii of 1.42 nm. According to the results of an X-ray structural analysis, both the matrix and the encapsulate were characterized by an amorphous structure, and the absence of a manifestation of the guest component could be caused by its nanodispersity. The electrical, conductive, and polarization properties of the encapsulate were studied with impedance spectroscopy. The nature of the changes in the frequency behavior of the impedance, dielectric permittivity, and tangent of the dielectric loss angle under normal conditions, in a constant magnetic field, and under illumination, was established. The obtained results indicated the manifestation of photo- and magneto-resistive and capacitive effects. In the studied encapsulate, the combination of a high value of ε and a value of the tgδ of less than 1 in the low-frequency range was achieved, which is a prerequisite for the realization of a quantum electric energy storage device. A confirmation of the possibility of accumulating an electric charge was obtained by measuring the I-V characteristic, which took on a hysteresis behavior. Nanotechnologies are being developed more and more intensively, not only in terms of the synthesis of and research into new nanomaterials, but new possibilities for their practical applications are also being studied to a greater extent ,2. NanosA careful study of such nanostructured materials has shown that a reduction in their physical dimensions from the microscopic to the meso- and nanoscopic scales leads to a change in most of their physical properties, such as their temperature and type of phase transitions ,6,7,8,9,Achieving a high dielectric constant due to the nanostructuring of the material will open up the possibility of accumulating electrical energy at the interphase heterojunctions. This essentially opens up the possibility of creating quantum analogs of autonomous power sources that will provide not only a significant increase in the energy-intensive characteristics of the latter, but will also allow for a significant increase in their speed and a direct incorporation into micro- and nanoelectronics. For example, researchers from the USA proposed using a thin dielectric layer with a large contact area, which was implemented in nanocomposites of ceramics and glass ,17. SpecSignificant progress on this path can be achieved by forming inorganic/organic nanocomposites based on the host<guest> principle. The research carried out in this paper shows the promising nature of this type of materials for electrical energy storage devices at a quantum level . The theIt makes it possible to obtain nanostructured materials with a wide range of controlled component sizes from ~1 nm to ~300 nm;It allows for the formation of nanostructured materials with different particle geometries and topologies: three-dimensional (3D) dendrites and regular structures, 2D film and layered structures, and 1D nanowires or 0D small nanoparticles;It has a large variability in the various heteroingredients for building nanostructured materials: metals, ferroelectrics, dielectrics, insulators, semiconductors, superconductors, and magnetic materials, as well as various organic compounds;It allows for the production of nanostructured materials in large quantities . This, in turn, opens up the possibility of using some experimental methods that require a large amount of the studied material .Presently, there are many methods for the synthesis of nanostructured materials. One such method is the introduction of materials into porous matrices. This technological approach has a number of advantages:2 matrix—MSM-41 (Mobil Composition of Matter No. 41 of the Sigma Aldrich trademark) was used. It is a matrix with ordered hexagonal channels that do not intersect. It is obtained by template technology. Its wall thickness ranges from 0.6 to 0.8 nm [As an inorganic dielectric matrix, a mesoporous regular structure based on the SiOo 0.8 nm ,22. Its The guest component was synthetic fulvic acid (SFA), which was obtained via the oxidation of pyrocatechol  with molecular oxygen in an alkaline environment (Ph ≈ 13). The peculiarity of this SFA is that it has a chemical composition that is close to the natural one, i.e., containing N and S atoms. It is obtained by introducing thiourea (Sigma Aldrich) into the reaction mixture, which is a source of nitrogen and sulfur in synthesized fulvic acid. The choice of pyrocatechin and thiourea as precursors was due to their ability to enter into a substitution reaction, the product of which simultaneously contains a phenolic nucleus and thiocarbamide residue.2 per 1 mol pyrocatechin per hour.To prepare the reaction mixture, a 2.2 g weight of pyrocatechin in a polyethylene bag was placed in a 1 l Kjeldahl flask, then a solution containing 3.2 g of NaOH, 0.76 g of thiourea, and distilled water was carefully added to the wall, with a total volume of 100 mL. The air was purged from the flask by slowly blowing away the excess pure electrolytic oxygen. After the reactor flask was filled with oxygen, it was sealed and shaken mechanically. The shaking led to the mixing of the reagents and started the reaction. The reaction was carried out until the oxygen consumption slowed down to less than 0.01 mol OAfter the completion of the oxidation reaction, the reaction mixture was passed through an ion exchange column filled with KU-2-8 cationite in H-form. The removal of the eluate started when a brown color appeared and was completed when the color changed to a light red. The volume of the obtained fraction was equal to the volume of the reaction mixture. The liquid was heated on an electric stove to remove the excess dissolved carbon dioxide. The resulting solution had a pH of 2.8.The substance was a black powder that was soluble in water. The aqueous solution had an acidic reaction (pH ≈ 3). From the point of view of the molecular structure, it was a complex mixture of oligomers containing –OH and –COOH groups. The average molecular weight was 860 g/mol. The substance gave an EPR signal, which indicated the presence of free radical centers in its structure, while the SFA synthesis technology was outlined in Ukrainian patents .The encapsulation of the SFA in the pores of the MSM-41 matrix was carried out by the method of a thermovacuum impregnation of the powdered material of the matrix with the obtained SFA solution. We have effectively used this technique for the formation of encapsulates in previous studies . The theThe pore size value of the MSM-41 matrix that was selected for the research was determined by the standard method of an isothermal adsorption/desorption of nitrogen at its boiling temperature (T = 77 K), using the Quantachrome Nova Touch LX2 (USA) automated analyzer. Before these measurements were taken, the material samples were pre-degassed in a vacuum at 473 K for 16 h. The adsorption parameters were calculated based on the BET model.In order to establish the structure of the obtained MSM-41<SFA> nanocomposite, several X-ray studies were conducted. The X-ray diffraction spectra were measured in scanning mode The impedance spectra were measured to study the conductive and polarization properties. For this purpose, the formed powdery nanocomposite MSM-41<SFA> was pressed into the form of a tablet with a diameter of 6.2 mm, a thickness of 0.96 mm, and a weight of 44 mg, using a mold. Ohmic contacts were deposited onto the parallel planes of the tablets using a silver contact paste.−3–106 Hz, using the measuring complex “AUTOLAB” of the Dutch “ECO CHEMIE” company, equipped with the computer programs FRA-2 and GPES. The amplitude of the measuring signal was 0.010 V. The ambiguous points were removed using a Dirichlet filter [−1 and, when illuminated by a solar radiation simulator for the standard AM 1.5 G solar spectrum, the total available power was 982 W/m2. The external fields were superimposed in the direction of the measurements of the impedance spectra. This measurement geometry was chosen in order to be able to omit the Lorentz force.Research utilizing the method of impedance spectroscopy was carried out in the frequency range of 10t filter . The imp−1. The measuring range and scanning speed were selected based on the experimental experience of studying these types of samples, in order to avoid damage.The current–voltage characteristics of the obtained structure were also measured in the range of −3 ÷ +3 V, with a potential change rate of 0.050 V·s15 Ohm.The structure of the energy impurity spectra was also studied by the method of a thermally stimulated discharge in the temperature range of −25–70 °C, with a constant heating rate of 5 degrees/min. The thermally stimulated discharge spectra were recorded in the mode of short-circuited contacts. To measure the current, the B7-30 (USSR) was used, which is characterized by an input impedance of 10Using the experimental method of nitrogen sorption/desorption, using the Quantachrome Nova Touch LX2 automated analyzer, it was established that the studied matrix MSM-41 is characterized by a high degree of monoporosity, with a maximum in the distribution of its pores by a radius of—1.42 nm .In the X-ray diffraction pattern that was measured for the MSM-41 matrix, we obtained the usual picture—a broad diffuse maximum, which is a characteristic of an amorphous material. In the case of the MSM-41<SFA> encapsulation, practically the same picture was obtained . The absThe next step was to study the conductive and polarization properties of the synthesized encapsulant MSM-41<SFA>. As a result of the conducted research, the impedance spectra were experimentally measured for the original MSM-41 matrix and for the MSM-41<SFA> encapsulate.That is, the frequency dependences of the total complex resistance In order to find out the conductive properties, we analyzed the frequency behavior of -41<SFA> .The corresponding dependencies are shown in wn ratio :(2)σ′=σdThus, the first term in (2) reflects the conductivity formed by the band carriers, which does not depend on the frequency, and the second term in (2) reflects the conductivity formed by the hopping conductivity, which depends on the frequency. Accordingly, from the dependence of the 4 Hz). The nature of the behavior of the The introduction of SFA into the pores of the MSM-41 matrix leads to an almost five-fold decrease in the In general, the behavior of the  the Z′ω .The imposition of a constant magnetic field leads to a significant decrease in the The effect of illumination also leads to a decrease in the In order to investigate in more detail the structure of the energy spectrum of the guest impurity subsystem, thermally stimulated discharge currents were measured . No therThe next step is to consider the behavior of the impedance hodograph, presented in the form of Nyquist diagrams that are shown in 1‖C1), which corresponds to the high-frequency part of the spectrum, represents the processes of current flow through the volume of the material’s particles, and the second semicircle (R2‖C2), which corresponds to the low-frequency part of the spectrum, represents the processes of current flow in the space between the particles. After conducting simulations in the ZView2 software environment of the Nyquist diagrams of the corresponding equivalent electrical circuits, the following element values were obtained  and (5), it is possible to determine the value of ε.−3 ÷ 3·10−1 Hz (curves 1 and 2 in 4. Such behavior of the ε(ω) most likely arises due to Maxwell–Wagner segmental polarization and the additional polarization that occurs when the charge carriers jump over the localized states near the Fermi level. The Maxwell–Wagner polarization occurs in the presence of macrodipoles that arise in the vicinity of charged defects, as well as at the heteroboundaries of the structure. In this case, instead of one group of particles being capable of relaxation polarization with one defined relaxation time τ, there is a large number of relaxers with different values of the time τ. This is confirmed by the dependence of the tgδ(ω) (curves 1 and 2 in The behavior of ε(ω), for a more detailed understanding of the polarization properties of the encapsulant MSM-41<SFA>, is analyzed in −3 ÷ 3·10−1 Hz) (curve 4 in −3 ÷ 3·10−1 Hz). 2–106 Hz, This is confirmed when a constant magnetic field is applied, as a result of which, the value of the ε increases strongly  = 1.77·10−7 (F/g) = 0.177 (μF/g).As a result of simple calculations, the specific capacitance C = 1.77·10Accordingly, we can calculate the specific capacity of our material by using the formula:nc = 0.053 μF/g, Cm = 1.260 μF/g, Cl = 0.56 μF/g. Therefore, from the data presented, we can see that our material has more than twice the specific capacitance that was theoretically proposed in [From the presented I-V current in posed in , but tha4 Hz). The conductivity of this encapsulate was formed not only by band carriers and jumps in localized states, but also by the process of the capture–retention–release of carriers from quantum wells.An MSM-41<SFA> encapsulation was formed for the first time. The introduction of SFA into the pores of the MSM-41 matrix led to an almost five-fold decrease in the The impurity energy spectrum of the MSM-41<SFA> encapsulate, in the temperature range of 247–315 K, represented narrow bands with a significantly higher density of states and a well-defined miniband character, where above 315 K, it became quasi-continuous. At the same time, a homocharge relaxation was observed.For the MSM-41<SFA> encapsulation, a magnetoresistive effect was observed, the maximum values of which reached: at a frequency of 0.001 Hz −3–3·10−1 Hz, the following values were reached: tgδ < 1, ε ≈ 104, which opens up the possibility of implementing the quantum storage of electrical energy. At the same time, the value of the ε noticeably increased under the influence of illumination, ensuring the functional hybridity of such a quantum battery.In the MSM-41<SFA> encapsulation, it was shown that, in the frequency interval of 2·10The I-V curve of the MSM-41<SFA> encapsulation assumed a hysteresis characteristic of devices with the capacitive storage of an electric charge. In our study, we showed that there is a separation between electric charges and their accumulation at interphase boundaries.


We are constantly bombarded by sensory information and constantly making decisions on how to act. In order to optimally adapt behavior, we must judge which sequences of sensory inputs and actions lead to successful outcomes in specific circumstances. Neuronal circuits of the basal ganglia have been strongly implicated in action selection, as well as the learning and execution of goal-directed behaviors, with accumulating evidence supporting the hypothesis that midbrain dopamine neurons might encode a reward signal useful for learning. Here, we review evidence suggesting that midbrain dopaminergic neurons signal reward prediction error, driving synaptic plasticity in the striatum underlying learning. We focus on phasic increases in action potential firing of midbrain dopamine neurons in response to unexpected rewards. These dopamine neurons prominently innervate the dorsal and ventral striatum. In the striatum, the released dopamine binds to dopamine receptors, where it regulates the plasticity of glutamatergic synapses. The increase of striatal dopamine accompanying an unexpected reward activates dopamine type 1 receptors (D1Rs) initiating a signaling cascade that promotes long-term potentiation of recently active glutamatergic input onto striatonigral neurons. Sensorimotor-evoked glutamatergic input, which is active immediately before reward delivery will thus be strengthened onto neurons in the striatum expressing D1Rs. In turn, these neurons cause disinhibition of brainstem motor centers and disinhibition of the motor thalamus, thus promoting motor output to reinforce rewarded stimulus-action outcomes. Although many details of the hypothesis need further investigation, altogether, it seems likely that dopamine signals in the striatum might underlie important aspects of goal-directed reward-based learning.  The brain constantly receives sensory input while governing motor output . SensoryExperimentally, in trial-based reward learning, subjects are typically presented with sensory information and need to perform a specific action in order to obtain a reward. The rewarded stimulus, the sensory context, and the required action might not be known to the subject in the first place and needs to be discovered through trial-and-error learning. Thus, there must be neuronal signals in the brain that encode the value of each trial outcome . If the outcome is unexpectedly positive/negative, then brain circuits should be modified to reinforce/reduce the link between these successful/unsuccessful sequences of sensory events and motor commands.1,2 due to its capacity to assign credit to sequences of events leading to reward and its sensitivity toward the temporal relationships between stimulus and outcomes.Interesting insights into the types of signals that could drive such bidirectional modulation come from the conversation between the fields of neuroscience and reinforcement learning. In the latter, rewards are used to learn associations between actions and outcomes and to use that information to maximize total future rewards. While many reinforcement learning methods are successful at learning such associations, temporal difference (TD) learning is of particular interest for neuroscience3,4 proposed that classical associations strengthen through the iterative computation of an error term that captures the difference between the reward an animal received at the current trial and what was experienced following previous presentations of the stimulusIn early models of reward-based learning, the primary focus was on establishing associations between conditioned stimuli and actions leading to reward. These models, initially formalized by Bush and Mosteller,where 5 but was limited in scope as it boils down to computing a weighted average of past rewards. As such, it is unable to account for a wide array of phenomena commonly observed in psychology, where various events occurring within a trial convey distinct information about reward availability. In particular, it fails to learn second-order conditioning, that is, learning that if a primary cue predicts reward and the occurrence of a secondary cue predicts the primary cue, then the secondary cue becomes predictive of reward and the appearance of the primary cue does not convey new information. A fundamental shift in understanding reward-based learning occurred with the introduction of TD learning by Sutton and Barto to address these problems.1,2 Temporal difference learning departs from Rescorla and Wagner’s approach in two ways. First, it breaks down the trial structure into a series of value of that state. After learning occurs, these predictions will converge to the expected sum of the currently expected reward and the discounted reward of all future time pointsThis formulation was famously employed in the Rescorla–Wagner model of Pavlovian conditioning,where reward prediction error (RPE) and serves as the teaching signal used to adjust The core concept in learning these values , but also the reward expected at the later step of delivery. In other words, this process allows the generation of RPEs not only when an unexpected reward is obtained but also when unexpected information signaling a reward is received, effectively propagating backwards in time the RPE of the rewarded state toward the predictive cue. As reviewed below, the functional role of RPEs as defined in TD learning closely aligns with the firing rate of dopaminergic neurons in the midbrain.10 surprise,11 or novelty.12It is crucial to note that while rewards can be delivered frequently in laboratory settings, typically in the form of food or water to hungry or thirsty animals, in nature, physical rewards can be very sparse, which makes learning difficult for animals as well as for artificial agents. Recently, there have therefore been efforts to expand the definition of reward and introduce other concepts that could serve as additional learning factors such as curiosity,6, AP firing rates of some SNc/VTA neurons rapidly and transiently increase in response to unexpected rewards  is specifically expressed in dopaminergic neurons by injecting Cre-dependent adeno-associated virus (AAV) into the SNc/VTA of dopamine reuptake transporter (DAT)-Cre mice  and the adjacently lying ventral tegmental area (VTA) . These m rewards  and moreCre mice . These mCre mice .818 Mice were also found to reinforce nose-poking when this triggered optogenetic stimulation of dopaminergic neurons.19 In an operant training behavior, mice would also learn to press a lever in order to self-stimulate dopamine neurons optogenetically, with some mice persevering with dopamine self-stimulation even when this was paired with footshock.20 In head-restrained mice carrying out a visual discrimination task in order to receive reward, stimulation of dopamine neurons appeared to enhance learning speed and prolonged session duration.21 Optogenetic stimulation of dopamine neurons was also found to evoke orofacial movements that share similarities to those time-locked to reward-predicting cues in animals trained in stimulus-reward association tasks.22 Finally, specific spontaneous movements occur more frequently in a behavioral session if they were previously paired with optogenetic stimulation of dopaminergic axons in the dorsolateral striatum (DLS).23 A large body of evidence therefore supports the notion that increases in dopamine are rewarding and act as positive reinforcers. Conversely, reduced firing of dopamine neurons, either by stimulating GABAergic inputs to the dopamine neurons24 or direct optogenetic inhibition of dopamine neurons25 can cause aversion, acting as a negative reinforcer.As discussed in the previous section, signals representing unexpected reward and RPE are useful for reinforcement learning, and dopamine neurons could therefore causally serve to deliver such learning signals. To test this hypothesis, it is essential to directly manipulate the activity of dopamine neurons. Current experimental data largely supports the notion that artificially induced transient increases in dopamine indeed act to positively reinforce behavior. By expressing ChR2 in dopamine neurons, dopamine concentrations can be increased through blue light stimulation, similar to the opto-tagging experiments described above. Optogenetic stimulation of dopamine neurons was found to induce place preference, such that a mouse would spend more time in the region of the test chamber in which the dopamine neurons were driven to fire at high frequency.6 If a sensory cue is repeatedly presented to a subject in a manner that anticipates reward delivery, then dopamine neurons will shift their responses to the earliest time point predictive of upcoming rewards. This computation is useful for optimally learning rewarded sensorimotor sequences.6,7,26,27 This shift in their response profile upon learning strongly reflects what one would expect from an RPE signal, according to the TD learning model. As mentioned in the previous section, in TD learning, RPE is obtained from the difference between the observed and predicted values of the current state. When we consider this computation performed moment-by-moment in a time-continuous setting, the result is that TD-RPE approximates the derivative of the value function. As unexpected rewards are presented, the sudden increase of value associated with that state generates a positive RPE, which propagates to the predictive cue over learning. At that stage, dopamine neurons will respond to the increase in rate of change of the value function associated with the presentation of the cue that is only resolved at the time of reward presentation.Interestingly, dopamine not only signals unexpected primary rewards, such as water for a thirsty animal, but, through learning, dopamine signals develop in response to cues that predict future reward .6 If a s6,8,9,28,29 If the sensory cue predicts upcoming reward with complete certainty, then the reward delivery itself becomes entirely expected, and thus, the dopamine signal at the reward delivery time decreases.7,26 This is well in accordance with the RPE signaling hypothesis and implies that with experience, the subject builds an internal model that associates an expected value (reward probability and size) to a given sensorimotor sequence in a specific context. Whereas in monkey studies dopamine signals for fully predicted reward completely disappear, this has typically not been observed in mouse experiments, perhaps because mice have noisier time estimation abilities or may have been less extensively trained and thus remain more uncertain about reward expectations.8 Indeed, even in monkeys, if the sensory cue is only partially predictive, then the partially predicted reward evokes a dopamine signal, although smaller compared to that evoked by delivery of the same reward at an unexpected time, and the sensory cue evokes a reduced dopamine signal compared to fully reward-predicting cues.7,26Indeed, as a reward becomes expected, the response of dopamine neurons to reward scales as a function of the difference between the obtained and the expected reward, that is, dopamine neurons increase their response to reward when the reward is bigger than expected and decrease when the reward is smaller than expected, even going below baseline firing rates in case of reward omission.30 GABAergic neurons in the VTA are more represented in the rostral and medial parts. Electrophysiological recordings from opto-tagged GABAergic neurons located in the vicinity of the VTA revealed that these inhibitory neurons appeared to encode expectation about rewards, but were not strongly affected by reward delivery or omission.8 Whereas dopamine neurons in VTA represent the difference between observed and predicted reward, the GABAergic neurons predominantly represent only the predicted reward. Optogenetic manipulations of the VTA GABAergic neurons directly showed that these neurons provided inhibition to the VTA dopamine neurons in a subtractive manner, such that the predicted reward is subtracted from the actual reward in order to compute RPE.31What drives RPE signals in midbrain dopamine neurons? One major input that dopamine neurons receive comes from nearby inhibitory GABAergic neurons. Dopaminergic neurons form approximately 60% of the neuronal population in VTA, and the rest are mostly GABAergic inhibitory neurons, with a smaller fraction of glutamatergic neurons.32 Applying this method to specifically trace inputs to midbrain dopamine neurons revealed many inputs, including from the cortex, basal ganglia, amygdala, hypothalamus, midbrain reticular formation, periaqueductal gray, superior colliculus, dorsal raphe, and the parabrachial nucleus.36 brainstem,37 midbrain,38 basal forebrain,39 and dorsal raphe nucleus,37 and GABAergic inputs from the lateral hypothalamus,40 brainstem,38 dorsal raphe nucleus,35 and ventral pallidum.41 Interesting differences were also observed comparing the inputs to SNc and VTA dopamine neurons.33,34 Whereas SNc dopamine neurons receive stronger input from the dorsal striatum, globus pallidus, subthalamic nucleus, substantia nigra pars reticulata (SNr) and sensorimotor neocortex, VTA neurons receive stronger input from ventral striatum, ventral pallidum, lateral habenula (LHb), prefrontal and orbital cortex, hypothalamus, and dorsal raphe. These differences in their inputs likely contribute to their differential activity patterns, as discussed later.In addition to input from local inhibitory neurons, dopamine neurons receive a multitude of inputs from other brain regions. These can be identified in a brain-wide manner by using monosynaptically restricted mapping of presynaptic neurons labeled by a modified rabies virus.42 In doing so, the LHb governs the activity of the downstream dopaminergic reward system, mainly through a disynaptic pathway relayed via midbrain GABAergic neurons of the rostromedial tegmental nucleus (RMTg), also known as the tail of the VTA.43 The RMTg neurons show phasic activation in response to aversive stimuli like footshocks, shock-predictive cues, food deprivation, or reward omission, whereas they are inhibited after rewards or reward prediction.44 As such, inhibition of LHb activity during reward stimuli would reduce the excitation of GABAergic RMTg neurons, leading to disinhibition of dopaminergic VTA neurons. This neuronal circuitry may play a key role in reward learning since habenula lesions were found to impair midbrain dopamine neurons from encoding reward omission in a reward-conditioning task.45 In support of these observations, it was recently reported that reward-predictive cues drive LHb inhibition mediated by fast GABAergic neurotransmission, which increases as reward-anticipatory behavior emerges.46 Although there is growing evidence that LHb participates in reward-based learning through a LHb-RMTg-VTA circuit,46,47 the upstream synaptic inputs onto LHb neurons active during these behaviors currently remain less clear.The neuronal circuitry and function of LHb inputs have been studied in some detail. Glutamatergic LHb neurons were found to be excited by reward omission, while reward-related stimuli inhibit their activity.48 or to directly image dopamine release,49,50 revealing interesting spatiotemporal dynamics of dopaminergic signaling in the striatum. These methods have largely replaced previous efforts to measure dopamine through microdialysis or voltammetry. Genetically encoded fluorescent calcium indicators, such as GCaMPs,51,52 can be expressed in midbrain dopamine neurons through combining mouse genetics and viral vectors, and the activity of individual axons can be imaged using two-photon microscopy together with invasive cranial windows or bulk signals can be measured using fiber photometry.48 Recently, it has also become possible to image dopamine release more directly through the development of genetically encoded fluorescent proteins sensitive to dopamine, such as dLight49 and GRAB-DA.50 Both of these dopamine sensors were engineered to couple a native dopamine receptor to a circularly permuted GFP, rendering fluorescence upon dopamine binding to the receptor. On the whole, the imaging data are consistent with a transient increase in dopamine in the ventral striatum (nucleus accumbens) following an unexpected reward and more generally signaling RPE,48,49,53 in good agreement with the electrophysiological measurements. However, other parts of the striatum seem to receive different dopamine signals. Indeed, movement, choice, and motor-related signals may dominate dopamine signaling in the DLS, with a smaller contribution of RPE signals.48,48,53,55 although important reward signals have also been reported in the dorsal striatum.57 Indeed, optogenetic stimulation of SNc dopamine neurons, which primarily innervate the dorsal striatum, enhanced movement initiation, whereas optogenetic inhibition of these neurons reduced the probability of movement initiation.55 Wave-like propagation of dopamine signals has also been reported through imaging across the dorsal striatum with the directionality changing depending upon task variables.58 Additionally, ramping dopamine concentration has also been reported to signal proximity to distant rewards,59 which may help enhance motivation and vigor, but these ramping signals can likely also be accounted for by careful consideration of RPE models.63 More recently, the tail of the striatum  has been identified as another region receiving distinct dopaminergic signals. The tail striatum receives dopamine innervation from the most lateral part of the substantia nigra. Rather than encoding reward or movement, the tail striatum dopamine signals seem to function as reinforcers for the avoidance of threatening stimuli.64 High-intensity unexpected sound stimuli, but not rewards, drove dopamine increases in the tail striatum, unlike the ventral striatum. Optogenetic stimulation of dopamine fibers in the tail striatum drove aversion, whereas optogenetic stimulation of dopamine fibers in the ventral striatum drove positive reinforcement.64 It is therefore clear that there are diverse dopamine signals in different parts of the striatum.As an alternative to the extracellular electrophysiological measurement of the somatic AP firing of dopamine neurons, it is also possible to image the activity of dopamine axons,65,66 This finding was supported by in vitro brain slice experiments in which the activation of metabotropic acetylcholine receptors prolonged the duration of spiking in layer 5 pyramidal neurons evoked by electrical stimulation, extending the time window for synaptic plasticity to occur.65 Thus, as described in more detail in the next section, metabotropic signaling by some neuromodulators seems to share common features of promoting synaptic plasticity and learning.Finally, it is important to remember that although dopamine is considered a key signal for reward learning, it is likely to function in a cooperative manner with several other neuromodulatory systems. For example, in the primary visual cortex of rats, acetylcholine has been shown to be necessary for the learning of the expected time of reward predicted by a visual stimulus during reinforcement learning.67 Striatonigral medium spiny neurons (MSNs) projecting from the DLS to the SNr express dopamine type 1 receptors (D1Rs) and define the so-called direct path. Striatopallidal MSNs projecting from DLS to the external segment of the globus pallidus (GPe) express D2Rs and form the basis of the so-called indirect path.The activity of dopamine neurons, at least in part, appears to serve as a signal that encodes RPE. In order to understand what impact these dopamine signals might have upon the brain, it is obviously important to consider where dopamine is released. The most prominent target of the axons of the midbrain dopaminergic neurons is the striatum, and it is presumably by releasing dopamine in the striatum that the midbrain dopaminergic neurons carry out an important part of their function. Two classes of striatal projection neurons make up the vast majority of neurons in the striatum, and these two classes of inhibitory GABAergic neurons can be distinguished through anatomical and molecular features, including the expression of different dopamine receptors .67 Stria68,69 Following LTP induction, enhanced efficacy of glutamatergic input increases the amplitude of excitatory postsynaptic potentials (EPSPs) in D1R-expressing MSNs, which can last for many hours. Such long-term synaptic plasticity likely contributes to learning.The striatum receives strong glutamatergic input from the cortex and thalamus. Glutamatergic input to MSNs can change strength through the induction of long-term synaptic plasticity . The pai70,71 ,72 inducing phosphorylation of cAMP-regulated phosphoprotein 32 kD (DARPP-32) and thereby turning PP1 off. A key impact of dopamine in D1R-expressing MSNs therefore seems to be in helping the activation of CaMKII by turning off its inactivation by PP1. Dopamine acting via D1Rs therefore enhances the activation of CaMKII, leading to the induction of LTP at the activated synapses. Interestingly, Haruo Kasai and colleagues70 found that the dopamine signals can arrive up to 1 s after the pairing of presynaptic and postsynaptic activity and can still retrogradely enhance LTP through inactivating PP1 to enhance CaMKII activity. This observation is important because rewards are typically delivered after the correct stimulus-response sensorimotor neuronal activity. In order for the dopamine reward signal to contribute to learning through the synaptic plasticity of sensorimotor circuits, it must therefore interact with traces of recent neuronal activity.73,74 This is often referred to as the “credit assignment problem” of identifying which synapses should be changed in order to learn and has led to the hypothesis of preferential synaptic plasticity of recently active synapses highlighted by an “eligibility trace,” sharing some similarity to the synaptic tagging hypothesis.75 The 1-s window of retrograde enhancement of LTP of recently activated synapses demonstrated in vitro in brain slices could help bridge the time between sensorimotor processing and reward feedback during the learning of simple stimulus-response-reward associations. Altogether, it seems plausible that a delayed dopamine reward signal might trigger plasticity at recently activated synapses, which might have been involved in the sensorimotor activity that gave rise to the reward, thus contributing to reinforcement learning. Similar observations have been made for other neuromodulatory signals, including the effects of norepinephrine and serotonin on the plasticity of cortical glutamatergic synapses76 and dopamine affecting synaptic plasticity in the hippocampus,77 with experiments showing that neuromodulatory agonists can change the effect of synaptic plasticity induction protocols carried out seconds before the application of the neuromodulatory agonists.The molecular signaling pathways engaged by LTP induction on D1R-expressing MSNs have been investigated in detail by Haruo Kasai and others.70,71 . At base71 D2R activation stimulates Gi/o subtypes of G proteins, which inhibit cAMP production and suppress PKA. Whereas increases in dopamine do not appear to drive reductions in PKA activity, decreases in dopamine do evoke increases in PKA activity in D2R-expressing MSNs.72 Such dopamine dips appear to be important for mice to carry out a task in which they learn to discriminate between reward-predicting and distractive auditory tones.71 The absence of reward in response to the presentation of distractor tones resulted in a reduction in dopamine in the ventral striatum during discrimination learning. The dopamine dip enhanced LTP of glutamatergic inputs onto D2R-expressing MSNs via increased PKA activity, provided concomitant NMDAR-mediated activation of CaMKII and co-activation of adenosine A2A receptors.71 Reward omission causing transient reductions in striatal dopamine  might therefore contribute to synaptic plasticity and learning through D2R-expressing MSNs.Interestingly, transient decreases in dopamine signals have been reported, especially after reward omissions and more generally in response to negative RPE. Unexpectedly bad outcomes are also important learning signals, and it is therefore also interesting to consider the effects of decreases in striatal dopamine concentration. Dopamine receptor subtypes differ in their affinity for dopamine, with D2Rs having an affinity approximately two orders of magnitude higher than D1Rs. It is thus possible that decreases in dopamine might not be sensed by D1Rs because they may be less activated under basal conditions, and further decreases in dopamine may be outside of the relevant dose-response range of receptor modulation. On the other hand, it may be that D2Rs are normally highly occupied with dopamine even during basal conditions because of their higher affinity. A reduction in dopamine concentration might then lead to a decreased activation of D2Rs. Haruo Kasai and colleagues investigated how such dopamine decreases might affect D2R-related signaling and learning in mice.+, K+, and Ca2+ channels,78,79 with a recent study suggesting that D1R activation increases excitability of striatonigral MSNs largely through voltage- and Ca2+-dependent K+ channels.80 Dopamine receptors are also prominently expressed on presynaptic terminals and other cell classes in the striatum, including the D2Rs on cholinergic interneurons81 and astrocytic glial cells.82 Finally, dopamine receptors are also found in other brain regions, including frontal cortex, which also receives dopaminergic innervation from VTA neurons. The overall functional role of dopamine signals is therefore likely to be complex.It is important to note that dopamine likely does more than modulate the induction of synaptic plasticity in the striatum. Dopamine receptors in MSNs regulate various ionic conductances, including voltage-gated NaThe hypothesis that dopamine signals might contribute to reward learning through modulating synaptic plasticity of specific neuronal circuits remains to be further tested in detail, but some experiments support the notion that striatal MSNs expressing D1Rs can contribute to driving goal-directed motor output and show enhanced fast sensory responses across learning, consistent with the dopamine hypothesis. Here, we will focus on a whisker detection task, which has been investigated in some detail with respect to D1R- and D2R-expressing MSNs across reward-based learning .83–85 Inm) recordings during the whisker detection task84,85 were obtained from neurons located in the region of the DLS, known to receive direct glutamatergic input from the primary whisker somatosensory cortex  optogenetic stimuli were delivered through an optical fiber inserted into the DLS. The optogenetic stimulus trials were randomly interleaved with whisker stimulus trials and no-stimulus catch trials. Stimulation of D1R-expressing neurons evoked licking, but not the stimulation of D2R-expressing neurons. Brief activity in D1R-expressing neurons therefore seems to be sufficient for task execution with the optogenetic stimulus readily substituting for the whisker stimulus. The fast sensory-evoked depolarization of D1R-expressing neurons found in expert mice  generates a dopamine signal. Some aspects of how thirst is represented in the brain are beginning to be understood, but it remains difficult to assemble an integrative view of how this impacts behavior. Blood osmolality is first sensed by neurons in the subfornical organ and the organum vasculosum of the lamina terminalis, structures that lack the blood-brain barrier. Interestingly, optical stimulation of a genetically defined subset of neurons in the subfornical organ  immediately triggers drinking behavior, and these neurons are also activated by thirst.In conclusion, remarkable progress has been made linking striatal dopamine signals to reward learning, but much remains to be learned.


The escalating crisis of antimicrobial resistance (AMR) warrants innovative therapeutic strategies. Fecal microbiota transplantation (FMT) and maggot debridement therapy (MDT) represent paradigm-shifting approaches, leveraging biological systems to mitigate AMR. FMT restores a healthy gut microbiome, providing a biotherapeutic counter to pathogenic bacteria, thereby reducing reliance on traditional antibiotics. Conversely, MDT, a form of bio-debridement, utilizes the antimicrobial secretions of maggots to cleanse wounds and eliminate resistant bacteria. Despite the promise these therapies hold, their broader clinical adoption faces multifaceted challenges including the need for rigorous scientific substantiation, standardized protocols, deepened understanding of mechanisms of action, and surmounting regulatory and public acceptance barriers. However, their potential integration with precision medicine could revolutionize disease management, particularly with antibiotic-resistant infections. The rising problem of drug-resistant infections calls for new treatment methods. Fecal microbiota transplantation (FMT) and maggot debridement therapy (MDT) offer innovative solutions. FMT uses healthy gut bacteria to fight harmful ones, while MDT employs maggots to clean wounds and kill resistant bacteria. Although promising, these methods face challenges like a lack of understanding, standardization and public acceptance. Their potential, however, could transform how we manage antibiotic-resistant diseases. Fecal microbiota transplantation (FMT) and maggot debridement therapy (MDT) present paradigm-shifting strategies to tackle antimicrobial resistance (AMR). Offering biological counters to pathogenic bacteria & antibiotic reliance. Promise & challenges ahead. #AMR #FMT #MDT. Antimicrobial resistance (AMR) is a formidable challenge that the global health community currently faces . This esIn response to this urgent need, this piece examines two unconventional yet potentially promising therapeutic approaches: fecal microbiota transplantation (FMT) and maggot therapy. FMT is a procedure which has been used since third-century China , involveThis paper aims to provide a comprehensive overview of these unconventional therapies, examining their historical context, the mechanisms underlying their action, their potential clinical applications and the challenges that need to be addressed to facilitate the transition of these therapies from the laboratory to the clinic. As we strive to push the boundaries in our battle against AMR, we must not hesitate to explore unconventional avenues. However, the journey from bench to bedside is fraught with challenges. The safety and efficacy of these therapies need to be thoroughly evaluated through rigorous scientific research ,11. ReguAs we stand on the precipice of a post-antibiotic era, it is imperative that we explore all possible avenues in our fight against AMR. The potential of FMT and maggot therapy represents a beacon of hope in this daunting scenario. Through rigorous research and open-minded exploration, we may yet turn the tide in our battle against antimicrobial resistance. The understanding and application of these therapies could open up new avenues in the fight against AMR, providing us with additional tools in our therapeutic arsenal.The vast and intricate assemblage of microorganisms that reside within the human body, known as the human microbiome, has become an increasingly significant area of focus in medical research ,13. ThisUnderstanding the relationship between the microbiome and AMR involves considering two interconnected facets. The first perspective regards the gut microbiota as a potential reservoir of antimicrobial resistance genes, known collectively as the resistome . This vaGiven these dynamics, the use of FMT has been proposed as a potentially effective intervention for restoring a balanced microbiome and thereby helping to curb AMR ,19. The Clostridioides difficile infection, a condition known to be linked to dysbiosis [The potential for FMT as a tool in the fight against AMR is a relatively new area of research that is beginning to gain recognition. FMT has been conventionally used for the treatment of recurrent ysbiosis . Howeverysbiosis ,15,18. Tysbiosis , and notysbiosis ,20.While the precise mechanisms by which FMT could help combat AMR are not fully understood, preliminary research indicates several possible avenues. For instance, the reintroduction of a diverse microbiota through FMT could outcompete resistant pathogens for resources and space, limit their proliferation, and reduce the likelihood of resistant infections ,20. FurtClostridium difficile Infection (rCDI), a condition intrinsically tied to antibiotic usage and resistance [In the realm of combating AMR, FMT stands as a promising frontier in microbiome therapy. Its proven efficacy in treating Recurrent sistance ,24, undesistance ,20. Sincsistance . FMT's psistance , irritabsistance , and metsistance . The hypIn a recent multicenter phase I trial, the potential of FMT was explored in combination with PD-1 inhibitors nivolumab or pembrolizumab in previously untreated patients with advanced melanoma . The stuFMT is positioning itself as a novel strategy in the fight against AMR ,20. Its Lucilia sericata, to necrotic or infected wounds [The concept of using maggots for therapeutic purposes, while repulsive to some, is not new. The healing properties of maggots have been acknowledged since antiquity, with their use in wound debridement dating back centuries ,11. Now,d wounds . The lard wounds . By seled wounds .MDT's potential in the context of AMR lies in its dual action: physical wound debridement coupled with the secretion of antimicrobial substances. The enzymes secreted by the maggots can break down biofilms  often found in chronic wounds that are notoriously resistant to antibiotics . The antet al. [The application of MDT has proven effective in managing chronic wounds such as diabetic foot ulcers , venous et al.  found siBiofilms are complex assemblies of microbial communities shielded within a self-generated matrix, rendering them resilient to traditional antimicrobial interventions . In addiHowever, as with any therapy, MDT is not without its challenges. Patient acceptance of MDT can be a significant hurdle in its widespread adoption. The utilization of maggots in medical treatment often elicits a natural repulsion or discomfort among many individuals. This psychological barrier may stem from cultural perceptions, personal phobias, or general unease with the unconventional nature of the therapy ,37. The The unconventional yet promising modalities of FMT and MDT, despite being disparate in their origins and applications, intersect in their potential to alleviate the AMR crisis. Each harnesses biological mechanisms in their approach, either by reinstating a protective microbiome or employing a biological debridement technique with additional antimicrobial effects. This shared principle of utilising biological means to target pathogenic bacteria could potentially reduce the dependency on antibiotics, thereby mitigating the proliferation of resistance. These therapeutic strategies represent a paradigm shift in the management of microbial resistance, symbolising the potential of intricate biological systems to transform healthcare. Their broader implications suggest a profound impact on the overall approach to disease management and health promotion. As the medical field moves toward precision medicine, the potential to customise these therapies to individual patients' needs presents an exciting frontier. For instance, the prospect of targeted FMT, using defined combinations of beneficial bacteria, and the potential fine-tuning of MDT to cater to specific wound environments, signal a future where these therapies can be personalized.In essence, FMT and MDT provide potential innovative pathways in the ongoing battle against AMR. As our comprehension of complex biological systems deepens, these therapies could pave the way for a more sustainable and efficient approach to managing AMR. While the path to realising their full potential will require dedicated scientific investigation, the initial promise warrants continued exploration and investment in these research areas.AMR continues to pose a formidable challenge to global health, necessitating innovative therapeutic strategies . Two intC. difficile infections represents more than a treatment for a specific disease [The advantages of FMT are anchored in its biological approach. Its effectiveness in recurrent  disease ; it demo disease . However disease .MDT's efficacy in wound healing is well-documented, offering a cost-effective and natural method of debridement. Its antibacterial properties provide a dual function, cleaning the wound while promoting healing ,10. ThisFMT and MDT in the context of AMR open promising avenues that are yet marked by a profound lack of comprehensive evidence. The advantages and limitations illuminated within this examination underscore a complex interplay between scientific potential and unresolved challenges. Our current understanding, although advancing, is nascent and overshadowed by the vastness of what remains uncharted. The field beckons for methodologically rigorous, systematic investigations that extend beyond anecdotal successes and delve into the core mechanisms, safety profiles and long-term outcomes. The guarded optimism surrounding FMT and MDT must be matched with a steadfast commitment to further research, embracing a multidisciplinary approach that fosters innovation, transparency, and a nuanced understanding of these novel interventions. This informed pursuit can turn experimental therapies into viable strategies in our fight against AMR, yet it demands we navigate with caution, guided by evidence and a recognition of the complexities involved.Emerging therapies such as FMT and MDT, promising as they are in combating AMR, bring with them a distinct set of regulatory challenges ,42. TheiA prominent regulatory issue for FMT revolves around its classification. The designation of fecal material whether as a drug, a tissue, or a unique entity significantly influences the regulatory requirements it must meet ,43. ThisMDT, though distinct, faces similar regulatory hurdles. As the therapy involves the introduction of living organisms (maggots) into open wounds, there are inherent safety and quality control concerns ,11,35. RMoreover, both FMT and MDT present challenges regarding the intersection of regulation and public perception is critical in determining patient acceptance of these therapies. Robust regulatory oversight that is transparent and clear can bolster public confidence in these treatments and increase their acceptance . As thesC. difficile infections. Conversely, the use of MDT has been shown to specifically target biofilms, which are key in AMR gene transmission. However, patient acceptance of MDT can be a significant hurdle, given the repulsion many feel toward maggots, and standardized protocols and guidelines for MDT use are still in development. Furthermore, the path to broader clinical adoption of these therapies is fraught with challenges, including a lack of deepened understanding of the precise mechanisms underlying their therapeutic effects, and the need for rigorous scientific substantiation, standardized procedural protocols, surmounting hurdles in regulatory landscapes and public acceptance. Successful advancement of these therapies will necessitate a coordinated multidisciplinary approach, involving the concerted efforts of clinicians, researchers, regulators and patients alike.The escalating threat of AMR underscores the pressing need for innovative therapeutic strategies. The biological underpinnings of FMT and MDT offer novel approaches that may help circumvent the rise of resistance by reducing reliance on traditional antibiotics. For example, a recent study demonstrated FMT's potential as a first-line treatment in patients with advanced melanoma, supporting its broader applications beyond recurrent Peering into the future, the intersection of FMT and MDT with precision medicine heralds intriguing prospects. The potential to customise these therapies to individual patients could precipitate a paradigm shift in the management of various health conditions, particularly those involving antibiotic-resistant infections. The introduction of a table delineating the multifaceted pros and cons of FMT and MDT provides a comprehensive overview to aid in future decisions. However, the transition from experimental therapies to routine clinical applications necessitates robust and multifaceted endeavors. A deeper understanding of the precise mechanisms underlying its therapeutic effects remains an area of ongoing exploration. While the benefits of MDT in wound healing and bacterial control are recognized, the specific biochemical and cellular processes that facilitate these outcomes are not fully elucidated. This gap in knowledge highlights the need for further scientific investigation to refine the methodology, enhance its efficacy and broaden its applicability in the fight against antimicrobial resistance. The formidable challenge posed by AMR is matched by the potential of innovative solutions to counter it, but there is still a pressing need for further research, especially considering the lack of substantial evidence supporting these therapies. As the field evolves, it is the hope that these therapies will offer fresh ammunition to curtail the burgeoning threat of AMR, contributing toward a more sustainable and effective strategy in managing infectious diseases. The potential benefits of FMT and MDT in the struggle against AMR warrant sustained investment in these research areas, as evidenced by the fact that drug-resistant infections are now once again a leading cause of death worldwide.Antimicrobial resistance (AMR) constitutes a global health emergency; fecal microbiota transplantation (FMT) and maggot debridement therapy (MDT) are pioneering therapeutic options to confront this mounting predicament.Delving into the biological foundations of FMT and MDT, these innovative methodologies may offer significant avenues to alleviate AMR, with implications for reducing reliance on traditional antibiotics.FMT is emerging as a promising intervention against pathogenic bacteria; however, comprehensive scientific validation is vital for its wider clinical application.Through the utilization of maggots' antimicrobial secretions, MDT promises a unique wound treatment strategy; however, standardized application and public perception remain challenges.The potential synergy between these therapies could represent a paradigmatic transformation in the management of antibiotic-resistant infections.While heralding significant potential, these therapies must traverse a multifaceted landscape of challenges, encompassing scientific substantiation, procedural standardization, mechanistic comprehension and regulatory and public acceptance.The regulatory environment poses significant hurdles; successful implementation will require coordinated efforts across clinical, research, regulatory and patient communities.The integration of FMT and MDT with precision medicine foreshadows a radical shift in the management of infectious diseases, particularly those characterized by antibiotic resistance; yet, the transition from experimental to routine clinical applications will necessitate robust, multifaceted efforts.