Mycobacterium bovis infection and spread. Selection and breeding of cattle resistant to M. bovis infection would greatly enhance the effectiveness of bTB eradication programs. Here, we have evaluated the potential of serum proteins as biomarkers of cattle resistance to bTB in Holstein-Friesian cows, 6–8-year-old, born and raised in similar conditions in herds with bTB prevalence >30%. Serum proteins obtained from uninfected cows  were compared to those from infected cows , defined by a negative or positive bTB diagnosis, respectively. bTB diagnosis included: (i) single intradermal  tuberculin test, (ii) whole blood IFN-gamma test, (iii) gross visible lesions in lymph nodes and lungs by inspection at the abattoir, and (iv) a bacteriological culture for M. bovis. Using 2D-GE and LC-ESI-MS/MS, we found higher expression levels of primary amine oxidase (AO), complement component 5 (C5), and serotransferrin (TF) in R cattle than S cattle. In-house developed and standardized ELISAs for these novel biomarkers showed the best sensitivities of 72, 77, 77%, and specificities of 94, 94, 83%, for AO, C5, and TF, respectively. AUC-ROC (95% CI) values of 0.8935 (0.7906–0.9964), 0.9290 (0.8484–1.010), and 0.8580 (0.7291–0.9869) were obtained at cut-off points of 192.0, 176.5 ng/ml, and 2.1 mg/ml for AO, C5, and TF, respectively. These proteins are involved in inflammatory/immunomodulatory responses to infections and may provide a novel avenue of research to determine the mechanisms of protection against bTB. Overall, our results indicate that these proteins could be novel biomarkers to help identify cattle resistant to bTB, which in turn could be used to strengthen the effectiveness of existing eradication programs against bTB.Eradication of bovine tuberculosis (bTB) continues to be a worldwide challenge. The lack of reliable vaccines dampens the control and eradication programs of  Mycobacterium tuberculosis complex are a group of pathogenic mycobacteria responsible for tuberculosis (TB) in mammals. The principal causative agent of bovine TB (bTB) is Mycobacterium bovis, which affects the livestock industry worldwide ; however, BCG is not currently used as the protection it confers is variable and interferes with the current diagnostic tests (M. bovis culture) and extensively evaluated in families and breeds exposed to M. bovis under field conditions , named as the Sp110 nuclear body protein (SP110), through transcription activator-like effector nuclease (TALEN) (DEFB103A) through plasmid transfection and somatic cell nuclear transfer has also achieved bTB-resistant cattle (SLC11A1), has produced cattle with increased resistance to bovine tuberculosis -associated protein 9 (Cas9) nickase (Cas9n) and somatic cell nuclear transfer (M. bovis and other intracellular pathogens (SLC11A1 gene influencing the NRAMP1 expression have been related to bTB-resistance in African Zebu cattle (Implementing immunogenomics with genome editing for the generation of transgenic-resistant cattle to bTB is currently being tested as a promising strategy –19. Gene (TALEN) . In compt cattle . The knorculosis , 19. SLCtransfer ; system athogens , 21. Theathogens , 21. Polu cattle  and Chinu cattle .M. bovis infects, resides, and replicates in monocytes-derived cells of infected cattle. Therefore, immunogenetic studies on those cells have been of particular interest for searching bTB-resistance candidate genes . Primary amine oxidase (AO), Complement component 5 (C5), and serotransferrin (TF), were found overexpressed in serum of bTB-resistant cattle compared to bTB-susceptible cattle. We then developed enzyme-linked immunosorbent assays (ELISAs) for each of these novel biomarkers and obtained an overall sensitivity higher than 77% and a specificity of at least 83% in field diagnosis. Our results provide potential novel targets for breeding purposes to improve the resistance of cattle to bTB and contribute to the success of eradication campaigns.te genes . Genomicte genes . Genome-te genes , Irelandte genes , 27, UK te genes , 14, andte genes . GWAS alte genes  and has te genes . Like gete genes –32. Furtte genes –35, havete genes –38, and te genes , 37, 39 M. bovis . Those herds were at active episodes of infection and were quarantined at the control campaign. All animals were tuberculin tested [single caudal fold  test—CFT or intradermal comparative cervical tuberculin test] at intervals of 60–90 days, with restriction from trading and permanent identification and subsequent culling of reactors. The prevalence of bTB within those dairy farms was greater than 30% . All herds had similar husbandry conditions: a semi-intensive system, housing in barns and grasslands after milking, twice a day, fed with forage, grains, and supplemented during the lactating period. The size of herds was 70–180 milking cows (median of 90) with average milk production of 53,795 kg.The present study included Holstein-Friesan dairy cows, 6–8-year-old, born and raised in eight -different dairy farms located in Jalisco, Mexico, a geographic area with known prevalence and active transmission of M. bovis . IndividM. bovis infected animals increases  or lungs and M. bovis positive culture. Lesions were processed for M. bovis culture on Lowenstein-Jensen and Stonebrink media at 37°C for 12 weeks. This conservative classification is consistent with the current formal definition of a potential bTB case of SENASICA from Mexico and the Animal and Plant Health Agency (APHA) from the UK. Cows with positive results for CFT or IGRA with no visible lesions and negative M. bovis culture were excluded from the study. Not infected animals (resistant) were cows that resulted negative for CFT and negative for IGRA (nonreactors). In addition, all these cattle were confirmed bTB-negative by detailed postmortem examination  to avoid inclusion of M. bovis-infected but unresponsive (anergic) animals (Infected cows (susceptible) were animals that had a positive CFT result (bTB reactors), and all these animals were also positive for the whole blood IFN-γ (interferon-gamma) release assay . When the IGRA is used in parallel with the CFT, the sensitivity for detecting ncreases . Further animals , 42.M. bovis infection from a reactor to an in-contact animal within a group can occur in a 12-mo period  with and without anticoagulant (lithium heparin). Samples were immediately protected from heat and sunlight after collection and processed within 2 h after sampling in the laboratory. After clot formation, serum was obtained by 15 mins centrifugation at 12,000 × M. bovis and derived from epidemiologically comparable herds  and cattle with bTB (n = 36), both diagnosed with or without any other infectious disease  or metabolic disease from the same herds described above. Two cases each of the most frequent diseases in the geographical region of the sampled cattle were included per group: (1) Mycobacterium avium sp. paratuberculosis (MAP)/Johne's disease, (2) Brucella abortus, (3) bovine herpesvirus 1 (BoHV-1), (4) bovine viral diarrhea virus (BVDV), (5) bovine parainfluenza virus 3 (BPIV-3), (6) Leptospira spp. infection (L. interrogans serovar hardjo type hardjoprajitno or L. borgpetersenii serovar hardjo type hardjo-bovis), (7) hypocalcemia, (8) ketosis, and (9) hypomagnesemia. Serum samples from 18 active clinical cases of cattle diagnosed with different infectious or metabolic diseases concomitant to bTB were included as a single group (bTB+IMD). While samples of 18 cattle matching the same diseases without bTB were grouped (bTB-IMD). The comparison included the samples of 18 cattle with bTB without any other clinical disease (bTB+) and 18 samples from cattle resistant to bTB (bTB–), used to standardize ELISAs. Additionally, blood samples of 18 healthy cattle (HS) from a bTB-free, paratuberculosis-free, and brucellosis-free herd  were also included.For biomarkers validation, a pilot study was performed individually with in-house ELISAs in another set of 72 serum samples collected from cattle without bTB , and quantified using the Pierce bicinchoninic acid microplate assay , following the manufacturer's instructions. For qualitative analysis, 10 μg of protein per well of crude serum, depleted serum (precipitate), and the albumin-rich fraction (supernatant) were resolved by 1.5 mm, 10% SDS-PAGE, carried out at a constant voltage set to 100 V for 1 h, and Coomassie blue stained  were used with a 12-well configuration following the supplier's protocol. The IPG gel strips  were rehydrated with IPG Strip Rehydration Solution in the assembled device with 40 μl per well for 15 min. Two hundred μg of serum proteins were diluted in protein OFFGEL solution  to a final volume of 1.8 ml and 150 μl of sample was loaded into each well. Proteins were submitted to isoelectric focusing (IEF) until reaching 50 kVh with a maximum voltage of 4000 V, 50 μA 200 mW, and a hold setting of 500 V. Twelve individual fractions were recovered and further resolved by molecular weight (kDa) through 1.5 mm, 4–20% gradient SDS-PAGE, under reducing conditions. To facilitate comparisons, fractions were loaded in SDS-PAGE (10 μg/well) organized by pairs matching resistant (R) with susceptible (S). SDS-PAGE was carried out at a constant voltage set to 100 V for 1 h. Gels were stained with Coomassie brilliant blue; images were recorded with a Gel Doc™ XR+ system  and analyzed by densitometry with Quantity One software . Single serum fractions that presented differences of two or more times the quantity of a band among paired lanes  were subt-test (p < 0.05). A factor greater than a 1.6-fold increase in the spot's average density in one group compared to the other was reported as overexpression.Individual sera fractions 2, 4, 6, 8, and 10 were respectively pooled (R or S) and processed to remove OFFGEL solution components  before 2D-GE. Pooled sera fractions containing 150 μg of protein each was diluted in a final volume of 350 μl of IPG rehydration buffer . The IPG gel strips 7 cm, pH 3-10  were rehydrated with this solution for 20 h using a DryStrip reswelling tray  covered with 1.6 ml of mineral oil. After rehydration, IEF was carried out on an IPGphor (Amersham Biosciences) to reach 52 kVh. Following IEF, gel strips were equilibrated in equilibration buffer  for 15 min. The strips were further transferred to a buffer containing 2.5% iodoacetamide for 15 min. After equilibration, the second dimension was performed on 1.5 mm, 12% non-gradient polyacrylamide gels in a Mini-Protean Tetra Cell chamber . SDS-PAGE was carried out at a constant voltage set to 120 V for 1.5 h. After protein fixation for 1 h with 10% methanol and 7% acetic acid, the gel was stained using colloidal Coomassie Blue . Gel images were taken with a CCD high-resolution camera using the Gel Doc™ XR+ system . To increase confidence in the results obtained from the 2D gel electrophoresis, gels were run in triplicates with samples from each pooled fractionated sera from R and S cattle, respectively, for quantitative analysis. Gel image analysis included image alignment, spot detection, background subtraction, spot measurement, and spot matching using PDQuest 2-D analysis software . Coomassie Blue-stained spots were quantified based on their relative volumes . Protein profiles were matched within each R and S gels, and then relative volumes of matched spots were compared between R and S samples for statistical analysis using the Student's Differentially expressed protein bands were excised from the Coomassie-stained gels, distained, and digested with sequencing-grade trypsin . Nanoscale liquid chromatography separation of tryptic peptides was performed with a nanoAcquity ultra-performance liquid chromatography (UPLC) system , equipped with a Symmetry C18 Trap Column  and a PST BEH C18  analytical column. The lock mass compound human [Glu1]-Fibrinopeptide B (Sigma-Aldrich)  was delivered at 0.5 μL/min at a concentration of 200 fmol/ml to the mass spectrometer. Mass spectrometric analysis (LC-MS/MS) was carried out in a Synapt-HDMS Q-TOF (Waters). The spectrometer was operated in V-mode, and analyses were performed in positive mode ESI. The acquisition window in MS mode was 400–2000 (m/z), and an MS/MS mode was 50–2000 (m/z). The charges of acquired ions were 2+, 3+, and 4+. The data acquisition was set at Data Dependent Acquisition (DDA). MS and MS/MS spectra were acquired at a fragmentation energy ramp of Low Mass Collision Energy (LM CE) of 15–45 volts and High Mass Collision Energy (HM CE) of 15–55 Volts. The Lock Mass reference sprayer was sampled every 30 s.http://www.matrixscience.com). Searches were conducted using the NCBIprot , the Bos taurus protein database , and the contaminants database (262 sequences). Trypsin was used as the specific protease, while one missed cleavage was allowed with a 0.6 Da tolerance set for the precursor and the fragment ion masses, while carbamidomethyl (C) and oxidation (M) were selected as variable modifications. Proteins were identified based on a minimum of two unmodified highly scoring unique peptides per protein at a false discovery rate (FDR) of 1%. Proteins identified were: (1) amine oxidase (EC 1.4.3.21), (2) complement component 5 (C5), (3) serotransferrin and (4) haptoglobin . Proteins were then identified using the Mascot search engine algorithm  emulsified in an equal volume of TiterMax Gold adjuvant ; rabbits were inoculated thrice at 2 weeks intervals. Antigens for injection were obtained as follows: complement C5 was isolated from bovine serum as previously described ; bovine blotting . Detectiblotting . The IgGCapture and detection antibodies were titrated in a range of standard concentrations as well as protein standards . All parameters were analyzed in triplicate. Other dose-response curves for the concentration of each standard in the sample matrix  were performed. Two standard dilution buffers were assayed: 10% blocking buffer with 0.05% Tween 20 in 1X PBS; and 2% (v/v) horse serum  0.05% Tween 20 in 1X PBS. Three blocking buffers were evaluated: 1% casein in 1X PBS ; 0.5% enzyme immunoassay-grade fish skin gelatin  in 1X TBS ; and 5% nonfat dry milk  in 1X TBS. The wash buffer was 0.05% Tween 20 in 1X PBS. The HRP substrate was 1% tetramethylbenzidine (TMB) dissolved in Dimethylsulfoxide (DMSO), diluted 1:100 in 0.1 M sodium acetate (pH 6.0) and 0.005% hydrogen peroxide (added just before using the substrate). The stopping solution was 1.5 N sulfuric acid. ELISAs were performed in 96-well clear flat bottom polystyrene High Binding microplates . The optimal concentration for each antibody pair was determined by the lowest signal-to-noise ratio of the mid and low standards compared to a top standard. The signal-to-noise ratio was obtained by dividing the average optical density (OD) of each standard by the average OD of its corresponding zero standards. The optimal blocking buffer was 0.5% fish skin gelatin, which produced the lowest OD reading difference between 0, 10, and 50% of the sample matrix plus standards for the entire tested interval.R ≥ 0.90 and the minimum of the linear range were higher than the LLOD. The LLOD was calculated by the mean plus two standard deviations (M+2SD) of the diluent OD measured 20 times.The precision of the assay was determined through the construction, modeling, and evaluation of the concentration curve of protein standards using all the combinations of reagents described above. Each detection was repeated ten times for three different days by two independent people. The four-parameter logistic (4PL) fit was performed as the reference model for the standard curves . The linR = C×(V0+V)−(C0×V0)/(V×CS)×100%, where V is the volume of sample A, V0 is the volume of sample B, C is the concentration of the mixture of A and B, C0 is the concentration of sample B, and CS is the concentration of sample A.The recovery rate was used to evaluate the accuracy of the ELISA tests. A sample of high-concentration standard (A) was added to a sample of low-concentration standard (B) with a volume ratio (A: B) ≤ 1:9. The recovery rate R was calculated as follows, M. bovis culture, with or without another infectious or metabolic disease, plus control healthy cattle, were included for the analysis. Flat bottom 96-well plates were coated overnight at 4°C with 50 μl of 2 μg/ml polyclonal rabbit capture antibodies against bovine AO, C5, or TF diluted in 50 mM sodium carbonate, pH 9.6. The ELISA components and serum samples were equilibrated at room temperature (RT) for 30 min before use. The detection procedure was as follows. Coated plates were washed with 0.05% Tween 20 in PBS, three times  using an automatic plate washer . Blocking buffer  was immediately added and incubated for 1 h, at RT shaking on an orbital shaker . After washing, 100 μl of standard sample dilutions and serum samples diluted 1:10, both in 0.05% gelatin-0.05% Tween 20 in 1X TBS, were added in triplicates. The plates were sealed with film and incubated for 2 h at RT, shaking. The plates were then washed, and 100 μl of 0.1 μg/ml HRP-conjugated detection antibodies diluted in 0.05% gelatin-0.05% Tween 20 in 1X TBS were added to wells. The sealed plates were left shaking for 1 h at RT, after which wells were washed once more. TMB substrate solution was added to each well (100 μl) and incubated in the dark without shaking for 15 min at RT. Finally, 100 μl of 1.5 N sulfuric acid was added per well, and the plate was gently mixed by hand. Plates were scanned with the iMark plate reader  at 450 and 595 nm, and the delta OD (OD450–OD595) was used as the OD data point.The concentration of AO, C5, and TF in cattle sera was measured using in-house-built and standardized indirect sandwich ELISAs. Equal numbers of serum samples (n=18 in each group) from cattle with or without bTB identified by skin test, IFN-gamma test, lesions, and The standard curves were provided from 7 dilutions of each purified standard and zero . The cut-off values were chosen by a percentile method at which the highest sensitivity and specificity are reached and ROC (receiver operating characteristic) evaluating the discriminative capacity of the diagnostic tests. The detection effect was evaluated according to the area under the ROC curve (AUC-ROC).t-test (resistant vs. susceptible) in the abundance of protein spots in 2D-GE determined by PDQuest 2-D analysis software and concentration of candidate biomarkers in individual serum samples quantified by the in-house ELISAs. Assessment of specificity, sensitivity, and area under the curve (AUC) was carried out using Receiver Operating Characteristic (ROC) curves. Kruskal-Wallis nonparametric test with Dunn's multiple comparison post hoc test was carried out to compare the mean rank serum levels of candidate biomarkers in each group of cattle  against the mean rank serum levels of the bTB-resistant cattle (bTB-). Nonparametric tests were selected after data were subjected to the D'Agostino-Pearson omnibus K2 normality test that showed that the data were not normally distributed. All statistical analyses were performed with Prism 6.0 . p < 0.05 was statistically significant.Comparisons between groups were conducted using the Student's To enhance the resolution of the comparative analysis, depleted sera were further fractionated by IEF with OFFGEL electrophoresis. Individual OFFGEL fractions of R and S sera were further paired and resolved by one-dimension SDS-PAGE. Afterward, densitometry of bands was performed with Quantity One software (Bio-Rad Laboratories). Protein patterns showing significant differences amongst bands of R and S fractionated sera are shown in p < 0.0001) (Representative images of 2D-GE of processed sera from R and S cattle are presented in  0.0001) . Three p 0.0001) .Before evaluating AO, C5, and TF, as potential biomarkers of resistance to bTB, there was a need to develop an assay that would allow the evaluation for the presence of these proteins in cattle samples. Accordingly, we developed and standardized capture and detection rabbit antibodies and individual ELISA tests to determine the concentration of these proteins in serum samples derived from 18 cattle resistant to bTB and 18 cattle with bTB.The performance of each ELISA against these novel biomarkers is summarized in M. bovis, infected (bTB+) and not infected (bTB-) and with and without a concomitant infectious or metabolic disease (IMD), as well as in healthy cattle (HS) not exposed to M. bovis. The mean serum concentration values (± interquartile rank) of AO, TF, and C5, in cattle resistant to bTB and cattle with bTB are summarized in p < 0.001). At the same time, the mean serum concentration of AO was significantly higher in cattle resistant to bTB without any simultaneous disease (bTB-) than in all other groups (p < 0.01). C5 mean serum values of the bTB- group were not distinguishable from C5 mean serum values of the healthy group (HS), although they were significantly higher than the rest of the animals in the other groups (p < 0.01). TF values in serum of bTB-resistant cattle, with or without an infectious or metabolic disease (IMD), were significantly higher than in sera of cattle with bTB (p < 0.001), but like those of HS (p > 0.05).In order to validate 2D-GE and LC/ESI-MS/MS data, we performed ELISA analyses for AO, C5, and TF in naturally exposed cattle to M. bovis infection is higher than the expression of the same proteins in cattle that have succumbed to bTB. These serum proteins are AO, C5, and TF and would appear to be promising biomarkers of resistance to bTB.In the present study, we have identified that the level of expression of some bovine serum proteins in Holstein-Friesian dairy cows that appear to be resistant to M. bovis infection  compared to cattle with M. bovis clinical infection  or not infected. Serum levels of fetuin, alpha-1-microglobulin/bikunin precursor protein (AMBP), alpha-1 acid glycoprotein, and alpha-1B glycoprotein were elevated exclusively in M. bovis infected animals compared to contemporary controls and M. paratuberculosis infected animals. Whereas transthyretin, retinol-binding proteins, and cathelicidin were identified exclusively in M. paratuberculosis infection . Our ELISA systems showed linear range of 6.25–400, 4.69–300 ng/ml, and 0.76–8.67 mg/ml, LLOD of 3.13, 2.34 ng/ml, and 0.51 mg/ml, as well as sensitivities of 72.2, 77.8, and 77.8% and specificities of 94.4, 94.4, and 83.3%, respectively, for AO, C5, and TF. Linear ranges showed correlation coefficients higher than 0.90, and LLOD could be separated from the background. The positive detection rate for AO, C5, and TF was 93.8, 94.3, 84.6%, respectively, showing the high sensitivity of these detection systems.While many efforts seeking to distinguish between cattle resistant and susceptible to orphisms , 22–25. M. bovis with a negative or positive diagnosis of bTB that were positive or negative to any unrelated infectious or metabolic disease. In addition, samples from healthy cattle without exposure to M. bovis (from a bTB-free herd) were included as controls. Overall, the OD values in our ELISA test of AO, TF, and C5 were significantly higher in cattle resistant to bTB than in those with bTB , regardless they were suffering or not from other unrelated infectious or metabolic diseases (p < 0.001). However, unlike TF levels, AO and C5 serum levels were depressed in cattle resistant to bTB with a simultaneous disease (bTB-IMD). Our observations suggest that AO, C5, and TF are reliable markers of a protective host response against bTB and that AO and C5 levels are negatively affected by other pathologies present in cattle resistant to M. bovis, like M. paratuberculosis infection, and other unrelated bacterial and viral infections or metabolic diseases. Seth et al. , an activator of the classical complement pathway, had no significant differences between M. bovis-infected cattle and uninfected controls. However, CRP levels in the PPD-B-stimulated blood of M. bovis-infected cattle were significantly higher than those in uninfected controls, while TF levels were lower than the unstimulated blood. These authors suggested that PPD-B may activate acute-phase proteins, increasing CRP and decreasing TF  between cattle resistant to bTB (bTB-) and the healthy group (HS), while AO levels in sera of these healthy animals were significantly lower compared to those of the bTB-group. Using the cutoff values of our ELISA tests for C5 and TF, more than half of HS animals would be resistant, despite not having been exposed to M. bovis. On the opposite, using the AO cutoff values, all HS animals would be susceptible. It is hard to interpret the variation in AO, C5, and TF levels in these not exposed cattle. Although HS cattle were randomly selected from bTB-free, paratuberculosis-free, and brucellosis-free dairy herd, the possibility of exposure to other viruses or bacteria, like environmental mycobacteria, may lead to these variations in the levels of AO, C5, and TF, cannot be excluded. As discussed above, non-specific responses may be related to particular mycobacterial infections or other pathogens. Previous studies have shown that the levels of specific serum proteins are dynamic along with the M. bovis infection (M. bovis infection) or are indicative of the intensity of the effector response, those are singularly related to the protective response against M. bovis infection, and their effect on bTB requires further investigation.Interestingly, in our study, C5 and TF serum values were similar  (Bovine plasma amine oxidase (AO), also known as bovine serum amine oxidase (BSAO), is a liver-expressed Cues (AOC) . Plasma es (AOC) . The higes (AOC) . AO prefes (AOC) . BSAO ales (AOC) . These, es (AOC) . Oxidaties (AOC) ; howeveres (AOC) . AOC3/VAes (AOC) . AOC3/VAes (AOC) , 63. Howes (AOC) . Bovine es (AOC) . HoweverM. bovis infection in dairy cows represents an attractive possibility to be investigated in the future.The complement system is a complex enzymatic cascade (consisting of more than 30 proteins in blood plasma) that functions in stepwise activation of several proteases produced by the liver, adipose tissue, leukocytes, and vascular cells . C5a andM. tuberculosis can acquire iron from both extracellular TF and endogenous macrophage sources, except that iron acquisition from macrophage cytoplasmic iron pools may be critical for the intracellular growth of M. tuberculosis (Bovine TF is a single-chain ß-globulin that is important for iron (Fe) transport in blood plasma and is mainly produced by hepatocytes, macrophages, and other cells . Intraphrculosis . TF in trculosis . Iron rerculosis . Such isrculosis . Howeverrculosis . Plasma rculosis .Haemophilus somnus or Trypanosoma vivax, were characterized by the relatively low TF serum levels in affected individuals compared to healthy controls (Bos taurus taurus) during heavier infestations of Rhipicephalus microplus. In contrast, there was no significant decrease in the serum concentration of TF in tick-resistant cattle  compared to susceptible cattle  is typically classified as negative acute-phase protein (APP) during acute infections and acute metabolic diseases , 74. Chrcontrols , 75, 76.controls . In agree cattle . Low base cattle . Increase cattle . TF overe cattle . Blood pe cattle .In conclusion, we have identified three significantly overexpressed serum proteins which could be used as potential biomarkers to determine resistance to bTB in diary Holstein-Friesian Mexican cattle. These proteins are involved in inflammatory/immunomodulatory responses to infections and may provide a novel avenue of research to determine the mechanisms of protection against bTB. Using validated serum biomarkers to identify and further enhance the resistance of cattle to bTB implemented as part of bTB control strategies could help eventually eradicate bTB from herds by reducing the susceptibility to infection. However, due to the low number of individuals gathered for validation, our results require further investigation to determine the extent to which these results could apply to cattle of different breeds and in different circumstances. In addition, it is clear that the ELISA requires further development and that, ideally, these markers could be evaluated in a multiplex system using perhaps monoclonal antibodies.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: ProteomeXChange MassIVE MSV000087778.The animal study was reviewed and approved by Institutional Ethics and Biosafety Committee. Written informed consent for participation was not obtained from the owners because owners gave their verbal consent for sampling of animals previous to slaughter at the abattoir facilities.JM-C, OA-L, BV-R, JG-P, and HE-S contributed to the conception and design of the study. HE-S acquired funding, coordinated the work, performed the statistical analysis, and prepared the final draft of the manuscript. OA-L and HE-S organized the database and conducted laboratory examinations. GL-R and HE-S collected clinical samples and conducted gross examinations. JM-C, OA-L, and HE-S performed the experiments. BV-R, JG-P, GL-R, and HE-S wrote sections of the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version, except OA-L and JM-C who lamentably departed before the final draft was achieved.This research was supported by the projects FOMIXJAL 2009-05-124324 and SEP-CONACYT CB-2009-132068.GL-R was employed by the Laboratorios Virbac México SA de CV . The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Compared with the rigid blue box, the first generation TxSB displayed considerably greater conformational flexibility and a relatively large central cavity, making it a good host for a variety of electron-rich guests. In this review, we provide a comprehensive summary of TxSB chemistry, detailing our recent progress in the area of anion-responsive supramolecular self-assembly and applications of the underlying chemistry to water purification, information storage, and controlled drug release. Our objective is to provide not only a review of the fundamental findings, but also to outline future research directions where TxSBs and their constructs may have a role to play.The design and synthesis of novel macrocyclic host molecules continues to attract attention because such species play important roles in supramolecular chemistry. However, the discovery of new classes of macrocycles presents a considerable challenge due to the need to embody by design effective molecular recognition features, as well as ideally the development of synthetic routes that permit further functionalization. In 2010, we reported a new class of macrocyclic hosts: a set of tetracationic imidazolium macrocycles, which we termed “Texas-sized” molecular boxes (TxSBs) in homage to Stoddart’s classic “blue box” (CBPQT In the rm MORFs . In cont1 n in solution and in the solid state  (MBAAm) containing both TxSB moieties  and alkylsulfonate subunits [Recently, we described the creation of information code arrays predicated on anion-recognition-based gels. A series of hydrogels were achieved via incorporating a poly(acrylamide) (pAAm) network cross-linked by subunits . Various1  and an amphiphilic copolymer containing carboxylate anions. The ratio of the hydrophilic part of the supra-amphiphilic polypseudorotaxane could be tuned by adding differing amounts of TxSB 1. This leads to a change in the overall self-assembled morphology, a feature that could be used to affect controlled drug release [Amphiphilic copolymers are of interest because they can stabilize a diverse variety of self-assembled structures in aqueous media. For instance, they can support the formation of inter alia spherical micelles, disklike micelles, cylindrical micelles, vesicles, and other interesting aggregates. We explored the use of TxSB-based anion receptor interactions to stabilize various morphologies. As shown in  release . The via1, 2, 3, 4, and 5). Their ability to recognize organic carboxylate led to the stabilization of a number of monomeric and self-assembled complexes. The binding modes inherent in these complexes and their aggregation features were found to be highly dependent on the choice of macrocycle and anionic substrate. It was also found that other inputs, including protons, competing anions, neutral molecules, and metal cations, could be used to regulate the nature of the structures formed. By judicious choice of the precursors and conditions, a variety of mechanically interlocked molecular architectures including, e.g., metal organic rotaxanes, MORFs, RSOFs, etc., could be obtained. Many of the complexes produced from the “Texas-sized” molecular boxes and the higher order aggregates formed from subsequent self-assembly were found to respond to changes in the environment, such as concentration, pH, temperature, presence of potentially competitive substrates, etc. The various TxSB-based hydrogels produced to date show promise in a number of application areas. For instance, this class of hydrogels has been used to remove surrogate anionic pollutants from aqueous source phases. Related systems were found useful for encoding information in a controllable pattern-based fashion.In summary, we have developed a series of tetracationic imidazolium macrocycles, colloquially known as “Texas-sized” molecular boxes (TxSBs On the basis of the work carried out to date, we believe that this versatile molecular receptor will continue to find application in the field of molecular recognition and self-assembly and will permit ultimately the construction of yet more complex molecular switches and externally triggered supramolecular arrays. To date, the “Texas-sized” molecular box and its congeners have allowed for the generation of a number of molecular frameworks with threaded structures. However, this is likely just the beginning. Given the facile synthesis of these macrocycles and the variety of molecular structures that they have been used to stabilize since the first generation system was introduced in 2010, we predict that “Texas-sized” molecular boxes and related cyclic imidazolium macrocycles will continue to attract attention within the self-assembly, anion recognition, cation coordination, and soft materials communities. Efforts to explore new opportunities associated with this class of receptors are currently ongoing in our laboratories.


In this review, we describe sex-specific differences in the regulatory systems for dopamine production in the brains of social insects, focusing on the honey bee. Dopamine has a crucial role in the promotion of reproduction in both sexes of the honey bee and is a key substance for understanding the mechanisms underlying the reproductive division of labor in females. Studies associated with dopamine regulation have been performed mainly in females, with less of a focus on its regulation in males. In social insects, males are specialized for reproduction and do not exhibit division of labor; however, they have evolved to adapt their social system and have acquired/discarded physiological and behavioral characteristics. Therefore, studies exploring the dopaminergic system in males can contribute to our understanding of social adaptation in males. We integrate findings related to dopamine in both honey bee sexes and provide insights into the physiology involved in dopaminergic systems in social insects.Dopamine has multiple functions in the modulation of social behavior and promotion of reproduction in eusocial Hymenoptera. In the honey bee, there are sex-specific differences in the regulation of dopamine production in the brain. These different dopaminergic systems might contribute to the maintenance of sex-specific behaviors and physiology. However, it is still not fully understood how the dopaminergic system in the brain is regulated by endocrinal factors and social stimuli in the colony. In this review, we focus on the regulation of dopamine production in queens, workers, and males in the honey bee. Dopamine production can be controlled by queen substance, juvenile hormone, and exogenous tyrosine from food. Queens can control dopamine production in workers via queen substance, whereas workers can manipulate the supply of tyrosine, a precursor of dopamine, to queens and males. The regulation of dopamine production through social interaction might affect the reproductive states of colony members and maintain sex-specific behaviors in unpredictable environments. The division of labor in eusocial insects is fundamental to the organization of insect societies. In highly eusocial hymenopterans, including the honey bee, females are differentiated both behaviorally and morphologically into reproductive individuals (queens) and infertile helper individuals (workers). These polymorphic individuals are called ‘castes’, and caste differentiation occurs initially based on nutrition during the larval stage ,2,3. SucMales in eusocial hymenopteran species do not exhibit division of labor and are cared for by workers. Honey bee males are fed by nurse bees during their early adult stage before sexual maturity ,12. EvenInsect hormones, including juvenile hormone (JH), contribute to caste differentiation via nutrition-sensing signal cascades during the larval stage and maintain the physiological state of the central nervous system and peripheral organs of the adults ,15,16,17Dopamine is a biogenic amine that has multiple roles in the regulation of behavior and reproduction in eusocial insects ,23,24. IAmth) and DOPA decarboxylase (Amddc) compared with newly emerged workers [Amddc are almost the same between castes [Amth and Amddc in the brain of the queen. However, this possibility has not been confirmed at the individual neuron level. Queens are also fed ‘royal jelly’ by nurse bees; this contains the dopamine precursor, tyrosine [Caste differences in the brain levels of dopamine have been reported in adult honey bees ,30. The tyrosine ,33,34. Ttyrosine . It has tyrosine . The higtyrosine ,31.Bombus ignitus [Caste differences in the dopamine levels in the brain have been reported in the bumble bee  ignitus . Newly eAmdop1, Amdop2, and Amdop3) [Amdat) [In workers in queenright colonies, the levels of dopamine in the brain increase with age ,37,38. T Amdop3)  and a do [Amdat)  increaseWorkers are usually infertile with inactivated ovaries in the presence of queens, but have the potential to become reproductive individuals with activated ovaries, laying unfertilized eggs that are destined to become males. This switch is a reproductive tactic, changing from cooperative altruistic behaviors for determining the success of the colony to selfish behaviors for determining their own reproductive success. In the honey bee, dopamine is involved in the reproductive maturation and reproductive behaviors of females . The braAmdop1 in the brain is enhanced in workers in cages without a brood [Amdop1 and Amdop2 is reduced [Amdop1 is enhanced, whereas that of Amdop3 is reduced in workers in queenless colonies [The gene expression of dopamine receptors in the brains of queenless honey bee workers is modulated by the presence of a queen in the colony ,47. In t a brood , whereasMale honey bees mate with a queen while flying; therefore, mating flight activity is an important factor for their mating success. Flight activity gradually increases with age  and maleAmdat increases progressively for at least 15 days after emergence [Amdop1, Amdop2, and Amdop3 is also enhanced up to 8 days of age [Amdop1, Amdop2, Amdop3, and Amgpcr19 (encoding the dopamine-ecdysteroid receptor) [Dopamine can contribute to the elevation of mating flight activity in males. Its levels in the brain, thoracic ganglia, and hemolymph increase up to 7–8 days of age . The expmergence . The exps of age , suggests of age . Flight-s of age . Given ts of age . In semieceptor) ,52, are eceptor) . HoweverIn terms of pheromones, the queen substance can control the behavior and physiology of workers in highly eusocial Hymenoptera. In the honey bee, a queen mandibular pheromone (QMP) inhibits ovarian activity in workers ,54 and hAmth and Amddc) involved in dopamine biosynthesis [Amdop1 in the male brain [Xylocopa appendiculata [Drosophila melanogaster [Lygus Hesperus [D. melanogaster, the roles of JH in reproduction with dopamine are different between sexes: the activation of the ovaries in females and the modulation of courtship behavior in males [The dopamine levels in the brain, thoracic ganglia, and hemolymph of males change in parallel with those of JH titer in the hemolymph or JH synthesis in the corpora allata ,60,61. Jynthesis , but notynthesis . Methoprle brain , suggestdiculata , suggestnogaster ,67,68 anHesperus . In D. mAmtbh (tyramine β-hydroxylase) in the brain, resulting in the promotion of foraging behavior [In workers under queenright conditions, JH enhances the octopamine levels via the expression of behavior ,70,71  individuals was calculated from previously published data ,74,75 T and comp of DOPA . These dHere, we have reviewed the dopaminergic regulation systems in the brains of honey bees. Dopamine has multiple functions in the nervous system that result in behavioral changes and in peripheral tissues that result in physiological changes. Reports of the behavioral effects of dopamine are accumulating in the honey bee and studies addressing dopamine targets in the brain are ongoing. Various peripheral tissues are potential targets of dopamine in reproductive individuals, but only a few studies on the peripheral targets of dopamine have been published ,49. GiveDopamine production can be manipulated in several ways through social interactions. Queens can inhibit dopamine increases in workers by queen substance, whereas workers can manipulate dopamine levels by feeding amino acid-rich food containing tyrosine to queens, males, and other workers. Such manipulations of dopamine production can adjust the physiological states required for reproduction and maintain sex-specific behaviors in response to unpredictable environments.


To generate on a computer molecules with optimized properties, scoring functions can be coupled with a molecular generator to design novel molecules with a desired property profile.In recent years, https://github.com/UnixJunkie/FASMIFRA. Experiments regarding speed, training set distribution matching, molecular diversity and benchmark against several other methods are also shown.In this article, a simple method is described to generate only valid molecules at high frequency ( In recent years, there has been a surge of methods developed for in silico molecular generation. Mostly using deep neural networks –18, but The hereby proposed method works directly at the SMILES level. It generates only valid SMILES and thus valence-correct molecules. Simplified Molecular Input Line Entry System (SMILES ) is a moIn the experiments, to quantify molecular diversity in a dataset and the molecules generated from it, the count of unique Bemis-Murcko scaffolds  Fig. 1)1) is monGuacaMol –36 consiValidity [1*]CC’. Where isotope numbers one and two are indexes into an atom type dictionary (a mapping from integer to atom type). In the following, if a fragment is a prefix of a SMILES string, it is called a “seed fragment”. All other fragments are called “branch fragments”.Fragmenting molecules with tagged cleaved bonds A SMILES string with tagged cleaved bonds can be transformed into a mixture of fragments. This is not strictly necessary, as long as the molecular fragment assembler can recognize tagged cleaved bonds. However, this is useful to visualize the fragments with a 2D molecular viewer. The SMILES ‘O[2*][1*]CC’ becomes ‘O[2*][1*].[2*][1*]CC’. When extracting a fragment from a SMILES with tagged cleaved bonds, care must be taken to not extend the fragment past (or shorter than) its SMILES branch nesting depth. i.e. the nesting of parentheses pairs inside the SMILES string must be taken into account  will determine under which cleaved bond type this fragment is registered. Our method uses randomized SMILES rather than canonical ones, in order to avoid any bias that could be introduced by the canonicalization procedure.Generating molecules In essence, some kind of Markov sampling of the previously created data structure is performed, until the generated string has no tagged cleaved bond left. The algorithm is: (i) a seed fragment is uniformly drawn from the array of seed fragments. (ii) as long as the string under construction contains tagged cleaved bonds, each of them is deleted and replaced by a uniformly drawn fragment from the array of fragments with the tagged cleaved bond as the key. When generating the right flavor of DeepSMILES, such a fragment assembly algorithm can be performed using almost exclusively string operations. However, when generating SMILES, an extra step renumbering ring opening and closure numbers is required, to avoid number collisions between rings from different fragments.Software implementation Typing atoms and bonds, selecting and tagging cleaved bonds is done by a Python script using RDKit Model traModel sampling frequency (Fig. ncy Fig. . AssemblGuacaMol benchmark (Table rk Table  In this Molecular diversity (Table ty Table . In termty Table . As expety Table . For exaTraining set distribution matching (Fig. ing Fig. . The metOn the positive side, this method is conceptually simple. Fragmenting molecules is reasonably fast (left of Fig. On the negative side, this method does require a training set. See the introduction for methods which don’t. Also, the method doesn’t create new rings. However, a medicinal chemistry technique is readily applicable in order to reasonably alter the generated rings . If the In this article, a simple method to generate molecules from molecular fragments was described. The method can work with any molecular fragmentation scheme, as long as rings are not opened/broken. Several experiments were presented, evaluating model training speed, molecular generation frequency, molecular diversity and training set distribution matching. The proposed method can be used as-is in genetic algorithm or simulated annealing fragment-based molecular generators. Our prototype software implementation is released under the GPL license. The technique may also be useful for dataset augmentation and demonstrates that DeepSMILES proposed useful simplifications to the SMILES syntax. We are not working on it and it might be difficult, but an interesting extension might be to support molecular fragmentation schemes which happen to open/break rings.


Matrices of morphological characters are frequently used for dating species divergence times in systematics. In some studies, morphological and molecular character data from living taxa are combined, whereas others use morphological characters from extinct taxa as well. We investigated whether morphological data produce time estimates that are concordant with molecular data. If true, it will justify the use of morphological characters alongside molecular data in divergence time inference.We systematically analyzed three empirical datasets from different species groups to test the concordance of species divergence dates inferred using molecular and discrete morphological data from extant taxa as test cases. We found a high correlation between their divergence time estimates, despite a poor linear relationship between branch lengths for morphological and molecular data mapped onto the same phylogeny. This was because node-to-tip distances showed a much higher correlation than branch lengths due to an averaging effect over multiple branches. We found that nodes with a large number of taxa often benefit from such averaging. However, considerable discordance between time estimates from molecules and morphology may still occur as  some intermediate nodes may show large time differences between these two types of data.Our findings suggest that node- and tip-calibration approaches may be better suited for nodes with many taxa. Nevertheless, we highlight the importance of evaluating the concordance of intrinsic time structure in morphological and molecular data before any dating analysis using combined datasets.The online version contains supplementary material available at 10.1186/s12862-021-01798-6. There is a growing interest in using morphological data to infer species divergence times in systematics by using it in morphological clock analyses e.g., –8) or co or co8])Nevertheless, utilizing morphological characters in a dating analysis is complicated , 19–23. Comparisons of tip-calibration methods applying different FBD models/priors e.g., , 18, 19), 1918, 1t4 and t7 is that both morphology and molecules produce concordant time trees. If true, the extrapolation applied in estimating t4 and t7 is appropriate because the molecular data are missing for extinct species. One way to evaluate this hypothesis is to assess node ages' concordance using the living species' data. So, we analyzed three empirical datasets from different species groups, Hemiptera (true bugs), Hymenoptera , and Spermatophyta (seed plants). Moreover, we evaluated the impact of incorporating morphological characters into a joint analysis with molecular on divergence time estimates.We have compared the intrinsic time structure offered by morphological and molecular data with and without internal node calibrations to avoid confounding the time structure introduced by calibrations. The concordance of time structure between morphological and molecular will be enhanced by the presence of calibrations, which has been the focus of tests by others e.g., , 18, 19), 1918, 1At the outset, we note that morphological information is often used to build evolutionary trees or place fossil taxa in the phylogeny, which usually requires a few diagnostic characters. However, this practice does not generally utilize any information on rates of evolution. Therefore, examining the concordance of intrinsic time structure in morphological and molecular data is independently useful. Its presence is expected to produce better time estimates for the nodes created by the fossil taxa and all other nodes in the molecular tree in the combined fossil and extant taxa analysis.r = 0.409, 0.131, and 0.363 respectively; Fig. r = 0.529) and Hymenoptera (r = 0.622). Spermatophyta showed a weak opposite trend (r = 0.028), suggesting discordance on intermediate and deep branches  estimates of branch lengths for the same tree topology were obtained separately for molecular and morphological data. The three datasets, Hemiptera, Hymenoptera, and Spermatophyta, showed a limited correlation and high variation between the molecular and morphological branch lengths , which is critical to learn about the intrinsic time structure in the data. In the Hemiptera dataset, the correlation between the time estimates obtained from molecules and morphology was significant (R2 = 0.801), but so was the variation , but the linear relationship was much tighter . Indeed, in the Hemiptera dataset, the correlation between the time estimates obtained from molecules and morphology became higher under this calibration strategy . , 8. 5, 8In this study, we explored the relationship of ML estimates from the molecular and morphological branch lengths for the same phylogeny Fig. a–c. We or = 0.708) was much higher than that for the branch lengths (r = 0.409), although the relationship was still noisy  compared to the branch lengths . The dispersion was lower for each dataset than that for branch lengths , rivaling the correlation between molecular and morphological time estimates . While we expected some correlation because of the same time prior and branching process, which results in drawing times from the same probability distribution of times for each node in the phylogeny [We examined if the high correlation between morphological and molecular time estimates could arise due to using the same topology and speciation tree prior in the Bayesian analysis (suggested by S. Guindon). We generated time estimates using a uniform BD branching process in MCMCTree , conditihylogeny , the magOverall, our study allows us to conclude that (1) relative ML branch lengths between morphological and molecular characters were very different, but relative node-to-tip distances were considerably more concordant, suggesting a much more concordant time structure in the morphological and molecular dataset than that captured in the comparison of branch lengths. (2) When no internal calibrations were applied, morphological and molecular clock produced time estimates with a high correlation, which may be caused by the same speciation tree prior applied in the joint consideration of molecular and morphological data in the Bayesian analyses. The concordance between time estimates was improved by applying multiple and/or narrow internal calibrations. (3) The combination of molecular and morphological data generally resulted in time estimates nearly identical to the ones from molecular data alone. Our study allows us to conclude that although there is a concordant time structure in morphological and molecular data, the interpretation of fossil ages is determinant for the agreement of time estimates from morphological and molecular clock analyses. We emphasize the importance of evaluating the concordance of time structure in morphological and molecular data before any dating analysis using combined datasets.For comparison between results from molecules and morphology, we composed three datasets consisting of extant species, for which both molecular and morphological data were available. Thus, extinct taxa were not included in our matrices. First, a dataset of 174 discrete morphological characters and 3731 molecular characters (base pairs—bp) from 44 Hemiptera species was obtained from Vea and Grimaldi (2016) . Second,Fossil calibrations are the foremost source of information for translating the distances between molecular/morphological sequences into estimates of absolute times and absolute rates in clock dating analysis. Thus, the calibrations' quality is expected to impact divergence time estimates significantly, even if a large amount of sequence data is available. Therefore, we employed three fossil calibration strategies simulating different quality of calibrations based on fossil evidence. Fossil calibrations were employed following justifications from the original studies , assigning a uniform distribution borrowed from calibration strategy C1 as described below Table . This sttL, tU), where tL is the minimum age bound and tU the maximum age bound, with mean tL) and mean (tM) for the uniform distribution so that the maximum bound was Calibrations were specified using a uniform distribution U. The offset value from the original exponential distribution was used as the minimum bound (tL), and the maximum bound was specified at tL) to be older . To model molecular evolution, we used the GTR general time-reversible (GTR) model [5). Unlike the model applied for morphological data (Mk + Γ5), the GTR + Γ5 model relaxes the assumption of equal character frequencies in molecular substitutions. A ML optimization of model parameters and branch lengths for the given topology was performed for both molecular and morphological data separately in MEGAX [To model morphological evolution, we used the Mk model , with thR) model , with diin MEGAX  and RAxMin MEGAX , respectin MEGAX . We also5 and the GTR + Γ5 substitution models, respectively. The combined subset was treated as two partitions under separate substitution models , with separate substitution-rate parameters assigned and estimated for each partition. Two sets of analyses were performed, using: a single clock for the entire morphological and molecular dataset (linked clocks) or unlinked clocks for morphological and molecular data [In each data analysis , we used one molecular subset, one morphological subset, and one combined  subset, and applied three calibration strategies, Cr, C1, and C2. All Bayesian analyses  were carried out with the program MrBayes . The seqlar data . The MCMIndependent Gamma Rate (IGR) model, in which the rates of evolution on branches varied independently from a gamma distribution [2/2} = 0.0023. The variance (Igrvarpr) was assigned an exponential hyperprior with a mean of 0.039. Analyses were performed with either linked or unlinked relaxed clock models ) or unlink Igrvar = ). For each analysis, four MCMC runs were performed, each consisting of 5 × 106 iterations, sampling every 200, with the burn-in set to 10%, resulting in a total of 9 × 104 samples from the four runs, which were used to obtain posterior time estimates.Analyses were performed on three subsets from 44 Hemiptera species, one of 174 morphological characters with 12.15% of missing data; one of 3731 molecular characters (bp) with 47.96% of missing data; and one combined subset  of 3905 characters with 46.36% of missing data. The rooted tree topology with seven calibration nodes Fig. a was fixribution , was use2/2} = 0.001. The variance of the gamma was assigned an exponential hyperprior with a mean of 0.027. Analyses were performed with either linked or unlinked relaxed clock models ) or unlink Igrvar = ). Four MCMC runs were performed, each consisting of 5 × 106 iterations, sampling every 200, with the burn-in set to 10%, resulting in a total of 9 × 104 samples from the four runs.Analyses were performed on three subsets from 56 Hymenoptera species; one of 353 morphological characters with 19.97% of missing data; one of 5096 molecular characters (bp) with 25.65% of missing data; and one combined subset  of 5449 characters with 25.28% of missing data. The rooted tree topology with seven calibration nodes ) or unlink Igrvar = ). Six MCMC runs were performed, each consisting of 1 × 107 iterations, sampling every 200, with the burn-in set to 10%, resulting in a total of 2.7 × 105 samples from all six runs.Analyses were performed on three subsets from 18 Spermatophyta species; one of 121 morphological characters with 27.5% of missing data; one of 19,870 molecular characters (bp) with 14.11% of missing data; and one combined subset  of 5449 characters with 14.18% of missing data. The rooted tree topology with seven calibration nodes  for (A) Hemiptera, (B) Hymenoptera, and (C) Spermatophyta. The slope, correlation coefficient (r) and p-values are shown. The black dashed line represents the best-fit linear regression through the origin. The solid grey line represents equality between estimates. Figure S2. Calibration densities (dark grey bands), 95% HPD-CIs in the time prior (light grey bands), and posterior (colored lines) for 47 nodes in the Hemiptera timetrees under calibration strategies (A) Cr-green, (B) C1-red, and (C) C2-purple. Figure S3. Calibration densities (dark grey bands), 95% HPD-CIs in the time prior (light grey bands), and posterior (colored lines) for 55 nodes in the Hymenoptera timetrees under calibration strategies (A) Cr-green, (B) C1-red, and (C) C2-purple. Figure S4. Calibration densities (dark grey bands), 95% HPD-CIs in the time prior (light grey bands), and posterior (colored lines) for 17 nodes in the Spermatophyta timetrees under calibration strategies (A) Cr-green, (B) C1-red, and (C) C2-purple. Figure S5. The posterior mean times (empty black dots) and 95% HPD-CIs under calibration strategies Cr (green lines), C1 (red lines), and C2 (purple lines) for the molecular subsets are plotted against the combined from Hemiptera, Hymenoptera, and Spermatophyta datasets using linked clock models.


These effects were countered by inhibition of midbrain dopamine neurons, and by activation of PV interneurons in pre-limbic cortex and ventral subiculum of the hippocampus. Sub-chronic ketamine reduced PV expression in these cortical and hippocampal regions. Pharmacological intervention with SEP-363856, a novel psychotropic agent with agonism at trace amine receptor 1 (TAAR1) and 5-HT1A receptors but no appreciable action at dopamine D2 receptors, significantly reduced the ketamine-induced increase in dopamine synthesis capacity. These results show that sub-chronic ketamine treatment in mice mimics the dopaminergic alterations in patients with psychosis, that this requires activation of midbrain dopamine neurons, and can be ameliorated by activating PV interneurons and by a TAAR1/5-HT1A agonist. This identifies novel therapeutic approaches for targeting presynaptic dopamine dysfunction in patients with schizophrenia and effects of ketamine relevant to its therapeutic use for  treating major depression.Patients with schizophrenia show increased striatal dopamine synthesis capacity in imaging studies. The mechanism underlying this is unclear but may be due to N-methyl-D-aspartate receptor (NMDAR) hypofunction and parvalbumin (PV) neuronal dysfunction leading to disinhibition of mesostriatal dopamine neurons. Here, we develop a translational mouse model of the dopamine pathophysiology seen in schizophrenia and test approaches to reverse the dopamine changes. Mice were treated with sub-chronic ketamine (30 mg/kg) or saline and then received in vivo positron emission tomography of striatal dopamine synthesis capacity, analogous to measures used in patients. Locomotor activity was measured using the open-field test. In vivo cell-type-specific chemogenetic approaches and pharmacological interventions were used to manipulate neuronal excitability. Immunohistochemistry and RNA sequencing were used to investigate molecular mechanisms. Sub-chronic ketamine increased striatal dopamine synthesis capacity (Cohen’s  This improves brain uptake [18F]-FDOPA by reducing peripheral metabolism of the radiotracer -FDOPA injection. Following cannulation, mice were transferred to the bore of an Inveon µPET/CT scanner . Mice underwent a 20 min CT scan for attenuation correction, and then received a bolus injection of ~4.5 MBq [18F]-FDOPA via the external jugular vein cannula at the start of the 120 min dynamic PET scan.One hour prior to scanning, mice were anaesthetised with isoflurane and underwent external jugular vein cannulation. During scanning, the respiration rate was monitored using the BioVet physiological monitoring software system  and body temperature was maintained at 37 °C. Mice received 40 mg/kg (i.p) entacapone , a catechol-O-methyl-transferase inhibitor, and 10 mg/kg (i.p) benserazide hydrochloride , an aromatic amino acid decarboxylase inhibitor, at 45 and 30 min before the -FDOPA PET imaging was performed. Sub-chronic ketamine treatment significantly increased striatal dopamine synthesis capacity compared to controls, with an effect size of d = 2.5   or saline for 5 consecutive days. Two days after the last ketamine or saline injection in vivo 8F-FDOPA hM4Di-mCherry fusion protein (AAV1-DIO-hM4Di-mCherry) into the VTA and the substantia nigra pars compacta (SNc) of DAT::Cre mice. Cre-dependent expression of hM4Di-mCherry showed ~98% specificity for dopamine neurons, and CNO-treatment silenced dopamine neuron firing in slice electrophysiology recordings, consistent with our previous findings  relative to saline controls  L-DOPA was the significant upstream regulator of the differentially expressed genes in ketamine vs. saline conditions . Collectively, these data support the hypothesis that sub-chronic ketamine increases dopamine synthesis capacity via a pathway that involves the inhibition of PV interneuron function. Furthermore, RNA Seq data on PLc tissue did not reveal changes in the expression of cholecystokinin (p > 0.05), nitric oxide synthase 1 (p > 0.05) and somatostatin (p > 0.05), which are proteins expressed in other GABAergic interneurons. Interestingly, consistent with a previous study, vasoactive intestinal peptide receptor 2 (VIPR2) expression was significantly increased suggesting that other GABAergic interneurons may be affected by sub-chronic ketamine administration.To investigate the molecular mechanisms underlying the effects of sub-chronic ketamine on dopamine synthesis we performed RNA Seq on PLc tissue. We hypothesised that sub-chronic ketamine would result in reduced PV expression, and changes in signalling pathways downstream of the NMDA receptor such as calcium signalling and the activation of brain-derived neurotrophic factor (BDNF) signalling. Consistent with our a priori hypotheses, RNA Seq data on PLc tissue revealed reductions in the expression of PV Fig. . MoreovehM3Dq-mCherry fusion protein were stereotaxically injected into the PLc and vSub of the hippocampus of PV::Cre mice    agonism. TAAR1 is a G-protein-coupled receptor that is expressed throughout monoaminergic brain nuclei including dopamine neurons . TAAR1 aeceptors , counter1A agonist, SEP-856. Our study demonstrates for the first time to our knowledge that an experimental model induces the same dopaminergic phenotype seen in patients with psychosis, and the potential of targeting PV interneurons and a novel TAAR1/5HT1A agonist to reverse the dopaminergic phenotype.Our results demonstrate that sub-chronic ketamine administration leads to elevated striatal dopamine synthesis capacity, and that this requires the activation of midbrain dopamine neurons. The ketamine-induced increase in dopamine synthesis capacity was attenuated by the activation of PV interneurons in the PLc and vSub, as well as by a novel TAAR1/5HTThe majority of studies show that acute ketamine significantly increases striatal dopamine levels –53, and p > 0.05), nitric oxide synthase 1 (p > 0.05) or somatostatin (p > 0.05) in our RNA seq data, suggesting that ketamine does not have major effects on these interneurons in the PLc, although we cannot exclude effects in other brain regions. In contrast, our RNA seq data revealed a significant increase in the expression of VIPR2 in the PLc. This extends a previous finding showing this following acute ketamine administration [Ketamine is a non-competitive NMDAR antagonist that binds with high affinity (Ki = 3.1 µM) to the same binding site as MK801 and PCP –57. PV istration , to indistration . This fiMoreover, there is evidence of direct glutamatergic projections from the PLc and other frontal regions to the substantia nigra/VTA that activate dopamine neurons and increase locomotor behaviour in an NMDAR dependent manner , 78. The2 receptors from PV interneurons induces hyperlocomotion [1 receptors on pyramidal cells in the prefrontal cortex and/or the action of a metabolite of ketamine on α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors might contribute to its long-lasting antidepressant effects [m) are considerably lower than its affinity for the NMDAR, and it is not clear if ketamine exhibits significant dopamine receptor occupancy in vivo at behaviourally relevant doses [Ketamine’s action on receptors other than the NMDAR, could also contribute to the observed effects . Evidenccomotion . In addi effects , 90. Hownt doses , 92. We Ketamine has a short half-life (13 min) in mice  and braiInterestingly, whilst CNO significantly reduced locomotor activity compared with baseline, it had no effect on dopamine synthesis capacity. It should be noted that locomotor activity was measured shortly after CNO administration whilst dopamine synthesis capacity was measured 2 days following CNO administration. Electrophysiology recordings show that dopamine neurons recover quickly upon washout of CNO from solution . Thus, o1A receptor (5-HT1AR), although with lower potency than for TAAR1 (EC50 of 2.3 μM) [1AR antagonist [1AR. Following our translational work, future pharmacology studies will help elucidate the molecular and circuit mechanisms by which SEP-856 attenuates the ketamine-induced increase in striatal dopamine synthesis capacity.A strength of our study is that it utilised a PET imaging approach that parallels the technique used in human studies , 30, 33, 2.3 μM) . Notwithtagonist , suggest1A agonist, reduces sub-chronic ketamine-induced elevation in striatal dopamine synthesis capacity provides a proof-of-concept for pharmacological attenuation of presynaptic dopamine dysfunction. The reduction in PV levels following ketamine in our model was large (Hedge’s g = −2.29), which compares to a moderate-large effect size (Hedge’s g = −0.61) reduction in PV-positive neuron immunoreactivity reported post-mortem in schizophrenia [PET imaging studies have repeatedly shown elevated dopamine synthesis and release capacity in schizophrenia (e.g. , 98 and ophrenia . Thus, oLastly, our data may also have implications for understanding ketamine’s antidepressant actions and its abuse potential. There is some evidence that major depression is associated with blunted dopaminergic function, including reduced levels of dopamine metabolites post-mortem and reduced dopamine synthesis capacity , 104. Mo1A activity.We demonstrate that sub-chronic ketamine leads to an increase in striatal dopamine synthesis capacity in the mouse, resembling the dopaminergic alteration seen in patients with schizophrenia. Our data suggest that ketamine’s effects on dopamine synthesis capacity are mediated by inhibition of PV interneurons in the cortex and vSub as well as activation of midbrain dopamine neurons, and that these alterations can be attenuated by a TAAR1 agonist with 5-HTSupplementary InformationSupplementary Figure 1Supplementary Figure 2Supplementary Figure 3Supplementary Figure 4Supplementary Figure 5Supplementary Figure 6Supplementary Figure 7