Besides the diagnosis of endometrial carcinoma (EC), the identification of EC subtypes is an important precondition for the effective treatment of the disease. Molecular factors have an important role in this context. Biomedical analysis is already very possible in experimental approaches. However, for clinical practice the procedures are often too complex, too expensive, and too time-consuming. In the present study, very good evaluated molecular markers were detected using an alternative method and compared with the original method. Analyses showed very good results. The important advantage of the new procedure is that the required molecular data can be obtained exclusively by sequencing. This may greatly simplify EC subtype classification and could be more easily incorporated into routine clinical diagnosis.Background: The Cancer Genome Atlas (TCGA) network  identified four molecular endometrial cancer (EC) subtypes using an extensive multi-method approach. The aim of this study was to determine the four TCGA EC molecular subtypes using a single-method whole-exome sequencing (WES)-based approach provided by MH Guide . Methods: WES and clinical data of n = 232 EC patients were obtained from TCGA. The four TCGA EC molecular subtypes designated as (i) Mutated Polymerase ε (POLE), (ii) Microsatellite Instability (MSI), (iii) Copy Number (CN) low and, (iv) CN-high were determined using the MH Guide software. The prognostic value of the subtypes determined by MH Guide were compared with the TCGA classification. Results: Analysis of WES data using the MH Guide software led to the precise identification of the four EC molecular subtypes analogous to the TCGA classification. Both approaches displayed high concordance in terms of prognostic significance. Conclusions: The multi-method-based TCGA EC molecular subtypes can reliably be reproduced by the single-method-based MH Guide approach. The easy-to-implement single-method MH Guide approach represents a promising diagnostic tool. Endometrial cancer (EC) is the sixth most common cancer in women worldwide, with more than 11,000 newly diagnosed cases and about 2700 cancer-related deaths in Germany per year ,4,5,6. ESeveral trials have shown that genomic profiling improves the risk assessment of patients with early-stage EC, thereby enabling individualized therapeutic approaches to optimize clinical outcomes ,11. The Moreover, the prognostic value of these four molecular EC subtypes with distinct clinical courses is of highest clinical relevance as it could be applied to therapy stratification. For instance, the POLE subtype was shown to have a significantly better progression-free survival (PFS) than the CN-high subtype, which was associated with the worst clinical outcome . CharactIn the present study, EC molecular subtypes according to the TCGA classification were reproduced using an innovative single-method analysis tool based on WES. Raw sequencing data from TCGA  were anaTCGA data processing: Raw sequencing data from n = 232 patients from the TCGA EC cohort were available from the Genomics Data Commons (GDC) for protected use under the project ‘Investigation of Cancer Subtyping Based on NGS Data’ (project ID 12045). Clinical data was downloaded from Firehose  and cBioportal ), and supplemented with molecular subtypes . Aligned reads (bam format) from whole-exome DNA sequencing (Illumina GAIIx or HiSeq 2000 platforms) of primary tumor and matched blood (preferred) or tissue-derived normal samples were downloaded from the GDC legacy archive. Paired-end sequencing reads were extracted from alignment files using PicardTools (version 1.115) and SamToFastq . Potentially existing unpaired reads were discarded.MH Guide classification: Read files (fastq format) from raw sequencing data from n = 232 patients from the TCGA EC cohort were classified using the oncology treatment decision support software MH Guide . MH Guide provides a full bioinformatics pipeline plus clinical annotation of variants called single nucleotide variants (SNV), short indels, somatic CN alterations, and MSI status. POLE and MSI tumors were classified in the same manner as TCGA classification , using STo reproduce the CN-high class of TCGA classification, we trained a supervised classification model to predict the CN cluster 4 of TCGA from the WXS-based copy number calls of MH Guide. Cross-validation accuracy, precision, recall, and f1 were used to evaluate how well the classification based on DNA NGS data reproduced the original three platform molecular subtypes of TCGA. CN clusters were extracted from the original data set  and binaA naïve Bayes classifier with the following features was selected: Number of CN gains/CN losses per sample, number of CN gains/CN losses per chromosome, number of CN gains/CN losses per gene for the 25 most abundant genes, and ploidy and length of CN alterations per mega base. Although our features violate the assumption of uncorrelated features for a naïve Bayes classifier, this model showed robust performance in predicting CN cluster 4 with average cross-validation accuracy, precision, recall, f1-score, and ROC-AUC of 91.25%, 83.51%, 85.77%, 83.98%, and 95.08%, respectively, for the 240 training samples.Statistics: The MH Guide classification results were evaluated using accuracy, precision, recall, and f1 scores with weighted averaging across sources to consider the imbalance of classes. The risk stratification performance of both classifications was assessed using Kaplan–Meier survival curves and multivariable log-rank tests. The chi-square test was used to test for a difference between class distributions of different classification methods. The MH Guide algorithm was applied to the publicly available TCGA EC cohort comprising n = 232 patients  12]. Ac. Ac12]. p = 0.9041).Application of the MH Guide algorithm to the TCGA EC cohort led to consistency with respect to identification of molecular EC subtypes in approximately 89% of the cases. There were slightly differing results in a few individual cases. In particular, among the EC cases with unfavorable prognosis, individual patients were assigned to different subtypes according to both classification systems, as indicated in Identification of the four molecular EC subtypes based on whole-exome sequencing data using the MH Guide algorithm resulted in good overall cross-validation accuracy, precision, recall, and f1-score of 88.78%, 91.92%, 90.76%, and 91.01%, respectively when using macro-averaging, and 88.78%, 89.61%, 88.78%, and 88.82%, respectively when using weighted averaging.To further evaluate the distributions of the different EC subtypes according to both methods, survival analyses were performed. In line with the results presented above, the Kaplan–Meier survival analyses demonstrated a very comparable overall pattern .Both PFS and OS demonstrated comparable values for POLE, MSI, CN-low, and CN-high subtypes. The 36-month PFS rates n = 220,  for the According to the WHO classification, seven histopathological EC types are differentiated: endometrioid, serous, clear cell, mixed, neuroendocrine, undifferentiated and dedifferentiated carcinomas, carcinosarcoma, and unusual carcinoma types . These hMolecular diagnostics are increasingly important in modern, individualized oncology as they enable therapy stratification to improve PFS and OS on the one side and to reduce therapy-related toxicity and to increase safety on the other side. The four TCGA EC molecular subtypes, namely POLE, MSI, CN-low, and CN-high, enable further stratification of EC patients allowing individualized treatment decisions. Patients with POLE hypermutated EC, typically displaying high-grade endometroid carcinomas, exhibit an excellent prognosis. In this setting, overtreatment by means of adjuvant chemotherapy can be avoided. In contrast, the CN-high subgroup, which comprises EC with serous-like histology and p53 gene mutations, exhibits a very poor prognosis and requires an extended therapy approach. Hence, it is critically important to identify the different EC subtypes in order to avoid under-treatment of high-risk patients and over-treatment of low-risk patients.With regard to prognosis, the EC subtypes MSI and CN-low are considered intermediate. The MSI subtype is characterized by high mutation rates, hypermethylation, and a frequently hypermethylated MLH1 promoter. Histopathologically, CN-low EC are endometrioid carcinomas with low copy number alterations including EC with nonspecific molecular profiles. For both subtypes, MSI and CN-low, prognosis is considered intermediate.For MSI patients, there is an increased risk of developing further cancer  ,19. In tIn the recently published PORTEC3 study, a multicenter, randomized, prospective phase III trial, molecular EC subtyping comparable to the TCGA classification was performed with regard to clinical outcomes. This study confirmed the strong prognostic value of molecular EC subtypes regardless of the histologic type. Especially, patients with POLE hypermutated tumors had excellent clinical outcomes ,20. ThesAs a consequence, EC subtyping is recommended in both the European EC guidelines and the EC guidelines of the National Comprehensive Cancer Network in the USA .The molecular EC subtypes defined by TCGA in 2013 mark a milestone in the understanding of this gynecologic malignancy and represent a step towards individualized therapy in EC patients. However, this classification requires an extensive multi-omics approach, making it impractical in routine clinical practice. Due to this not inconsiderable effort, the original molecular subtypes have not yet been evaluated in prospective trials. Molecular subtyping of EC, i.e., prognostic evaluation of the methodology and its introduction into routine diagnostics, would benefit considerably if it could be performed with a single routine laboratory method. In current clinical routine, molecular EC subtypes are determined by molecular surrogate markers analogous to the TransPORTEC or ProMisE, algorithm . Both alThe implementation of a simple single method approach would not only facilitate evaluation of large international EC cohorts, but it is also the more feasible procedure in clinical routines at EC therapy centers. In addition, the sequencing approach of the MH Guide classification has the advantage of determining the mutation status of further possible target genes in terms of individualized therapy as BRCA-1/2, CTNNB1, or PTEN. This could further improve molecular subtyping and, since no additional laboratory procedures need to be installed, simplify molecular diagnostics in EC. Of note, identification of possible druggable mutations might delineate targeted treatment determination in later therapy lines of EC. This is a clear advantage over the currently used classification systems TransPORTEC and ProMisE.The four molecular subtypes of EC according to TCGA classification are determined using a multi-method approach. The present study shows that this can also be performed based on WES data using MH Guide software. All four molecular TCGA EC subtypes (i) POLE, (ii) MSI, (iii) CN low, and (iv) CN high could be accurately identified after MH Guide analysis. The prognostic deductions from MH Guide analysis were also highly consistent with the original TCGA classification. The multi-method molecular subtypes of the TCGA classification system can be reliably reproduced by the single-method MH Guide approach. Thus, the easy-to-implement MH Guide approach represents a promising diagnostic approach for clinical use.


Lipomatous atrial septal hypertrophy (LASH) with atrial septal defect (ASD) is a rare congenital anomaly. Although LASH is a histologically benign cardiac lesion characterized by excessive fat deposition in the interatrial septum that spares the fossa ovale, it has been associated with supraventricular arrhythmias or sick sinus syndrome. Application of multimodal imaging is crucial for accurate diagnosis, appropriate treatment of LASH with ASD, and follow-up.A 68-year-old female patient presented with recurrent chest tightness and palpitation. Multimodal imaging revealed the characterizations of LASH and ASD. Two-dimensional transesophageal echocardiography showed a “dumbbell”-shaped involvement of the cephalad and caudal regions with sparing of a single secundum ASD. The septum with a brightness feature is an uncommon condition characterized by the deposition of unencapsulated fat cells in the atrial septum. Real-time four-dimensional transesophageal echocardiography reflected the lipomatous hypertrophy of the atrial septum and an oval-shaped ASD. Cardiac computer tomography angiography later confirmed this finding. The patient achieved a good clinical response with an ASD percutaneous occlusion guided by intracardiac echocardiography (ICE).This case demonstrates a LASH combined with ASD. Multimodality imaging can provide an accurate diagnosis and may guide the procedure for precise occlusion. Lipomatous atrial septal hypertrophy (LASH) with atrial septal defect (ASD) is a rare abnormality. LASH is a histologically benign cardiac lesion characterized by excessive fat deposition in the interatrial septum that spares the fossa ovale –3. The pA 68-year-old female patient with a 10-year history of hypertension was hospitalized in our department due to recurrent chest tightness and palpitation for half a month, accompanied by limb edema and dizziness. She had a history of multiple ground-glass nodules in both lungs, pulmonary bullae in the lower lobe of the right lung, esophageal papilloma, reflux esophagitis, gastric body submucosal eminence, and erosive gastritis. On presentation, she was afebrile, with a heart rate of 75 beats per minute, a blood pressure of 175/90 mmHg, and normal oxygen saturation.An electrocardiogram documented that the patient had sinus arrhythmia and intermittent atrial premature beats, with short atrial tachycardia. The levels of serum N-terminal pro-B-type natriuretic peptide (NT-proBNP) (20.59 pg/ml) were elevated. The glycosylated hemoglobin level was 6.5%. Transthoracic echocardiography showed a secundum ASD with a size of 12 mm × 13.2 mm, and the interatrial septum was significantly thickened (19.4 mm). The right atrium and right ventricle were dilated, with a pulmonary-to-systemic flow ratio (Qp/Qs) of 2.0. There was mild tricuspid regurgitation, and the systolic pulmonary artery pressure was ≈49.8 mm Hg. The global left ventricle ejection fraction measured using the Simpson method was 63.4%, and grade I diastolic dysfunction was detected.Two-dimensional transesophageal echocardiography revealed a “dumbbell”-shaped involvement of the cephalad and caudal regions with sparing of a single secundum ASD with a size of 9 mm × 13.2 mm. The maximal thickness of the interatrial septum was 20.7 mm, and the thinnest atrial septum was 1.6 mm from the view at 0°–180°. The atrial septum presented with a brightness feature, a rare condition characterized by the deposition of unencapsulated fat cells in this area . There wA cardiac computer tomography angiography (CCTA) scan was performed to further characterize the atrial septum and evaluate the status of the coronary artery. The CCTA images demonstrated a 9 mm × 13 mm defect on the atrial septum . The volMyocardial perfusion imaging was performed to evaluate metabolic activity. Results showed no hypermetabolic lesions or abnormal myocardial blood perfusion in the heart. The left ventricular systolic and diastolic functions were normal.The patient underwent an ASD percutaneous occlusion under the guidance of intracardiac echocardiography (ICE) . During The patient was examined using multimodality imaging, including two-dimensional TEE (2D-TEE), RT4D-TEE, and CCTA. The diagnostic features of LASH with ASD include the following: (1) initial 2D-TEE imaging showed a “dumbbell”-shaped involvement of the cephalad and caudal regions with sparing of a single secundum ASD, (2) RT4D-TEE demonstrated lipomatous hypertrophy of the atrial septum and an oval-shaped ASD with a significant left-to-right shunt, and (3) CCTA images demonstrated the defect structure of the atrial septum and showed a non-enhancing, smooth, well-marginated mass with an appearance similar to subcutaneous fat.The pathognomonic “dumbbell” shape is due to hypertrophy of the septum primum and secundum with sparing of the fossa ovalis . EchocarIn addition, the images could be misinterpreted as a tumor or other structural abnormalities –23. MassIn this case, TEE played an important role in diagnosing LASH with ASD. Multiple sectional views at 0°–180° could be presented during TEE examinations , which hIn conclusion, in this case, the multimodality imaging techniques, especially for RT4D-TEE imaging, are pivotal for diagnosing a rare LASH with ASD and for deciding to perform the occlusion of ASD guided by ICE.


We investigated destination memory, defined as the ability to remember to whom a piece of information was previously transmitted, for emotional destinations  in Korsakoff’s syndrome (KS). We asked patients with KS and control participants to tell facts to neutral, positive, or negative faces. On a subsequent recognition task, participants had to decide to whom they told each fact. Compared with control participants, patients with KS demonstrated lower recognition of neutral, emotionally positive, and emotionally negative destinations. Patients with KS demonstrated lower recognition of emotionally negative than for emotionally positive or neutral destinations, but there were no significant differences between recognition of neutral and emotionally positive destinations. Our study demonstrates a compromised ability to process negative destinations in KS. Our study highlights the relationship between memory decline and impaired emotional processing in KS. Korsakoff’s syndrome (KS) is the chronic phase of acute Wernicke disease as caused by thiamine deficiency, malnutrition, and prolonged heavy ingestion of alcohol ,2. KS isDestination memory refers to the ability to remember to whom a piece of information was previously transmitted  ,4,5,6. TDestination memory can be influenced by emotion, and research has demonstrated that the ability to remember to whom a piece of information was previously told can be influenced by the emotional characteristics of the destination. One study investigated this issue by asking heathy young and older adults to tell neutral facts to a neutral, an emotionally positive, and an emotionally negative face . AfterwaBesides being based on research demonstrating how destination memory can be influenced by emotion, the current study is based on research demonstrating the negative effects of KS on both destination memory and emotion. The effects of KS on destination memory were investigated in a study in which patients with KS and control participants were invited to tell proverbs to pictures of celebrities, and in a subsequent recognition test, they were invited to indicate to which celebrity they had previously told the proverbs . The resThe impairment in recognition of facial emotional expressions in KS was investigated by Oscar-Berman et al. , who invTo summarize, we investigated whether emotion would influence destination memory in patients with KS. Although previous research has demonstrated a decline in destination memory in patients with KS , they we2 = 0.61, p > 0.10), age (t(51) = 1.35, p > 0.10), and educational level (t(51) = 0.72, p > 0.10). The diagnosis of KS was made by experienced psychiatrists using the DSM V [The study included twenty-five patients diagnosed with KS  who were recruited from several alcohol-dependence or psychiatric medical units and day-care facilities at Lille and Nantes. As for the control group, we recruited 28 participants without previous or current substance addiction and without psychiatric or neurological history . The control group was matched with the KS group according to sex ratio  and those of the control participants (p > 0.1). With these precautions taken, the performance of our participants was likely to reflect the processing of the emotional valences of faces rather than issues of gender or age bias. Concerning facts, they were judged as neutral  by a separate sample of eight middle-aged adults  on a five-point scale . Each fact was printed in black Times New Roman 48-point font under one (16 × 16 cm) colored face on white paper of A4 format.We used twenty-six facts  and twenty-six high quality pictures. The pictures contained unfamiliar faces and were taken from the FACES database . Among tThe assessment of destination memory included three phases: study, distraction, and recognition. The study phase included 24 trials. In each trial, the experimenter presented a fact that participants were invited to tell the paired face. Immediately after the study phase, the participants were engaged in the distraction phase, which consisted of reading strings of three-digit numbers aloud for one min. Immediately after the distraction phase, they proceeded to the recognition phase, in which the previously-exposed 24 face–fact pairs  were presented, one at a time. For each pair, participants were invited to decide, with no time constraint, whether they had previously told the particular fact in the pair to the associated face or not. Encoding was intentional, and participants were instructed that their memory for the facts and destinations would be tested later. In order to ensure that participants could comfortably execute the task, we invited them to perform a training trial prior to the experiment, during which they were asked to tell two facts to two faces to subsequently decide whether they previously told the particular fact in the pair to the associated face or not. As recommended for analyzing recognition memory , performd [d was calculated for nonparametric tests according to the recommendations of Rosenthal and DiMatteo [p ≤ 0.05; p values between 0.051 and 0.10 were considered to be trends.We investigated the differences between patients with KS and control participants regarding mean destination recognition as well as regarding recognition for each category of destination . We also investigated differences across the three categories of destination memory  within each population. Because data were not distributed normally, as observed by Kolmogorov–Smirnov tests, the Mann–Whitney U test was used for intergroup comparisons, and Wilcoxon’s signed-rank test was used for intragroup comparisons. We provided effect sizes using Cohen’s d : 0.20 = DiMatteo  and ElliDiMatteo . For allZ = −5.25, p < 0.001, Cohen’s d = 2.08). Compared to control participants, patients with KS also demonstrated lower recognition of neutral , emotionally positive , and emotionally negative destinations . Patients with KS demonstrated lower recognition of emotionally negative destinations than of emotionally positive  or neutral destinations , but we did not find any significant differences between recognition rates of neutral and emotionally positive destinations . Control participants demonstrated higher recognition of emotionally negative destinations than of emotionally positive  or neutral destinations  as well as higher recognition of emotionally positive than of neutral destinations .Data is provided in Z = −4.23, p < 0.001, Cohen’s d = 1.90). KS patients showed higher false alarms compared to control participants with means of 0.20 (SD = 0.21) and 0.07 (SD = 0.02), respectively . In addition, to investigate potential gender differences, we compared the scores of women vs those of men for mean destination recognition; analysis demonstrated no gender differences between patients with KS  or between control participants and KS patients .Considering hits vs false alarms, the analysis demonstrates lower hits in patients with KS compared to control participants with means of 0.47 (SD = 0.52) and 0.88 (SD = 0.02), respectively (We investigated the effects of emotion on destination memory in KS by asking patients with KS and control participants to remember whether they previously told information to a neutral, emotionally positive, or emotionally negative destination. Compared to the control participants, patients with KS demonstrated lower mean destination recognition as well as lower recognition of neutral, emotionally positive, and emotionally negative destinations. Patients with KS demonstrated lower recognition of emotionally negative than of emotionally positive or neutral destinations, but we did not find any significant differences between recognition of neutral and emotionally positive destinations. Control participants demonstrated higher recognition of emotionally negative than of emotionally positive or neutral destinations as well as higher recognition of emotionally positive destinations than of neutral destinations.To begin with the low mean destination recognition in patients with KS, these findings mirror previous research demonstrating a decline in destination memory in KS, which was attributed to the compromised ability to integrate information with its encoding context . There iIn our study, patients with KS demonstrated lower recognition of emotionally negative than of emotionally positive or neutral destinations, but we did not find any significant differences between the recognition of neutral and emotionally positive destinations. These findings suggest a compromised ability to process negative information in KS. The difficulty to process negative information in KS was reported by research demonstrating a compromised ability of patients to recognize negative facial expressions  and to dThe difficulties of patients with KS to process emotional, especially negative, information can be interpreted in light of the dual-process model ,70, whicUnlike patients with KS, control participants demonstrated higher recognition of emotionally negative than of emotionally positive or neutral destinations as well as higher recognition of emotionally positive than of neutral destinations. The high memory for emotionally negative destinations in our control participants does not fit with the well-known positivity bias in healthy aging, although this bias may explain the higher destination memory of emotionally positive than of neutral destinations in these participants. Research using several methodological approaches supports the notion that older adults tend to prioritize emotionally positive compared to emotionally negative information ,72. ThisOne limitation of our study is that we only used neutral information. It would be of interest to investigate whether patients with KS would demonstrate diminished memory when relaying negative information. However, and regardless of potential limitations, this study has the merit of answering the open question of the relationship between the decline in memory and decline in emotional processing in KS. KS has been mainly investigated with regard to memory decline, and relatively little research has investigated emotional and affective processing in the syndrome. This is not surprising, as in his original work, Korsakoff  did not In summary, our study demonstrates how memory in KS can be influenced by emotion; more specifically, it shows how the emotional attributes of interlocutors may influence destination memory in KS. By doing this, our study contributes to the understating of emotional processing in KS and, more specifically, to the understanding of the relationship between the decline in memory and decline in emotional processing in the syndrome.


In 2011, the Chilean bovine tuberculosis (bTB) program was launched by the Livestock and Agriculture Service (SAG) as a compulsory countrywide program based on testing and culling of bTB reactors at herd-owners expense. This review outlines the rationale and key components of the bTB program, and the dynamic changes that have occurred since 2011. The paper also examines the problems identified by stakeholders and the initiatives put in place to address the constraints to achieving progress.To date, the program has shown progress in controlling bTB. However, in order to achieve bTB eradication it will be essential to improve the commitment of stakeholders, and to develop a framework of strong and workable regulations that will help to manage bTB outbreaks, particularly where clusters of bTB infection are recorded. Bovine tuberculosis (bTB) is a worldwide-distributed disease with incidence levels that vary depending on a range of geographical and environmental factors, as well as production systems and a host of animal and herd risk factors . In ChilIn order to deal with differences in prevalence and production systems throughout the country, regional zoning was applied to provide specific and effective strategies to control and eradicate bTB . The ‘erAccording to the latest livestock census in 2021, there are 125,408 bovine herd owners in Chile and a total population of 3,718,532 cattle, with 71%  of the cattle population located in the southern eradication zone [Due to the high costs of the testing and culling borne by producers in the control area , the implementation of the program has experienced difficulties in maintaining surveillance measures and cleaning up of infected herds . TherefoTo carry out this review, the official data was extracted from the Livestock and Agriculture Service (SAG) annual reports of animal diseases surveillance and directly from the Animal Health System (SSA), the web platform on which veterinarians  record laboratory and skin test results. The objective of this review is to identify and clarify the various components of the program and evaluate the surveillance data in order to understand the successes and constraints of the Chilean bTB program.Official inspection teams work in the country's 62 slaughterhouses, inspecting carcasses and verifying their suitability for human consumption. All visible lesions (VL), compatible with bTB are sent to the official laboratory for PCR, culture, and histopathology. This is the principal way in which the largest number of infected herds have been detected. Elsewhere, official and accredited veterinarians carry out the tuberculin skin test in the field, where all herds with reactor animals are investigated, and skin test positive animals are sent to the slaughterhouse for post-mortem inspection for lesions. The skin tests are carried out as part of the surveillance program within dairy farms, as well as providing certification for export from bTB free farms.During the 1980s, the public and private sectors managed the first action points for bTB control; the development of a bTB—free herd certification scheme; a voluntary binding agreement with dairy farms where SAG certifies that bTB is absent, and the milk industry pays a bonus price per liter to the producer. This is a key part of the program since it is the only major incentive for dairy producers to screen and manage the bTB infection risk, and also because this certification process covers approximately 70% of the tuberculin skin tests that are performed in the field.In 2011 the bTB program was launched with a package of compulsory rules to notify, control and screen herds for bTB, depending on the epidemiological risk zone. The regional zoning took into account the regional disease incidence and bovine population data by using a clustering model to support the decision-making processes of the two epidemiological zones as part of the National Tuberculosis Management Plan , where there has been active surveillance with negative results .Surveyed: 1,025 (5.3%), those that have been surveyed (2011–2021) either at post- mortem inspection or by checks in the field. Although surveyed farms have negative bTB results, the information is not sufficient for them to be classified as negative farms.Suspect: 89 (0.5%) farms under investigation due to positive bTB surveillance in the field or at post-mortem.Infected: 403 (2.1%), farms where bTB infection has been confirmed. It is notable that 65 (16.1%) of these infected farms are dairy enterprises, and 338 (83.9%) are breeding and/or fattening farms.No information: 6,141 (31.8%) i.e., those farms that haven’t had bTB surveillance records, but may have other disease surveillance records, for example, brucellosis infection data.The distribution of bTB classifications at farm level is presented in Fig. If all the farms in the livestock census were included in this classification, from the total number of farms that own cattle, 10.5% have no record of bovine tuberculosis surveillance (Table 1).The bTB heat map in Fig. Due to the limited coverage of bTB surveillance, the total number of surveillance farms that were considered infected, expressed as a percentage, was used to assess the annual dynamic changes in bTB rates. The results are shown in Figs. In other studies, the vaccination field trial has shown that BCG could be a useful tool for reducing the bTB incidence in highly infected herds, where the rates in vaccinated and non-vaccinated heifers at the end of the trial were 0.76% and 2.27%, respectively . This prThe annual reduction in bTB carcass condemnation rates is an important positive indicator of the program’s success, since it is a direct measure that impacts on the financial security and wellbeing of producers. As the program has progressed, visible lesions are now rarely seen in many herds. However, as a consequence of reduced infection levels, the economic losses due to carcass condemnation have also decreased for the farmers, which may serve to reduce incentives to continue and strengthen the bTB control programs, despite the continual presence of infection.The progress of bTB control in the southern eradication zone in Chile has been similar to that experienced in the dairy production areas of Australia at the beginning of their brucellosis and tuberculosis eradication campaign (BTEC) . In the The initial regulatory framework of the program tried to adopt the practicalities of implementation without interfering or challenging the livestock business and trade, while also recognising that the high bTB prevalence in dairy farms could result in herd depopulation at the producers’ expense. However, after ten years of the program, it is likely that the regulations may need to be updated, commensurate with the farmers’ commitment to disease control, and also to include beef breeders, since animal movements from this economically important sector may require stricter and additional rules.The limited surveillance coverage in the control area and low level of herd classification are not unexpected given that regulations and incentives have not been sufficient to monitor herds, or indeed bTB herd classification in breeding systems or in dairy enterprises.The commitment of the dairy industry, incentivized by the bonus paid per liter of milk to certified bTB free farms has not been sufficient to encourage dairy farms in the control area to maintain comprehensive disease control measures. Likewise, due to the fact that the largest proportion of infected farms is located in the area of breeding and fattening enterprises, it has become difficult to encourage their commitment to increased disease control measures, as it is a sector with varied interests that does not easily recognize bTB as its only problem.During the first 10 years of the bTB program in Chile, there has been progress in establishing rules and regulations to control the disease through defining protocols for the surveillance and classification of herds, as well as the use of the official traceability system as a tool to support the implementation of the program.In the eradication zone, effective infection control has been achieved, mainly due to bTB-free farm certification, government support to clean up infected farms, as well as the beneficial interest of farmers exporting products or live animals. Despite the important progress achieved in the eradication zone, it will be a challenge to achieve eradication without compensation, not least because a much greater level of diagnostic coverage is required to identify all infected herds.Within the control zone there is a contrasting epidemiological situation and no evidence of successful disease control. Although the dairy sector is aware of the impact of bTB, the high prevalence and the lack of compensation has driven the reluctance of producers to engage with the program. The farmers continue with a business-as-usual style of behavior. Given this lack of full commitment to the program, vaccination may provide an additional option to reduce bTB incidence. It may also provide a means to convince farmers of the benefits that may arise from increasing their commitment to disease control.There is also a need to strengthen the agreement between the industry and the state, and to involve medium-sized farming enterprises who provide replacements for fattening. These enhanced agreements should address each of the following: a) reinforcing the implementation of bTB risk-based regulations, b) implementing movement controls to include beef breeders, both within and between areas; c) seeking joint industry-government schemes to financially compensate farmers for losses due to reactors  or herd depopulation.


We use a formulation of Noether’s theorem for contact Hamiltonian systems to derive a relation between the thermodynamic entropy and the Noether invariant associated with time-translational symmetry. In the particular case of thermostatted systems at equilibrium, we show that the total entropy of the system plus the reservoir are conserved as a consequence thereof. Our results contribute to understanding thermodynamic entropy from a geometric point of view. Geometric methods have become indispensable in modern theoretical physics. A primary example is Noether’s theorem, which in its most famous version, relates the symmetries of a mechanical system to the corresponding conservation laws. In thermodynamics, Noether’s theorem has not had thus far the same success as, for instance, in mechanics. This is certainly to be ascribed to the fact that, despite many attempts, there is still no consensus on a variational or geometric formulation of thermodynamics, especially in the irreversible case  state of a contact Hamiltonian system. Specifically, we show that this entropy can be seen as the average of the Noether invariant associated with the symmetry under time translations. In the particular case of systems in equilibrium with a heat bath, we show that the identity becomes a conservation law for the total entropy of the system plus the reservoir. To this end, we rely on recent results showing that a large class of systems that do not preserve energy can be modelled through contact Hamiltonian dynamics ,13,14, wTo carry out what we described above, we have organized this paper as follows: We present a summary of ref.  in SectiEntropy appears as a Noether invariant when considering thermally isolated systems in the quasistatic regime. This was proven by Sasa and Yokokura from a variational perspective . In thisN particles interacting only through conservative forces. Such a system can be described by a Lagrangian function Consider a system of To prove that entropy is a Noether invariant, the authors of ref.  start wiTheorem 1.Let Then, the transformation where S being the thermodynamic entropy of the system and b a dimensionless constant. As the number of particles N is fixed, this entails that the entropy is the conserved quantity associated with the symmetry . Thecontact Hamiltonian vector field contact Hamiltonian.The dynamics of a contact manifold is usually given in terms of a Locally, on any contact manifold there are “canonical” (Darboux) coordinates s, all the results of standard symplectic Hamiltonian dynamics such as conservation of the Hamiltonian and Liouville’s theorem are recovered. On the other hand, when s, the Hamiltonian is not conserved and the evolution n is the number of degrees of freedom of the system, and the divergence of a vector field Y, £ denotes the Lie derivative.The main characteristic of these systems is the fact that, when the Hamiltonian does not depend explicitly on Starting from the results of the previous section, one can prove a contact version of Noether’s theorem ,27,28,29We begin with the following definition.Definition 2.An infinitesimal dynamical symmetry of a contact Hamiltonian system Now, by analogy with the standard Noether theorem, we can state the following relation between infinitesimal dynamical symmetries and conserved quantities in the contact case.Theorem 3.Let Y be an infinitesimal dynamical symmetry of the contact Hamiltonian system is conserved along the solutions of .(Contact Noether)Proof. Let On the other hand, substituting Equation  in CartaFinally, since It is worth observing that, typically, the quantity  is the pt, the system is invariant under time translations and the corresponding generator is just s, then the conservation of .  systems , it follf H only  16)  showt, and the second one is, using 21), we hMoreover, since given by . This imgiven by  holds, yTheorem 6.For a system of n degrees of freedom at thermal equilibrium with a reservoir—a system described by —the cons(Main)Proof. From , we can For H as in , using . □For a system described by , thermal Moreover, since the system and the heat bath are allowed to exchange energy, the entropy of the former alone is not constant (there is a heat flow from and into it).The fact that the total entropy above is conserved at equilibrium is trivial. What we consider important is that, in light of Noether’s theorem, this can be regarded as a consequence of a symmetry under time translations. Indeed, we could rephrase Theorem 6 informally in the following way: Using an appropriate geometric description for the microscopic dynamics of mechanical systems in thermal equilibrium with a heat bath, we have derived a relationship between Noether’s theorem and the conservation of the total entropy of the system plus the reservoir. We stress that in this case the symmetry related to the conservation of the entropy is just the standard time-translational invariance. Therefore, we conclude that thermodynamic equilibrium emerges as a consequence of time-translational invariance.Our results are in line with recent findings deriving the thermodynamic entropy as the Noether invariant associated with a symmetry under (non-uniform) time translations. Indeed, it should be noted that in our setting the temperature of the system is constant . Therefore, the non-uniform time translation of Equation  found inOur work also provides an answer in a deterministic setting to the question formulated in ref. , concern


Kaposi sarcoma (KS) is a low-grade vascular neoplasm associated with human herpesvirus 8 (HHV-8) infection. The disease has various phases, and the morphology of the lesion may vary, especially in the early course of the disease, where the morphological features may not be even suggestive of Kaposi sarcoma. The authors take this opportunity to report a case of Kaposi sarcoma where the diagnosis was established because of HHV-8 staining rather than its histopathological features. In the era of AIDS and solid organ transplantation, some fatal tumors have emerged. Kaposi sarcoma (KS) is one such tumor. KS is a vascular neoplasm, usually low grade but may tend to be locally aggressive. KS has a very strong association with human herpesvirus 8 (HHV-8) infection, to the extent that it is considered a causative agent for the same . KS has A 72-year-old male with known type 2 diabetes mellitus (DM), maintaining good control with oral hypoglycemic drugs, was referred to the dermatology department of our hospital. The patient had consulted another dermatology clinic outside our hospital about five months ago. A provisional clinical diagnosis of vasculitis was given there, and a skin punch biopsy was done, which favored the diagnosis of vasculitis. However, there was no response to the treatment, and the patient was referred to our hospital. The patient gave a history of multiple non-pruritic patches over his body at various locations for one year. On examination, the patient was well oriented with normal vitals . Local examination showed multiple reddish-bluish patches of variable sizes over the right upper arm, both ears, and lower legs, with some having nodular configurations  macules and papules, which later clustered together to form a linear or serpiginous pattern. Oral mucosa was uninvolved. No organomegaly or lymphadenopathy was identified. There was no history of any discomfort with swallowing, swollen legs, respiratory or gastrointestinal symptoms. A clinical differential diagnosis of Kaposi sarcoma was suggested.The previous biopsy slides were retrieved from the dermatology clinic outside our hospital and were then reviewed intra-departmentally at our hospital, and none of the histopathologists favored Kaposi sarcoma. The H&E stained slides showed superficial dermal perivascular moderate inflammatory infiltrate with extravasation of red blood cells  and visceral organs . ClinicaHHV-8 DNA has been detected in virtually all forms of KS but not in the perilesional unaffected skin of patients with KS . HHV-8 iUsing polymerase chain reaction (PCR), HHV-8 is found in about 95-100% of Kaposi's sarcoma lesions ,12. HoweThe histological diagnosis may also be further verified by immunohistochemistry (IHC) for HHV-8 latent nuclear antigen-1 (LNA-1). LNA-1 helps viral DNA bind to the host chromosomes, and hence immunoreactivity stains in the pattern of nuclear stippling. The IHC for HHV-8 is considered positive when the staining is nuclear with a granular appearance and has no concomitant cytoplasmic staining , as was The detection of HHV-8 in tumor cells can be done using reverse transcriptase (RT)-PCR as well as by immunohistochemistry. IHC is clearly less labor-intensive and time-consuming, easier, inexpensive, less complex, and more practical as it allows one to correlate with the morphologic features and definitely less prone to false positive results or erroneous interpretations in view of contamination, in comparison to RT-PCR . TherefoTherapeutic options for KS are variable, including local therapy like topical imiquimod or cryotherapy, radiation therapy, chemotherapy, and immunotherapy. The appropriate selection for each patient depends on the general health of the patient as well as the number, location, and size of the KS lesions . Early Kaposi sarcoma may not show the typical characteristic histological picture but rather reveal nonspecific features. This may create a dilemma for the pathologist as well as the dermatologist and eventually lead to a delay in the diagnosis of KS and further proper management. HHV-8 immunostaining is 99% sensitive and 100% specific for the diagnosis of KS, and therefore cases where histological features do not favor KS, as was seen in our case, HHV-8 immunostaining can play a vital role in early diagnosis and treatment. However, it is not practical to do HHV-8 staining in all skin biopsies without any context. Clinical suspicion of Kaposi sarcoma is definitely required to go forward with HHV-8 staining in cases with nonspecific histopathological findings.


Antimicrobial resistance (AMR), a rising global health crisis causing about 700,000 deaths annually and potentially 10 million deaths by 2050, disproportionately impacts marginalised populations. Due to socioeconomic, ethnic, geographic, and other barriers, these communities often have restricted healthcare access, compounding the AMR threat. Unequal access to effective antibiotics, inadequate living conditions, and a lack of awareness exacerbate the crisis in marginalised communities, making them more susceptible to AMR. A broader, inclusive response is needed to ensure equitable access to antibiotics, improved living conditions, education, and policy changes to challenge the root socio-economic disparities. Ignoring marginalised populations in the fight against AMR is both a moral and strategic failure. Therefore, inclusivity must be a central tenet in combating AMR. This article not only critically dissects this prevailing oversight but also urgently calls for comprehensive action to address this significant shortcoming in our response efforts. In the realm of public health, there is an inherent interplay between the global community and the individual, the majority, and the minority. This interplay is most visible when we are faced with a crisis that is both global and personal in its impact. The current situation of increasing antimicrobial resistance (AMR) is a case in point, causing about 700,000 deaths annually, and projections suggest that by 2050, AMR could lead to 10 million deaths per year, surpassing the current 8.2 million annual deaths from cancer [Marginalised populations, in this context, are those who experience systemic exclusion, often resulting in reduced access to healthcare due to factors such as socioeconomic status, ethnicity, geographic location, immigration status, and disability. These groups can include rural and remote communities, urban slums, ethnic and racial minorities, indigenous communities, and immigrant or refugee populations, each facing unique and compounded barriers that exacerbate the challenges associated with AMR . This diFurthermore, we must also recognize the role of social determinants of health . OvercroS. aureus [Escherichia coli (DEC) was detected in 58% of the children residing in the slum area, while it was found in only 17% of the control group (children who attended a private school of the same city) (P = 0.001) [Indigenous communities globally are more likely to live in poverty and have less access to quality healthcare . This, a. aureus . A syste. aureus . An exte. aureus . Notably. aureus . The pro= 0.001) . Further= 0.001) . In addi= 0.001) . All theThe WHO's Global Action Plan on Antimicrobial Resistance , while oThe issue of AMR is not just a biological problem to be solved by scientists in labs. It is also a social problem that requires us to challenge our existing systems of health and economic inequality . If the To create an effective global response to AMR, we must ensure equitable access to antibiotics and healthcare in general. We need to engage with marginalised communities to provide education and awareness, building antibiotic stewardship from the ground up. Investments need to be made in improving living conditions and sanitation, reducing the need for antibiotic use in the first place. On a broader level, there is a need for policy changes that acknowledge and challenge the socio-economic disparities at the root of this problem. Policy changes can be implemented effectively through active collaboration between government bodies, healthcare organisations, and community leaders. Public health initiatives should be designed with cultural competence, incorporating local customs and language into educational materials to ensure understanding. For example, a "train-the-trainer" model can be employed, where community health workers are educated about AMR and antibiotic stewardship, who then pass on this knowledge to members of their communities, thereby ensuring a culturally sensitive and community-focused approach to public health education. Furthermore, mobile health clinics and telemedicine could also be utilised to reach remote and underserved communities. Addressing barriers to vaccination and promoting vaccination programs to prevent infections that typically necessitate antibiotic treatment can significantly mitigate the need for antibiotic usage, thereby reducing the risk of AMR. Alongside these efforts, it is imperative to encourage and prioritize research that focuses on AMR within marginalised populations, as this can provide critical insights into their unique challenges and inform the development of customized, effective interventions to tackle AMR in these communities.If the goal is a healthier world where effective antibiotics are available to all, we must not forget that the 'all' includes the marginalised and the disenfranchised. Their exclusion from the global AMR response effort is not just a moral failing but also a strategic one, threatening to undo whatever gains we might achieve. Therefore, inclusivity must be at the heart of the fight against AMR.


Dopamine neurons are essential for voluntary movement, reward learning, and motivation, and their dysfunction is closely linked to various psychological and neurodegenerative diseases. Hence, understanding the detailed signaling mechanisms that functionally modulate dopamine neurons is crucial for the development of better therapeutic strategies against dopamine-related disorders. Phospholipase Cγ1 (PLCγ1) is a key enzyme in intracellular signaling that regulates diverse neuronal functions in the brain. It was proposed that PLCγ1 is implicated in the development of dopaminergic neurons, while the physiological function of PLCγ1 remains to be determined. In this study, we investigated the physiological role of PLCγ1, one of the key effector enzymes in intracellular signaling, in regulating dopaminergic function in vivo. We found that cell type-specific deletion of PLCγ1 does not adversely affect the development and cellular morphology of midbrain dopamine neurons but does facilitate dopamine release from dopaminergic axon terminals in the striatum. The enhancement of dopamine release was accompanied by increased colocalization of vesicular monoamine transporter 2 (VMAT2) at dopaminergic axon terminals. Notably, dopamine neuron-specific knockout of PLCγ1 also led to heightened expression and colocalization of synapsin III, which controls the trafficking of synaptic vesicles. Furthermore, the knockdown of VMAT2 and synapsin III in dopamine neurons resulted in a significant attenuation of dopamine release, while this attenuation was less severe in PLCγ1 cKO mice. Our findings suggest that PLCγ1 in dopamine neurons could critically modulate dopamine release at axon terminals by directly or indirectly interacting with synaptic machinery, including VMAT2 and synapsin III. Dopamine release is significantly increased in mice lacking the phospholipase Cγ1 (PLCγ1) enzyme in their dopamine neurons, according to a study by researchers at the Ulsan National Institute of Science and Technology. The team found that PLCγ1 conditional knockout mice exhibited normal dopamine neuron development and cellular structures, but showed enhanced dopamine release. This was potentially due to increased localization of vesicular monoamine transporter 2 (VMAT2) and synapsin III in dopaminergic axons. The findings suggest that PLCγ1 plays a critical role in regulating dopamine release by potentially modulating synaptic vesicle trafficking in dopamine neurons. Dopamine is largely synthesized from dopamine neurons in the midbrain and released into the synaptic cleft, binding to dopamine receptors and activating dopamine receptor-mediated signaling in both presynaptic terminals and postsynaptic neurons4. Although a small set of neurons constitute dopamine neurons primarily in the substantia nigra pars compacta (SNc) and the ventral tegmental area (VTA) in the rodent and human midbrain5, they exert powerful modulatory effects on synaptic transmission and neuronal activity through extensive axonal arborization with numerous synaptic boutons7. These dopaminergic axons innervate diverse brain regions, such as the striatum, globus pallidus, cortex, and limbic areas, via nigrostriatal and mesocorticolimbic pathways8. Due to these structural and functional attributes, dysfunction of dopamine neurons can lead to severe neurological and psychiatric illnesses, including Parkinson’s disease10, schizophrenia12, attention deficit hyperactivity disorder (ADHD)13, depression14, and addiction15. In particular, selective degeneration of dopamine neurons in the SNc and their axon terminals decreases dopamine levels in the basal ganglia, which causes the main symptoms of Parkinson’s disease, such as tremor, muscle stiffness, and bradykinesia10. Many previous studies on dopamine and dopamine neurons have centered on the presynaptic and postsynaptic effects of dopamine through dopamine receptors and their downstream signaling4. However, there is a critically unmet need to better understand the intracellular signaling and molecular events by which dopamine neurons and their normal physiology are maintained.The monoamine neurotransmitter dopamine regulates various cognitive and psychomotor functions, including voluntary movement, reward-related learning, emotion, and motivation20. PLC is generally activated by extracellular signal-mediated receptors, and it hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) to generate diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3). These second messengers in turn regulate key cellular functions, including proliferation, differentiation, migration, and survival25. Mammalian PLC comprises 13 isozymes that are differentially expressed in bodily organs. Among them, PLCγ1, which is activated by receptor tyrosine kinase (RTK), is ubiquitously distributed in several organs and abundantly expressed in embryonal cortical structures, neurons, oligodendrocytes, and astrocytes in the brain16. PLCγ1 has been known to mediate pivotal neuronal functions in the brain, including neuronal development, synaptic transmission, and synaptic plasticity28. Interestingly, accumulating evidence indicates that PLCγ1 appears to modulate the functions of dopamine neurons. Chronic treatment with morphine, one of the opiates facilitating dopamine release, significantly increased PLCγ1 expression and phosphorylation in the ventral midbrain30. In addition, VTA region-specific overexpression of PLCγ1 was shown to regulate mood-related behaviors in rodents, such as sucrose preference, forced swimming, nociceptive stimuli, and elevated-plus maze behavior31. Despite these findings, our understanding of the physiological role and significance of PLCγ1 in dopamine neurons is far from complete, and the cell type-specific function of PLCγ1 in vivo remains unknown in dopamine neurons.Several intracellular signaling pathways have been implicated in the development and function of dopamine neurons. Among them, phospholipase C (PLC) is one of the most important intracellular signaling molecules in a variety of biological processesIn this study, we focused on the physiological function of PLCγ1 in dopamine neurons in vivo by utilizing dopamine neuron-specific PLCγ1 conditional knockout mice. We found that genetic deletion of PLCγ1 does not negatively affect the development and cellular structures of dopamine neurons. Interestingly, while intrinsic properties were unaffected, the acute and chronic disruption of PLCγ1 in dopamine neurons markedly facilitated dopamine release, potentially through indirect interaction with VMAT2 and synapsin III. Furthermore, the possible role of VMAT2 and synapsin III in this enhanced dopamine release was confirmed by the knockdown of VMAT2 and synapsin III in dopamine neurons. Thus, our findings demonstrate that PLCγ1 critically affects dopamine release by potentially regulating synaptic vesicle trafficking in dopamine neurons.Plcg1F/F mice  were generated as previously described27. To produce dopamine neuron-specific PLCγ1 conditional knockout mice, DAT-Cre mice (B6.SJL-Slc6a3tm1.1(cre)Bkmn/J, Jackson stock No. 006660) were crossed with Plcg1F/F mice, generating DAT-Cre;Plcg1F/F mice. DAT-Cre mice were used as the control group. In optogenetic experiments, DAT-Cre;Plcg1F/F mice were crossed with Ai32 mice (B6.Cg-Gt(ROSA)26Sortm32(CAG-COP4*H134R/EYFP)Hze/J, Jackson stock No. 024109) to selectively express channelrhodopsin-2 in dopamine neurons, producing DAT-Cre;Ai32;Plcg1F/F mice. DAT-Cre;Ai32 mice were used as the control group in optogenetic experiments. Adult (2–5 months old) male and female mice were used in most of the experiments. Young (3–4 weeks old) male and female mice were used for whole-cell patch clamp recording due to the difficulty in making healthy whole-cell configurations from midbrain dopamine neurons in adult mice. Mice used for this study were maintained under consistent housing conditions on a 12-h light/dark cycle (lights on from 6 a.m. to 6 p.m.) in groups of up to 5 mice per cage. Food and water were available ad libitum. The mice were bred under standard pathogen-free housing conditions in the animal facility of the Ulsan National Institute of Science and Technology (UNIST). The experimenter was not blind to the genotypes of the mice. All experimental procedures were conducted in accordance with protocols approved by the Institutional Animal Care and Utilization Committee of UNIST (UNISTIACUC-20-18).32, TH , DAT , synapsin I , VMAT2 , synapsin II , synapsin III , β-actin , and GAPDH . After washing, membranes were incubated with secondary HRP-conjugated antibodies  for 1 h at room temperature. After washing three times, protein bands were visualized by Clarity Western ECL Substrate  with an Amersham Imager . Protein bands were quantified by ImageJ software. Before reprobing, membranes were stripped for 30 min at 65 °C in a stripping buffer . After washing three times, the membranes were blocked and sequentially reprobed with antibodies.Mice were anesthetized with isoflurane inhalation  and decapitated. Brains were immediately dissected in 4 °C cold ACSF solution. Coronal striatum and midbrain slices were prepared at 1000 μm thickness using a vibratome . Using dissection tools, we specifically isolated the dorsal striatum from coronal striatal slices. Proteins were extracted from brain slices using RIPA Lysis and Extraction Buffer . Halt™ Protease and Phosphatase Inhibitor Cocktail  was also freshly added. A Pierce™ BCA Protein Assay Kit  was used to measure the protein concentration. Proteins with 5X SDS‒PAGE loading buffer  were denatured at 95 °C for 5 min. Protein samples were loaded in the gel, electrophoresed, and transferred to a 0.45 μm nitrocellulose membrane . The membranes were blocked with 5% skim milk in TTBS and incubated with primary antibodies at 4 °C overnight. Primary antibodies used for western blotting targeted PLCγ1 33. Coronal striatum slices were homogenized in lysis buffer . The homogenates were centrifuged at 1000×g for 10 min at 4 °C. The supernatants were collected and centrifuged at 12,500×g for 20 min at 4 °C. The supernatants containing the cytosolic and light membrane fractions were discarded, and the pellets containing the crude synaptosomal fraction were resuspended in lysis buffer for western blotting.The synaptosomal fraction was prepared as previously described32, TH , DAT , VMAT2 , D2R , synapsin I , synapsin II , synapsin III , bassoon , GABAA receptor α1 subunit , VGAT , Gephyrin , and PIP2 . The secondary antibodies were Alexa Fluor 405-, 488-, 594-, and 647-conjugated antibodies (Thermo Scientific). After washing with PBST and PBS, sections were mounted with ProLong™ Gold Antifade Mountant . For quantitative analysis, images were captured by using an FV1000 confocal laser scanning microscope (Olympus) and an LSM 880 confocal laser scanning microscope with Airyscan (Carl Zeiss). To quantify the dopamine neurons in the SNc and VTA, an FV1000 confocal microscope with a ×20 or ×63 lens was used. The images were analyzed using ImageJ software (NIH) to quantify the fluorescence-positive area and mean intensity. Confocal 3D images were acquired using the z-stack function of an LSM880 confocal laser scanning microscope with Airyscan (Carl Zeiss). Images were collected at 2 μm intervals with a ×63 oil-immersion objective, ×3 optical zoom, and a resolution of 1248 × 1248 pixels. 3D images were reconstructed using IMARIS 9.6 software. Acquired images were further analyzed by Zen software (Carl Zeiss), ImageJ program , and MATLAB . For DAB  staining, brain slices were mounted on slides and allowed to dry overnight at RT. The slides were washed in PBS for 10 min, followed by incubation in 0.3% H2O2 in PBS for 10 min to block endogenous peroxidase activity. After two additional PBS washes, the slides were permeabilized in 0.5% PBST for 10 min and subsequently blocked with a blocking solution for 1 h. Next, the slides were incubated with TH antibody  in blocking solution at 4 °C overnight. Following rinsing, the slides were incubated with HRP-conjugated secondary antibody  for 2 h at RT. After rinsing in PBS, the sections were exposed to DAB solution  for 2 min, followed by dehydration in a graded series of ethanol (70–100%) for 30 sec and allowed to air dry. Finally, cover slips were applied using a permanent mounting medium . For quantitative analysis, images were captured using a virtual microscope (Olympus).Mice were anesthetized with a zoletile  and rompun  mixture solution and transcardially perfused with 0.1 M PB, followed by 4% paraformaldehyde (PFA) in 0.1 M PB. The dissected brains were further fixed in 4% PFA overnight, followed by cryoprotection in 30% sucrose in 0.01 M PB overnight at 4 °C. For the staining of PLCγ1, brains were fixed in 4% PFA for 4 h. Sections (40 μm thickness) were coronally cut using a microtome . Free-floating sections were washed in PBS for 10 min and incubated in 0.5% PBST (0.5% Triton X-100 in PBS) for 10 min at room temperature (RT). Sections were blocked in 2% bovine serum albumin and 10% normal goat serum in PBST for 1 h at RT and incubated with primary antibodies diluted in a blocking solution overnight at 4 °C. Following three washes in PBST, the sections were incubated with Alexa Fluor-tagged secondary antibodies at a 1:1000 dilution for 2 h at room temperature. Primary antibodies used in this study targeted PLCγ1 35. The images were captured by using a THUNDER Imager Live Cell (Leica). Images were collected with a ×20 lens and a resolution of 1248 × 1248 pixels.The number of TH-positive fluorescent cells was quantified using the ImageJ cell count plugin. We counted the TH-positive dopamine neurons co-labeled by DAPI. The number of dopamine neurons in the midbrain dramatically varies depending on the rostral-to-caudal location of the coronal slices. In our data, we chose a single slice from each coronal location , counted the number of dopamine neurons from each slice, and then summed these numbers . For stereological counting of dopamine neurons, the stereological analysis method was used as described previously36. Briefly, the tissue was fixed in a solution of acetone–methanol (1:1) at −20 °C for 10 min. The slides were rinsed in RNase-free 1% PBST. The sections were covered with 200 μl of PBST containing tyrosine hydroxylase antibody , along with 400 U/ml of RNaseOUT™ Recombinant Ribonuclease Inhibitor . After a 15 min incubation, the slides were briefly rinsed in PBS. Next, the tissue was covered with 200 μl of HRP-conjugated secondary antibody diluted 1:100 in PBST with 400 U/ml ribonuclease inhibitor and incubated for 10 min. After rinsing in PBS, the sections were treated with DAB solution  for 2 min, followed by dehydration in a graded series of RNase-free ethanol (70–100%) for 30 s and allowed to air dry. Using the PALM MicroBeam laser microdissection system (Carl Zeiss), more than 1000 TH-positive dopamine neurons were collected from a single mouse.Mice were anesthetized with isoflurane inhalation  and decapitated. The brain was then extracted and embedded in OCT compound, followed by freezing on dry ice and storage at −80 °C. Slicing was performed using a cryostat at a thickness of 20 μm, and the sections were mounted on PEN membrane slides . For TH antibody staining, we referred to a previous studyIsolated dopamine neurons were used for total RNA extraction using the PicoPure™ RNA Isolation Kit . Reverse transcription of the extracted RNA was carried out using the High-Capacity RNA-to-cDNA Kit . Two hundred nanograms of total RNA was used as input for cDNA synthesis. Quantitative real-time PCR was performed using the LightCycler 480 II Real-Time PCR system (Roche) and Prime Q-Mastermix . Specific primers for each target mRNA were used in the qPCR. The expression level of each mRNA was normalized to that of GAPDH, which was used as the internal control. We used the following primers: PLCβ1 , PLCβ2 , PLCβ3 , PLCβ4 , PLCγ1 , and PLCγ2 .37. Brain slices were maintained in the AcX solution overnight. After washing with PBS, brain slices were incubated in a gelling solution for 30 min at 4 °C. These slices were then mounted onto the gelation chamber and incubated for 2 h at 37 °C. The gels were then trimmed and submerged in a digestion buffer overnight. Finally, the gels were expanded in water for 20 min three times and were mounted onto coverslips coated with poly-l-lysine for imaging. For quantitative analysis, images were captured by using an LSM 980 confocal laser scanning microscope (Carl Zeiss). Images were collected with a ×40 water-immersion objective, 2 × 2 stitched images, and a resolution of 4894 × 4894 pixels. The expansion factor of the images was measured as 5. Acquired images were analyzed using Zen software (Carl Zeiss), ImageJ (NIH), and MATLAB .For immunofluorescence staining, brain slices were stained following the immunohistochemistry protocol described above until they were ready to mount to slide glasses. Expansion of brain slices was performed as previously described38. Coronal brain slices containing the DStr-NAc (300 μm thick) or SNc-VTA (250 μm thick) were obtained for whole-cell patch clamp recording and fast-scanning cyclic voltammetry (FSCV). Mice were anesthetized with isoflurane , decapitated, and the brain was briefly exposed to ice-chilled artificial cerebrospinal fluid (ACSF) containing 125 mM NaCl, 2.5 mM KCl, 1.25 mM NaH2PO4, 25 mM NaHCO3, 1 mM MgCl2, 2 mM CaCl2 and 15 mM glucose oxygenated with 95% O2 and 5% CO2. Acute brain slices were prepared using a tissue vibratome  in ice-cold ACSF. Brain slices were first maintained in ACSF for 30 min at 34 °C and then another 30 min at room temperature. After recovery, slices were transferred to a submerged recording chamber perfused with ACSF. The temperature of the ACSF was maintained at 30–31 °C by an in-line solution heater (Warner Instruments) with a flow rate of 2–3 ml/min. Brain slices were used for electrophysiological recording within 5 h after recovery.Brain slices were prepared as previously described38. Dopamine neurons were visualized using a conventional infrared (IR)-differential interference contrast (DIC) microscope . Whole-cell voltage-clamp recordings were performed using borosilicate glass pipettes (2.5–3.5 MΩ) filled with Cs+-based low Cl− internal solution containing 135 mM CsMeSO3, 10 mM HEPES, 1 mM EGTA, 3.3 mM QX-314, 0.1 mM CaCl2, 4 mM Mg-ATP, 0.3 mM Na3-GTP, and 8 mM Na2-phosphocreatine . To measure inhibitory and excitatory synaptic currents from the same neuron, the membrane potential was first held at +0 mV  to measure inhibitory synaptic currents and then continuously held at −70 mV  to measure excitatory synaptic currents under the voltage clamp mode . The access resistance was 10–20 MΩ, and only cells with a change in access resistance <20% were included in the analysis. The signals were collected using Multiclamp 700B (Molecular Devices), low-pass filtered at 2 kHz, and digitized at 10 kHz . Recording data were monitored and acquired by WinWCP  and further analyzed offline using Clampfit 10.7 software (Molecular Devices) and OriginPro 2017 (OriginLab). For mEPSC and mIPSC measurements, tetrodotoxin  and D-AP5  were included in ACSF. The amplitude and frequency of mEPSCs and mIPSCs were analyzed using Mini Analysis software (Synaptosoft); spontaneous events were first detected by Mini software, and then individual spontaneous events were manually checked again to increase the accuracy of the analysis. To examine the intrinsic properties of dopamine neurons, the potassium-based internal solution containing 135 mM KMeSO3, 3 mM KCl, 10 mM HEPES, 1 mM EGTA, 0.1 mM CaCl2, 8 mM Na2-phosphocreatine, 4 mM Mg-ATP, and 0.3 mM Na3-GTP  was used under the current clamp mode. Spontaneous firing of dopamine neurons was measured for 1 min, and the average firing frequency was calculated. To measure input resistance and detect voltage sag from dopamine neurons, a stepwise hyperpolarizing current  was injected for 1 sec , and the amplitude of voltage sag was measured. Voltage sag was determined as the difference between the instantaneous voltage (peak hyperpolarization) and steady-state voltage during 1 sec of current injection. To examine the membrane excitability of dopamine neurons, a stepwise depolarizing current  was injected for 1 sec , and the total number of action potentials was counted. To induce hyperpolarization-activated current (Ih current) in dopamine neurons, voltage-clamp recording was carried out, and holding potentials were continuously held at different negative potentials  for 1 s. Ih is calculated as the current difference between the instantaneous current and steady-state inward current during 1 s. To stimulate ChR2-expressing dopaminergic axons, blue laser light  from a diode laser  was focused on the back focal plane of the objective to generate wide-field illumination. GABA (IPSC) and glutamate (EPSC) cotransmission from DA axon terminals was recorded from the medium spiny neurons (MSNs) in the DStr by optogenetically activating DA terminals.Electrophysiology was performed as previously described38. To examine the morphology, dendritic arborization, and number of dendritic spines in a single dopamine neuron, the potassium-based internal solution with 0.2% biocytin  was loaded into dopamine neurons in the SNc and VTA for 30 min. After the delicate removal of the glass pipette, brain slices were fixed with 4% PFA overnight at 4 °C. On the following day, slices were washed with PBS, permeabilized with PBST, and blocked with PBST solution containing 10% normal goat serum and 2% bovine serum albumin for 1 h. Brain slices were then incubated with Alexa 594-conjugated streptavidin  in a blocking solution for 2 h at room temperature. After washing with PBST and PBS, brain slices were mounted on slides using a mounting medium containing DAPI . Images were obtained by using an FV1000 confocal laser scanning microscope (Olympus). Images of dendritic spines were acquired with a ×100 oil-immersion lens and ×2 optical zoom. We chose one secondary dendrite with the highest number of spines in each neuron for the analysis of spine density. We only included spine-like structures in our analysis to calculate spine densities. Sholl analysis was performed by using the ImageJ Sholl Analysis plugin.Biocytin labeling was performed as previously described38. Extracellular dopamine release was recorded by FSCV using carbon-fiber microelectrodes  at the DStr and NAc core. To detect dopamine release, a triangular scanning waveform was applied to the carbon fiber electrode from −0.4 V (vs. AgCl reference electrode) to +1.3 V and back (8.5 ms waveform width) at a scan rate of 400 V/s (repeated at 10 Hz). The carbon fiber electrode was held at −0.4 V between scans. Cyclic voltammograms were background-subtracted by averaging 10 background scans. To evoke dopamine release by optogenetic stimulation, ChR2-expressing axon terminals of dopamine neurons were stimulated by blue laser light . We used 50% saturation power under the objective . For electrical stimulation, a bipolar concentric electrode  was placed on the surface of the slice close to the carbon fiber electrode. Dopamine release was evoked by an electrical current (2 ms pulse duration for one pulse stimulation and five pulses stimulation) controlled by an ISO-flex stimulus isolator  or DS-3 stimulus isolator . We used the current intensity by which 50% of maximal dopamine release could be induced. The oxidation current by optogenetically or electrically evoked dopamine release was detected and monitored using the TarHeel CV system. Changes in dopamine concentration were quantified by plotting the peak oxidation current of the voltammogram over time. The carbon fiber electrode was calibrated at the end of each experiment to convert the oxidation current to dopamine concentration using dopamine  in ACSF. U73122 , reserpine , and quinpirole  were used in the corresponding experiments.FSCV was performed as previously described39. We tested three different pSicoR vectors encoding VMAT2 or synapsin III-specific shRNA expression cassettes for their ability to knock down target gene expression in HEK293T (ATCC® CRL3216™) cells; the pSicoR-control served as a nontarget control shRNA containing nonhuman or mouse shRNA (5′-TCGCA TAGCG TATGC CGTT-3′). Three kinds of shRNA were synthesized by the following method: Two kinds of oligos were purchased . The sequence information of the three candidates for the shRNA is as follows: mVMAT2-sh1 (5′-TATCT CATTG GAACC AATAT T-3′), mVMAT2-sh2 (5′-TCCTT GGCTC ATGAC AATTA T-3′), mVMAT2-sh3 (5′-ACAGC ATCTT CTCTT ACTAT A-3′), mSyn3-sh1 (5′-ACCCA GGATC TTGTT GGTTA T-3′), mSyn3-sh2 (5′- GAGCT GAACC TGGCT GCTTA T-3′), and mSyn3-sh3 (5′-TCCTT GGGTC CTGAG AAATT T-3′). The two oligos were annealed with annealing buffer  and incubated at 95 °C for 5 min and 70 °C for 10 min. The annealed double-stranded oligo was inserted into HpaI-XhoI restriction enzyme sites within the pSicoR vector and verified by sequencing.VMAT2, synapsin III-specific, and control shRNAs were expressed in the pSicoR vector 40. Among the three shRNA candidates, we selected the most effective shRNA candidate to achieve target gene knockdown for AAV production.Quantitative real-time polymerase chain reaction (qRT-PCR) assays for gene silencing that targeted VMAT2 and synapsin III candidates were used. All shRNAs were transfected into HEK293T cells. Approximately 24 h after transfection, total RNA was isolated from HEK293T cells using an RNA extraction kit  according to the manufacturer’s instructions. Complementary DNA (cDNA) was synthesized from 1 μg of total RNA, and reverse transcription was performed using a SuperScript cDNA synthesis kit  according to the manufacturer’s instructions. qRT-PCR was performed using a SYBR Green expression assay . Primer sets for target genes were purchased from Macrogen: VMAT2  and synapsin III . GAPDH is a housekeeping gene that was used as a control. The delta-delta Ct method was used to calculate fold changes in gene expression14 genome copies/ml), AAV5-U6-mVMAT2-sh3-CMV-mCherry (1.09 × 1014 genome copies/ml), and AAV5-U6-mSyn III-sh1-CMV-mCherry (1.02 × 1014 genome copies/ml).For adeno-associated virus (AAV)-based shRNA expression, pSicoR-shRNA vectors with XbaI/HindIII-containing enzymatic sites were inserted into the pAAV-minCMV-mCherry  vector by the same enzymatic site. AAV virus was thereafter purified by iodixanol gradient ultracentrifugation by the KIST Virus Facility. The virus titers were as follows: AAV5-U6-scr-CMV-mCherry  and performed using a stereotaxic system . Before surgery, mice were deeply anesthetized by intraperitoneal injection of a zoletil  and rompun  mixture solution . A total volume of 300 nl 6-OHDA solution  was injected unilaterally into the left MFB . A glass micropipette with a long narrow tip (size: 10–20 μm) was made using a micropipette puller  to deliver 6-OHDA. The glass pipette slowly reached the target area and was left for 5 min before 6-OHDA injection. The 6-OHDA solution was injected at an infusion rate of 100 nl/min and withdrawn 10 min after the end of the injection. After injection, the scalp was sutured, and the mice were returned to their home cages. 6-OHDA-injected mice were used for electrophysiology and immunohistochemistry experiments 3 days after injection.Stereotaxic 6-OHDA injection was conducted on over 10–20-week-old male and female mice (Plcg1F/F) and performed using a stereotaxic system . Before surgery, mice were deeply anesthetized by intraperitoneal injection of a zoletil  and rompun  mixture solution . A total volume of 700 nl Cre virus solution  was injected bilaterally into the SNc . The virus titer was 6.5 × 1012 virus molecules/ml for AAV8-hsyn-GFP-Cre. A glass micropipette with a long narrow tip (size: 10–20 μm) was made using a micropipette puller  to deliver the virus. The glass pipette slowly reached the target area and was left for 5 min before the virus injection. Virus solution was injected at an infusion rate of 100 nl/min and withdrawn 7 min after the end of injection. After injection, the scalp was sutured, and the mice were returned to their home cages. Virus-injected mice were used for electrophysiology and immunohistochemistry experiments 4 weeks after injection.Stereotaxic virus injection was conducted on 8- to 12-week-old male and female mice  and performed using a stereotaxic system . Before surgery, mice were deeply anesthetized by intraperitoneal injection of a zoletil  and rompun  mixture solution . A total volume of 1 μl of control virus solution (AAV5-U6-scr-CMV-mCherry for VMAT2 and synapsin III knockdown) was injected unilaterally into the left SNc , and a total volume of 1 μl of KD virus solution  was injected unilaterally into the right SNc . A glass micropipette with a long narrow tip (size: 10–20 μm) was made using a micropipette puller  to deliver the virus. The glass pipette slowly reached the target area and was left for 5 min before the virus injection. Virus solution was injected at an infusion rate of 100 nl/min and the micropipette was withdrawn 10 min after the end of injection. After injection, the scalp was sutured, and the mice were returned to their home cages. Virus-injected mice were used for electrophysiology and immunohistochemistry experiments 4 weeks after injection.Stereotaxic virus injection was conducted on 6- to 8-week-old male and female mice . The appropriate statistical methods for each experiment are described in the figure legend. Summary graphs are all shown as the mean ± SEM. Unpaired Student’s DAT-IRES-Cre;Plcg1F/F, PLCγ1 cKO) by crossing DAT-Cre mice with Plcg1F/F mice  and PLCγ1 antibodies and checked the expression of these proteins in dopamine neurons  normally expressed PLCγ1, but PLCγ1 expression was significantly abolished in the cell bodies of dopamine neurons in both the SNc and VTA regions of PLCγ1 cKO mice, clearly demonstrating that PLCγ1 was selectively eliminated in the dopamine neurons of DAT-Cre;Plcg1F/F mice  and nucleus accumbens (NAc) core by using fast-scan cyclic voltammetry (FSCV) Fig. , which cice Fig. . This enice Fig. , indicat52. To determine whether this increased dopamine release in the striatum of PLCγ1 cKO mice was affected by electrical stimulation, we produced and checked DAT-Cre;Ai32;Plcg1F/F mice that express channelrhodopsin-2 (ChR2) with the H134R mutation exclusively in dopamine neurons. As in the case of electrical stimulation, dopamine release elicited by either 1 pulse or 5 pulses of optogenetic stimulation (450 nm wavelength) was markedly elevated both in the dorsal striatum and NAc core of DAT-Cre;Ai32;Plcg1F/F mice  and ice Fig. . We founrus Fig. .To further determine whether acute pharmacological inhibition of PLC can elevate dopamine release from dopaminergic axon terminals, we incubated striatal slices with a nonselective PLC inhibitor  for 30 min and then monitored the release of dopamine. However, acute PLC inhibition by U73122 did not alter dopamine release in the dorsal striatum  monitored in the SNc dopamine neurons of PLCγ1 cKO mice were not different from those in control mice  as an inhibitory presynaptic marker and the colocalization of TH and gephyrin as an inhibitory postsynaptic marker Fig. . Interes63, we hypothesized that the increased dopamine release in PLCγ1 cKO mice would attenuate the degeneration of dopamine neurons in an animal model of PD. To validate this possibility, we injected 6-OHDA (6-hydroxydopamine) unilaterally into the medial forebrain bundle (MFB) of DAT-Cre and DAT-Cre;Plcg1F/F mice  and their premature degeneration during disease progressionDAT-Cre and DAT-Cre;Plcg1F/F mice  is a key presynaptic regulator in dopamine release, packaging cytosolic dopamine into synaptic vesiclesAT2 Fig. . Indeed,AT2 Fig. . We alsoAT2 Fig. . We founAT2 Fig. .Fig. 5En68  considerably attenuated dopamine release in both control and PLCγ1 cKO mice Fig. . However71. GABAA α1 subunit-containing receptors are highly expressed in the striatum, and dopamine neurons mainly express the α1 subunit of GABAA receptors73. Moreover, PLCγ1 seems to regulate the surface expression of the GABAA α1 subunit75. Therefore, we evaluated the localization of the GABAA α1 subunit on dopaminergic axons in the dorsal striatum but found that disruption of PLCγ1 does not affect the localization of the GABAA α1 subunit on dopaminergic axons, indicating that a GABAA receptor-mediated mechanism may not be responsible for enhanced dopamine release in PLCγ1 cKO mice  is commonly expressed in dopaminergic axon terminals and provides negative feedback to regulate excessive extracellular dopamine levelsain Fig. . We founain Fig. . To provain Fig. . These r79. Synapsins are encoded by three genes in mice and humans: synapsin I, II, and III. In addition, alternative splicing of these genes can generate several synapsin isoforms. Synapsin isoforms are differentially expressed in the nerve terminals of various types of neurons, acting as key regulators of presynaptic vesicle release. Synapsin I, II, and III begin to be highly expressed during the developmental stages in the brain80. Interestingly, synapsin III is reported to modulate the release of dopamine82. Furthermore, PLCγ1 seems to interact with synapsins84. Given these previous findings, it is natural to speculate that any alterations in synapsin may contribute to the increased localization of VMAT2 on dopaminergic axons in PLCγ1 cKO mice. Thus, we first assessed the expression of synapsin II and synapsin III in the striatum by conducting western blot analysis , which controls the docking, priming, and fusion process of synaptic vesicles94. In addition to these functions, PLC can limit synaptic dopamine availability by controlling the endocytic process of receptors and transporters. Activation of PKC, which is a major downstream molecule of PLC, can promote DAT and D2R internalization in cultured dopamine neurons96. Moreover, overexpression of PLCγ1 increases clathrin-mediated endocytosis of EGF receptors in PC12 cells98. However, in our data, the expression of DATs in dopaminergic axons was not altered by deletion of PLCγ1, whereas D2R expression on TH-positive dopaminergic axons was downregulated. Overall, it is possible that the role of PLC in the regulation of neurotransmitter release greatly varies depending on both the particular cell types where PLC is expressed and the specific isoform of PLC.Previous studies have suggested that PLC is implicated in the exocytosis of neurotransmitters. Glutamate release induced by BDNF is mediated by PLC/IP101, while one other study reported a contrasting result102. In our study, the elevated dopamine release observed in PLCγ1 cKO mice does not appear to be caused by alterations in dopamine synthesis and reuptake. Instead, elevated localization of VMAT2 and synapsin III in dopaminergic axons might underlie the enhancement of dopamine release caused by the deletion of PLCγ1. Synapsin is one of the critical regulators that govern the mobilization of synaptic vesicles to release sites103. Synapsins are encoded by three genes  in mammals, and among them, synapsin III has been shown to exert a regulatory effect on dopamine neurons82. Double knockout of synapsin I and synapsin II led to decreased glutamate and GABA transport into synaptic vesicles, but vesicular uptake of dopamine was unaffected104. Noticeably, synapsin III single knockout mice showed increased dopamine release81, which is in contrast to our findings, and the reasons for this discrepancy are not clear. When we acutely knocked down synapsin III in dopamine neurons, we found that the remaining colocalization of TH and synapsin III over the TH-positive axonal area was significantly higher in PLCγ1 cKO mice than in control mice. Importantly, contrary to the previous finding, we found that the knockdown of synapsin III in dopamine neurons markedly weakens DA transmission in the DStr. Furthermore, the remaining DA transmission in PLCγ1 cKO mice was significantly higher than that in wild-type control mice. Our findings suggest the following possibilities. First, in addition to the previous finding about the role of synapsin III in dopamine transmission81, the functional roles of synapsin III in regulating DA transmission can be multifaceted; the specific roles of synapsin III in DA terminals and transmission remain poorly understood. Synapsin III has also been shown to have versatile functions across various neuronal cell types and differentially regulates neurotransmitter release in excitatory and inhibitory neurons106. According to one study, synapsin III can enhance neurotransmitter release probability, which partially supports our findings106. In our experiment, we acutely knocked down the expression of synapsin III, which subsequently led to a reduction in dopamine transmission. Thus, it is likely that the regulatory role of synapsin III can vary depending on the physiological circumstances at dopamine terminals. Moreover, there are notable differences in the experimental conditions between our findings and the previous one. The previous study used whole-body knockout mice in which synapsin III was widely knocked out during brain development, while we acutely knocked down synapsin III in the dopamine neurons of adult mice81. In addition, the genetic background of control and PLCγ1 cKO mice in our study was C57BL/6J, but the previous study did not provide specific information on the genetic background of synapsin III knockout mice; it is well known that the genetic background of mice can critically affect the physiological functions of many synaptic proteins expressed in both presynaptic and postsynaptic sites. In summary, all of these factors could contribute to the discrepancy between our findings and the previous report. It is worth mentioning that similar to the results from the VMAT2 knockdown experiment, the attenuation of dopamine transmission by synapsin III knockdown was less severe in PLCγ1 cKO mice than in control mice. These results indicate that the elevated levels of both VMAT2 and synapsin III in dopamine terminals might contribute to the facilitation of dopamine transmission in PLCγ1 cKO mice. It has been demonstrated that both synapsin I and synapsin II have PLCγ1 binding domains and directly bind to the SH3 domain of PLCγ184, while direct or indirect interactions between PLCγ1 and synapsin III are unknown. Our study suggests that PLCγ1 might be a key regulator of dopamine release through potential interaction with synapsin III in dopaminergic axons. The detailed mechanisms by which PLCγ1 interacts with these synaptic molecules need to be investigated in further studies. Additional works are also required to clarify the detailed role of PLCγ1 in the regulation of dopamine release via vesicle trafficking by synapsin III.It is important to note that brain-derived neurotrophic factor (BDNF) and its receptor TrkB (tropomyosin receptor kinase B), both of which are upstream activators of PLCγ1, are related to dopamine release. According to several studies using in vivo microdialysis and slice voltammetry, extracellular dopamine levels and dopamine release were increased together in BDNF heterozygous mice2, which is known to interact with various presynaptic molecules important for synaptic release107. In our study, we observed that PIP2, which is hydrolyzed by PLC, is significantly upregulated in PLCγ1 cKO mice. It was shown that presynaptic molecules such as RIM or Piccolo that can interact with PIP2 are present in presynaptic active zone-like sites of dopamine boutons69. These results imply that PIP2 might be also critical for dopamine release in dopamine boutons. Future studies are needed to reveal the role of PLCγ1 in regulating dopamine release through PIP2. Taken together, our findings provide a fundamental understanding of intracellular signaling pathways critical for dopamine release.Finally, we focused our attention on PIPSupplementary Figures


Molecular probes, specialized tools or substances meticulously designed to bind to specific molecules or biomarkers within cells, tissues, or biological samples, play a pivotal role in various domains such as biomedical research, diagnostics, and medical treatments . They arThis editorial features a study by Zheng et al., highlighting the potential of molecular subtyping in guiding diagnosis and personalized treatment decisions for esophageal squamous cell carcinoma (ESCC). This study identified two distinct molecular subtypes of ESCC, S1 and S2, and linked them to the tumor microenvironment, distinct immune pathways, and clinical outcomes . Ye et aIn this Special Issue, we have explored the evolving role of advanced molecular probes. These specialized tools have become increasingly precise and sensitive, aiding in the detection and manipulation of specific molecules within cells and tissues. The articles cover various diseases, highlighting how molecular probes are reshaping diagnosis, treatment, and therapeutic possibilities. Collaborations between scientists, healthcare experts, and technologists promise to drive continued progress in this field, offering new avenues for disease management and improved patient outcomes.