We also measured spontaneous activity and learning/memory ability because these deficits could confound the assessment of impulsivity. We found small but significant effects of D5 receptor knockout on home cage activity only at specific times of the day. In addition, an analysis using the q-learning model revealed that D5KO mice displayed lower behavioral adjustment after impulsive actions. However, our results also showed that baseline impulsive actions and the effects of an anti-impulsive drug in D5KO mice were comparable to those in wild-type littermates. Moreover, unlike previous studies that used other D5 receptor-deficient mouse lines, we did not observe reductions in locomotor activity, working memory deficits, or severe learning deficits in our line of D5KO mice. These findings demonstrate that D5 receptors are dispensable for impulse control. Our results also indicate that time series analysis and detailed analysis of the learning process are necessary to clarify the behavioral functions of D5 receptors.Major psychiatric disorders such as attention-deficit/hyperactivity disorder and schizophrenia are often accompanied by elevated impulsivity. However, anti-impulsive drug treatments are still limited. To explore a novel molecular target, we examined the role of dopamine D Clinically available anti-impulsive drugs vary from country to country, but at present, the major ones include amphetamine, methylphenidate, and lisdexamfetamine as psychostimulants, and atomoxetine, guanfacine, and clonidine as adrenaline-related drugs. However, psychostimulants pose a risk of abuse and dependence, and adrenaline-related drugs are often difficult to recommend due to their cardiovascular and autonomic side effects. For example, atomoxetine, a noradrenaline reuptake inhibitor, might not be appropriate in some patients since it can exacerbate hypertension, which is often comorbid with ADHD, bipolar disorder, and borderline personality disorder4. Although guanfacine and clonidine are hypotensive agents, their sedative side effects could interfere with work or study. Therefore, further development of novel anti-impulsive drugs is required.Various psychiatric disorders, such as attention-deficit/hyperactivity disorder (ADHD), schizophrenia, substance use disorder, bipolar disorder, and borderline personality disorder, have been associated with increased impulsivity5 receptors play an important role in the control of impulsivity. We have previously demonstrated that dopamine D1-like receptors in the ventral part of the medial prefrontal cortex play a critical role in the anti-impulsive effects of milnacipran, duloxetine, and atomoxetine6. There are two types of dopamine D1-like receptors: D1 receptors and D5 receptors. Dopamine D1 receptors are densely expressed in the nucleus accumbens, where impulsivity is enhanced by increased extracellular dopamine levels8, while dopamine D5 receptors are sparsely expressed in the region9. In comparison, in the medial prefrontal cortex, where impulsivity is inhibited by increased extracellular dopamine levels11, both dopamine D1 and dopamine D5 receptors are expressed9. Furthermore, dopamine D5 receptors have a tenfold higher affinity for dopamine than dopamine D1 receptors12. Thus, we hypothesized that the anti-impulsive effects of the above drugs might be exerted via stimulation of dopamine D5 receptors.To this end, we examined whether dopamine D5 receptor agonist might resolve problems encountered with the current stable of anti-impulsive drugs by enabling us to selectively manipulate the medial prefrontal cortex without affecting the nucleus accumbens. To our knowledge, however, there are so far no drugs that clearly distinguish between dopamine D1 and D5 receptors. Given that psychostimulants primarily facilitate addiction through the modulation of the nucleus accumbens13, a selective dopamine D5 receptor agonist might not induce this process, unlike psychostimulant-based anti-impulsive drugs. Furthermore, a selective agonist for dopamine D5 receptors would not likely exacerbate hypertension since dopamine D5 receptor knockout (D5KO) mice are hypertensive14. Spontaneous motor activity in D5KO mice is generally normal or reduced, implying that a selective dopamine D5 receptor agonist will not induce sedation. However, in the absence of selective D5 receptor agonists, examining dopamine D5KO mice is a reasonable way to determine whether D5 receptors could be a promising target for anti-impulsive drugs.The development of a selective dopamine D15 instead of traditional D5KO mice14 because of three reasons. First, the traditional D5KO mice could express truncated transcripts that might alter the expression of related genes17, while the alternative line of D5KO mice would not express them because the entire dopamine D5 receptor gene region, including the promoter region, is removed. Second, previous studies have shown that different lines of transgenic mice or different background strains of transgenic mice could alter baseline behavioral phenotype19. To clarify the role of a molecule in brain functions, we are better off testing not only a specific line or background strain but also another line or background strain. Third, some studies have reported lower spontaneous motor activity and deficits of learning and working memory in traditional D5KO mice22. These phenotypes make it difficult for researchers to assess impulsivity because most tasks evaluating impulsivity assume a certain level of spontaneous activity and learning/memory ability. We speculated that these phenotypes are due to the above reasons, but not due to the lack of D5 receptors.In the present study, we used an alternative line of D5KO mice5 receptors were not transcribed as expected and whether compensatory changes in dopamine D1 receptors did not occur, (2) measured locomotor activity in two different environments: a novel environment and a familiar environment, (3) conducted a Y-maze test to assess working memory, and (4) employed the 3-choice serial reaction time task (3-CSRTT)23 to assess learning ability and impulsivity. To evaluate possible learning deficits or bias, we modeled the learning process within the 3-CSRTT using a q-learning model.In this study, using an alternative line of D5KO mice, we conducted quantitative PCR to confirm that dopamine D5 receptor gene is not transcribed and that a compensatory overexpression of D1 receptors does not occur, we conducted quantitative PCR tests. As expected, the Drd5 gene expression levels were below the detection limit in the D5KO mice , medial prefrontal cortex (mPFC) , and striatum  . There was a significant interaction between time and genotype . Other main effects and interactions were not detected   .Simple main effects analyses for each time point revealed that D5KO mice were significantly more active than wildtype littermates between 7:00 and 9:00 (36) Fig. a. D5KO m11) Fig. a. No difF3.45, 169 = 119.1, p < 0.0001, with Geisser-Greenhouse correction). However, we found no significant main effects or interactions in other factors . However, we found no significant main effects or interactions in other factors . However, we did not find other main effects or any interactions . A multiple comparison with Bonferroni's correction revealed that the 0.3 mg/kg, 1.0 mg/kg, and 3.0 mg/kg dose of duloxetine significantly decreased the percentage of premature responses compared to vehicle administration. However, we did not find any other main effects or interactions , correct response latency , and reward latency , but not on omission . However, we did not find any other main effects or interactions  but lower locomotor activity at the end of the dark period (15:00–17:00) in their home cage, a familiar environment  was almost the same between D5 receptor KO mice and wild type littermates. Based on these results, we suggest that dopamine D5 receptors do not play an important role in impulsivity. It should be noted that other parameters were also affected by duloxetine. Accuracy, a measure of attentional function, was decreased when 3 mg/kg duloxetine was injected in both genotype and both sexes. In addition, 1 mg/kg and 3 mg/kg duloxetine administration prolonged the mean correct latency and reward latency in both genotype and both sexes. These measures represent motivation and motor function. The percentage of omissions, which represents attentional function and motivation, was not affected by duloxetine. Therefore, the prolonged latencies would reflect a decrease in motor function, indicating that higher doses of duloxetine would be inappropriate in the evaluation of anti-impulsive effects. However, we still conclude that dopamine D5 receptors have a negligible role in impulse control because these side effects were equally observed in either genotype and the anti-impulsive effects of a low or moderate dose of duloxetine did not disappear in D5KO mice.We replicated the dose-dependent anti-impulsive effects of duloxetine previously found in male rats6 indicating that drugs suppress impulsivity by stimulating dopamine D1-like receptors in the mPFC? There are at least two possibilities. First, dopamine D1 receptors may be more involved in impulsivity suppression, since the involvement of D5 receptors has been ruled out. However, since dopamine D1 receptors are also densely expressed in the nucleus accumbens, where impulsivity is enhanced by their stimulation8, they will not be an appropriate molecular target for anti-impulsive drugs. The second possibility is that previous studies have largely examined nonselective effects of dopamine D1-like receptor antagonists, where SCH23390 is frequently used, although its selectivity for D1-like receptors is not high enough to completely exclude effects on other receptors and channels28. In either case, the development of a selective dopamine D5 receptor agonist would not resolve the current problems encountered in current anti-impulsive drugs. Interestingly, a recent study showed that striatal dopamine D5 receptors are involved in the pathophysiology of levodopa-induced dyskinesia29. Therefore, dopamine D5 receptors might play a role in pathological but not physiological situations.In light of these results, how should we interpret previous studies15 or wildtype littermates (8–28 weeks old) were used. The B6.129-Drd5 < tm1Mok > mouse strain (RBRC01084) was provided by RIKEN BRC through the National Bio-Resource Project of the MEXT, Japan. In the D5KO mice used in this study, the entire dopamine D5 receptor gene region was removed and replaced with a neomycin resistance gene. These mice were backcrossed to the C57BL/6N strain for more than 13 generations. C57BL/6N mice were supplied from Nippon SLC Co. Ltd . Animals were group-housed before starting behavioral experiments at 25 °C ± 2 °C and relative humidity of 40%–50%. Food and water were provided ad libitum except for the mice undergoing the 3-choice serial reaction time task. The lights of the animal rooms were turned on from 19:00 to 07:00. All tests were performed during the dark period except for the home cage activity test. All procedures followed the guidelines for the Care and Use of Laboratory Animals from the Animal Research Committee of Hokkaido University and were approved by the Animal Research Committee of Hokkaido University . We conducted all experiments in compliance with the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines. Mice received one or several behavioral tests as summarized in Table Adult male and female D5KO miceDuloxetine hydrochloride  was dissolved in saline and administered intraperitoneally at a volume of 10 mL/kg. Doses reported here are based on the molecular weight of the salt.Drd1 and Drd5 were quantified by reverse-transcription quantitative PCR (RT-qPCR) using the respective cDNA fragment as a standard and were normalized to mouse Gapdh mRNA levels. Briefly, 5 μg of total RNA were reverse transcribed using ReverTra Ace® qPCR RT Master Mix with gDNA Remover . Real-time quantitative PCR was performed on a fluorescence thermal cycler Step One™ Real-time PCR System  by using TaqMan® Fast Advanced Master Mix + probe set (Thermo Fisher Scientific). The PCR conditions were 50 °C for 2 min, 95℃ for 20 s, followed by 40 cycles of 95 °C for 1 s, and 60 °C for 20 s. Primer sequences for Drd1  and Drd5  were chosen based on a previous study30. Gapdh was used as a control . The results were analyzed using the StepOne Software ver.2.3 (Thermo Fisher Scientific).Mice were deeply anesthetized with urethane (2 g/kg) intraperitoneally and sacrificed by decapitation. Brain tissue including hippocampus, medial prefrontal cortex (mPFC), and striatum (Str) were dissected on ice. Each sample was weighed, placed in a tube, immediately frozen in liquid nitrogen and kept frozen at − 80 °C until analysis. Total RNA was extracted from tissue using NucleoSpin RNA reagent . The mRNA expression levels of 31. The sensor detected a change in the intensity of infrared energy radiated from an animal . The amount of movement was recorded every minute with computer software Analysis98 .Animals were individually housed in a Plexiglas cage (18 cm × 26 cm × 12 cm) for at least 1 week before this test. Spontaneous movements were measured by a passive infrared sensor that detected changes in animal thermal radiation due to movementA mouse was placed in an acrylic box (45 × 45 × 45 cm) for 70 min. The inside of the box was covered by rough-surfaced polypropylene sheets. The light intensity in the box was adjusted to 20 lx. The movement of each mouse was monitored through a CCD camera and was tracked using a software package . We considered the total distance traveled and the number of total crossings (defined by crossings of the lines made by the division of the chamber into 7.5 cm × 7.5 cm squares) as measures of locomotor activity. Moreover, we considered the percentage of time spent in the central area (15 cm × 15 cm square) as a measure of anxiety-like behavior.32. Briefly, a mouse was placed in an apparatus consisting of three arms  for 8 min. The light intensity in the apparatus was adjusted to 20 lx. The number of entries into an arm was as a measure of locomotor activity. The percentage of spontaneous alternation was used as a measure of working memory.The details of the Y maze test have been described in our previous studies33. We purchased aluminum operant chambers from Med Associates Inc. . The main sequence of the 3-CSRTT is briefly described below. When a mouse entered the food magazine, a 5-s inter-trial interval (ITI) began. After the ITI, one of the three hole lights was turned on (stimulus duration (SD) in experimental sessions: 1 s (SD1)) with a pseudo-random order. Nose poking before turning on a hole light was recorded as a “premature response,” which is a measure of impulsive action. Nose poking into the lit hole was recorded as a correct response and resulted in delivery of a palatable food pellet . Nose poking into an unlit hole was recorded as an incorrect response. When the animal did not nose poke into any holes, we recorded it as an omission. A 5-s time-out period started after premature responses, incorrect responses, and omissions. We also recorded the premature response latency (the time between the ITI onset and a nose poke into a unlit hole), the correct response latency (the time between stimulus onset and a nose poke into the lit hole), and reward latency (the time between reward delivery and a nose poke into the food magazine).Mice were trained to perform the 3-CSRTT as described previouslySession data in the 3-CSRTT were used for two purposes Fig. a. TrainiWe focused on ITI with 5-s (ITI5) training session data from the first session after the pre-training process to the tenth session Fig. a. In theThe q-learning model in this study attempts to capture the mechanisms of premature behavior, which represents an impulsive action. To model the memory state in the mouse brain, we assumed two types of time-dependent probability distribution functions (p.d.f.) to represent “experience” and “non-reward” memory states. The experience distribution represents the memory of all trials that mice have completed, and a non-reward distribution represents the memory of trials with premature results. Combining these two mechanisms, mice decide when to nose poke into a hole. Parameters of both distributions are updated based on the q-learning process. Since incorrect results were thought to be caused by different mechanisms and the number of incorrect results was small, incorrect results were treated the same as correct ones in this analysis.We firstly define random variables for “experience” and “non-reward” distributions, which follow a normal distribution. With q-learning theory, these two random variables were updated based on a result type and elapsed time from a start timing of the previous trial. Rates of updating each parameter are controlled by learning rates of the experience distribution, Experience and non-reward distributions are used to calculate the probability of confidence with a softmax function. An inverse temperature, Detailed explanations of model derivation, parameter specification, estimation procedures, and simulation procedures are in 5 receptors play an important role in the enhancement of impulse control, we administered duloxetine  intraperitoneally to D5KO mice and their wildtype littermates 30 min before the 3-choice serial reaction time task session. We did not use higher doses of duloxetine (> 3.0 mg/kg) because higher doses induced sedation in our preliminary study. Drug treatments were carried out using a Latin square design and were administered with at least a 2-day interval between injections. During the testing phase of this study, the duration of the ITI was prolonged to 9 s (ITI9) because the mice made only a few (< 10) premature responses during the task using a 5-s interval (ITI5). Each testing session with ITI9 was conducted for 70 min or until 100 trials were completed, whichever came first, while sessions with ITI5 were conducted for 60 min or until 100 trials were completed, whichever came first. When the mice experienced 10 ITI5 sessions, they were habituated to ITI9 sessions 6 times with 2-day intervals.To determine whether dopamine DPercentage of premature responses: [premature responses/(premature + correct + incorrect responses)] × 100, a measure of impulsive actionAccuracy (percentage of correct responses): [correct responses/(correct + incorrect responses)] × 100, a measure of attentional functionPercentage of omissions [ × 100], a measure of attentional function and motivation for the taskCorrect response latency (s), a measure of attentional function, motivation for the task, and motor functionReward latency (s), a measure of motivation for reward and motor functionThe following behavioral measures in the 3-CSRTT were analyzed:p < 0.05. SPSS (version 23.0) and GraphPad Prism (version 8.4.2) were used for statistical analyses.For the measurement of locomotor activity in the home cage and in the open field test, we used a three-factor mixed analysis of variance (ANOVA) with time as a within-subjects factor and genotype and sex as between-subjects factors. For the effect of genotype in the Y maze test, we used a two-factor mixed ANOVA with genotype and sex as between-subjects factors. For the 3-CSRTT, each measure was analyzed separately by a three-factor mixed ANOVA with drug as a within-subjects factor and genotype and sex as between-subjects factors except for the assessment of learning ability. If Mauchly's sphericity test was significant, a Greenhouse–Geisser correction was used. Multiple comparisons with Bonferroni's correction were also conducted in cases where ANOVA revealed a significant main effect. All results except for the assessment of learning ability are presented as mean ± standard error of the mean (S.E.M.). The results were considered statistically significant when Supplementary Information.


Perfusion of dopamine (100 μM) in the extracellular solution significantly reduced peak transient Na+ currents by approximately 20% of control. A decrease in Na+ current amplitude was also seen with extracellular application of the D2 dopamine receptor agonist quinpirole, whereas the D2 receptor antagonist eticlopride largely abolished the response to dopamine. Inclusion of the phosphatase inhibitor okadaic acid in the patch electrode solution occluded the response to dopamine. The reduction in calyx sodium current in response to dopamine suggests efferent signaling through D2 dopaminergic receptors may occur via common mechanisms to decrease excitability in inner ear afferents.Inner ear hair cells form synapses with afferent terminals and afferent neurons carry signals as action potentials to the central nervous system. Efferent neurons have their origins in the brainstem and some make synaptic contact with afferent dendrites beneath hair cells. Several neurotransmitters have been identified that may be released from efferent terminals to modulate afferent activity. Dopamine is a candidate efferent neurotransmitter in both the vestibular and auditory systems. Within the cochlea, activation of dopamine receptors may reduce excitotoxicity at the inner hair cell synapse via a direct effect of dopamine on afferent terminals. Here we investigated the effect of dopamine on sodium currents in acutely dissociated vestibular afferent calyces to determine if dopaminergic signaling could also modulate vestibular responses. Calyx terminals were isolated along with their accompanying type I hair cells from the cristae of gerbils (P15-33) and whole cell patch clamp recordings performed. Large transient sodium currents were present in all isolated calyces; compared to data from crista slices, resurgent Na Intraperitoneal injections of ketamine (70 mg/kg) and xylazine (3 mg/kg) in sterile saline were used to induce anesthesia with supplemental doses given when necessary. Following decapitation the cristae of the semicircular canals were dissected out and immersed in Leibovitz’s L-15 medium . Procedures were in accordance with NIH guidelines and protocols approved by the University of Colorado’s Institutional Animal Care and Use Committee.Mongolian gerbils  and differential interference contrast (DIC) optics on an Olympus microscope (BX51). Isolated cell pairs of single type I hair cells enveloped by calyx terminals or calyces within slices were selected for recordings. Electrodes were pulled from electrode glass  on a multistep horizontal puller  and electrode tips were polished by heat using a Narishige MF 830 microforge . SYLGARD 18  was applied close to the tip of each electrode to reduce stray capacitance.+ currents (+ currents the electrode solution contained (in mM): KF (115), KCl (10), NaCl (2), HEPES (10), D-glucose (3), MgCl2 (2), EGTA (10), pH 7.4 adjusted with KOH (27 mM). For all other experiments the electrode solution contained (in mM): CsF (120), CsCl (10), NaCl (2), HEPES (10), D-glucose (3), MgCl2 (2), EGTA (10), pH 7.4 adjusted with CsOH (24 mM). To further isolate Na+ currents from other conductances, recordings for investigating resurgent currents (INaR) were obtained in an extracellular solution containing in: (mM): NaCl (120), CsCl (5.4), MgCl2 (2.5), tetraethylammonium Cl (30), CaCl2 (1.3), glucose (10), HEPES (5). For all other experiments the external solution was Leibovitz’s L-15 medium.Calyx terminals express several different types of voltage-gated conductances including Nacurrents . For expNaT) and resurgent (INaR) Na+ currents. Data were acquired using pCLAMP 10 software with a patch amplifier  connected to a PC via an AD converter . Signals were low-pass filtered online at 5 or 10 kHz and sampled at 20–50 kHz. Correction for liquid junction potentials was made in the analysis.Electrode resistance was 2.3–5 MΩ prior to seal formation on calyces. Recordings were made at room temperature (21 – 24°C) with voltage protocols designed to probe for transient  at a flow rate of 0.5-1.0 ml/min. Drugs were typically perfused for a minimum of 4-9 min before measurements of drug effects on Na+ currents were taken. The length of the wash (return to control solution) was between 6 and 10 min.Dopamine was dissolved in deionized water or L-15 on each experimental day to make a 10 mM stock solution which was then added to L-15 for a final concentration of 100 μM. In experiments with Cst-tests.Clampfit 10 (Molecular Devices) and Sigmaplot 11  were used to analyze voltage clamp data. Mean values ± SD are reported and values were compared with paired + channels are a target for neuromodulation in many cell types. In cochlear afferent neurons transient Na+ currents are decreased by the application of dopamine  was measured during perfusion of 100 μM dopamine in a P21 isolated calyx  and decreased markedly in the presence of dopamine shortly after the perfusion onset. K+ current amplitude was measured at the end of 40 ms voltage steps to +20 mV and remained relatively unchanged throughout the course of dopamine application. In a group of calyces (ages P21-27) mean IK was 2,161 ± 1,501 pA in control conditions, which was not significantly different from the mean value of 1,976 ± 1,454 pA in dopamine . These experiments suggest that the effect of dopamine is specific to Na+ currents in calyx terminals.To test if dopamine affected voltage-dependent outward Ked calyx . The stacordings . The pea+ currents were not significantly affected by dopamine, we performed additional experiments using Cs+ in the patch electrode solution to inhibit voltage-dependent K+ currents. In the experiments with internal Cs+ outward currents were greatly reduced . The effect of dopamine was tested in four calyces and perfusion with 100 μM dopamine reversibly reduced peak INaT at the test step of –40 mV as shown for one cell in + current amplitude following dopamine exposure and subsequent washout is shown for cells with either K+ or Cs+ as the major cation in the patch electrode solution in + currents were measured at –40 mV and averaged –2,151 ± 1,685 pA in control; following dopamine perfusion mean peak current was reduced to –1,732 ± 1,718 pA (n = 9 cells). The decrease in amplitude was significant and a partial or complete washout was observed in all but two cells on return to normal extracellular solution  and five receptor subtypes (D1-D5) have been described . Based o% n = 6, . Althougdopamine . Eticlop+ current through a change in the phosphorylation state of Na+ channels and inhibition of phosphatase might be expected to produce an inhibition of Na+ currents analogous to activation of dopamine receptors. We therefore tested the effect of okadaic acid, a known inhibitor of phosphatase 1 and 2A, by perfusing cells with extracellular 100 nM okadaic acid. However, we saw no consistent change in amplitude of INa in 3 cells tested with external perfusion of okadaic acid. Next we studied the effect of including 100 nM okadaic acid in the patch electrode solution followed by extracellular dopamine perfusion  were abolished by tetrodotoxin (TTX) (NaR) and persistent currents (INaP), which occurred frequently, but contributed much smaller components of the TTX-sensitive Na+ current than INaT  was observed in greater than half of calyces studied in crista slices at ages P13 and older , INaR was only detected in 2 out of 15 isolated calyces  . We subshan INaT . Dopamin neurons , but we  channel . Calycesnel  in auditory and vestibular afferents, where it acts to shift the activation to more positive potentials and increase the degree of current available around the resting potential  that mediate the upward phase of the action potential. In addition to INaT, resurgent (INaR) and persistent Na+ currents also manifest in certain cell types  and several of these are expressed in vestibular ganglion neurons (+ currents (NaR and INaT were detected together in the majority of mature calyx terminals in crista slices (NaT was present in all isolated calyces studied here, but INaR was rarely observed. In both vestibular calyces and spiral ganglion neurons INaR expression increased with postnatal development suggesting a role for this current in afferent firing in adult inner ear epithelia (NaR expression (NaT, Ih and a variety of K+ currents (NaR generation. Spike generation likely occurs in afferent terminals at the initial unmyelinated segment close to hair cells (+ channel subtypes remains to be determined.Voltage-gated Nall types . Dopamincurrents . Large aodotoxin , 2020. N neurons . Nav1.6 currents . A specia slices . In contpithelia . Navβ4 spression , were depression  and immupression . Althougcurrents , these sir cells , but the+ channels.Vestibular hair cells and inner hair cells make synapses with afferent terminals to convey sound and balance information to the brain as action potentials, but the auditory and vestibular systems also receive extensive efferent input from the brain. Outer hair cells and type II hair cells are contacted directly by efferent terminals, but efferent fibers also terminate on unmyelinated vestibular calyx terminals and auditory afferents postsynaptic to inner hair cells. The strategic placement of efferent synapses in the initial segment zone of the axon and calyx outer face could allow modulation of afferent firing close to the site of action potential initiation through receptor-mediated influence on current components mediated through voltage-gated ion Na2+ influx through α9-containing nicotinic receptors activates SK channels producing a hyperpolarization of type II vestibular hair cells and a decrease in membrane resistance and hair cell sensitivity (+ currents in calyx afferents of turtle and rat crista (B receptor-mediated inhibition of K+ channels (+ currents in vestibular hair cells and afferents, but direct effects of potential neuromodulators on Na+ channels linked to firing have not been reported until now. Our data suggest dopamine release could impact firing in vestibular afferents through activation of D2 receptors and associated intracellular signaling mechanisms resulting in Na+ channel inhibition within calyx terminals. As described in cochlear afferents, dopamine-mediated reduction of INaT would result in a decrease in firing in vestibular calyx-bearing neurons.Gerbil cristae receive substantial efferent innervation with distinct efferent groups projecting to ipsilateral and contralateral cristae . Althougsitivity , 2020. Asitivity . Converst crista . Other tt crista . In rat channels . TherefoDopaminergic efferents are present in the cochlea and may also be present in vestibular epithelia. Tyrosine hydroxylase is required for the synthesis of dopamine and + current inhibition and the involvement of other dopamine receptor subtypes remains to be elucidated. Disruption to dopaminergic efferent pathways could be associated with auditory and vestibular dysfunction.Dopamine may operate as a neuroactive substance at the hair cell/afferent synapse in mammalian auditory and vestibular systems. Through a reduction in sodium channel activity via changes in phosphorylation, dopamine released from efferents may reduce action potential firing in both auditory and vestibular afferents. In the cochlea, dopamine appears to exert its inhibitory effect on afferent firing via two distinct dopaminergic signaling pathways. Our data support a role for D2 receptors in NaThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by University of Colorado Denver Institutional Animal Care and Use Committee.FM and KR designed the experiments, performed the research experiments, analyzed the data, and prepared the figures. KR wrote the first draft of the manuscript. Both authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.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.


Vibrio vulnificus FJ03-X2 strain with high pathogenicity and strong virulence were established in zebrafish (Danio rerio) fibroblast cell line (ZF4 cells) and zebrafish. Zebrafish tnfb (Zetnf-b) gene was cloned and expressed by Escherichia coli BL21 (DE3), and Zetnf-b polyclonal antibody was prepared. Pre-injection of Zetnf-b polyclonal antibody and AG-126 before infecting with V. vulnificus could increase the survival rate of zebrafish by 36.6 and 46.7%, respectively. Pre-injection of Zetnf-b polyclonal antibody could effectively decrease the mortality of zebrafish infected by V. vulnificus. Thus, TNF polyclonal antibody therapy could be considered as an effective strategy to control V. vulnificus in fish.Tumor necrosis factor (TNF) plays an important role in an inflammatory cytokine storm. Over-secretion of TNF by the host in response to infection aggravates the disease. TNF expression level is positively correlated with the mortality caused by some bacterial infections. Therefore, using TNF antibody may alleviate the inflammation to resist bacterial infections. The function of fish TNF-b antibody in bacterial infection is still unclear. In this study, infection models of  Danio rerio) (Larimichthys crocea)  (Epinephelus coioides) (E. coioides) (Oncorhynchus mykiss) (Oryzias latipes) (Mycobacterium marinum (Aeromonas hydrophila and Edwardasiella tarda) and viral  infections (Fish tumor necrosis factor (TNF) family members are cytokines with multiple effects, which play vital roles in cell growth, differentiation, proliferation and immunity, apoptosis, macrophage activity and autoimmune diseases . Fish poo rerio) , large y crocea) , Japanesivaceus)  and grouoioides) . In addioioides) , rainbow mykiss) , zebrafilatipes) . There a marinum . It playfections . Zebrafifections –17. HoweVibrio vulnificus is the main pathogen of important economic fish, such as European eel (Anguilla Anguilla), large yellow croaker and grouper. As one of the most serious aquatic pathogens, it can cause hemorrhagic sepsis and the death of aquatic animals. Also, it can infect people through wounds or foodborne pathogenic infections . The result presented that the polyclonal antibody could significantly improve the survival rate of the immunized group after being infected with V. alginolyticus  were developed in the form of TNFα antibodies to treat many diseases, including osteoporosis, psoriasis, arthritis and Crohn disease. The TNF superfamily is considered as an active target for human drug development  in immune-related tissues of zebrafish in response to the intraperitoneal infection of V. vulnificus and that in the infected ZF4 cells at different time points. V. vulnificus infection models of ZF4 cells and zebrafish were developed to study the role of Zetnf-b during infection. Finally, the Zetnf-b gene was cloned, and the effect of its polyclonal antibody on resistance to V. vulnificus infection was determined in zebrafish. The results provide a perspective of TNF antibody therapy for the prevention and treatment of V. vulnificus in fish.This study aimed to investigate the function of zebrafish TNF-b polyclonal antibody in fighting against All zebrafish were anesthetized with tricaine methane sulfonate , and surgeries were performed. All animal experiments were conducted in accordance with the guidelines and regulations of the Institutional Animal Care and Use Ethics Committee of Fujian Academy of Agricultural Sciences (Permit Number: BI-AEC-2021055006).2 cell incubator. Sprague Dawley (SD) rats of Specific-pathogen-free (SPF) grade were purchased from the Laboratory Animal Center of Fujian Academy of Traditional Chinese Medicine, Fuzhou, China. The fish bacterial pathogen, V. vulnificus FJ03-X2 strain, which possesses strong virulence and high pathogenicity, was isolated from the sick European eel (Anguilla anguilla) and maintained in our laboratory. Its half-lethal dose (LD50) for elvers (15–30 g/tail) at a water temperature of 28°C was 2.3–7.1 × 103 colony forming unit (CFU)/tail. Tryptic soy broth (TSB) medium  was used to cultivate V. vulnificus FJ03-X2 strain.The zebrafish (AB strain) and ZF4 (ATCC CRL-2050) were purchased from the China Zebrafish Resource Center . The zebrafish with an average weight of 0.2 g were raised in a flow through pure water aquaculture system. No disease was observed during breeding. The ZF4 cells were maintained at 28°C in DME/F12 medium  with 10% fetal bovine serum  containing 10 U/ml penicillin and 10 μg/ml streptomycin in a 5% COZetnf-b-F and Zetnf-b-R primers (tnf-b gene (Accession no.: NM_001024447). Total RNA from liver and kidney of 3 healthy zebrafish was extracted using Trizol Reagent solution  according to the manufacturer's protocol. Zetnf-b was amplified by reverse transcription-polymerase chain reaction (RT-PCR) and cloned into pMD18-T vector . The positive clone plasmid was subjected to double enzyme digestion and sub-cloned into the expression vector pET-32a  to get recombinant expression plasmid pET32a–Zetnfb. The recombinant proteins of Zetnfb (rZetnfb) were obtained by Escherichia coli BL21 (DE3) containing pET32a–Zetnfb and confirmed by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). primers  containiThe rZetnfb was purified by cutting the target band from the SDS-PAGE gel and quantified. T 10 basic ULTRA-TURRAX ®  was used to mix the target protein and adjuvant in a volume ratio of 1:1. Complete Freund's adjuvant  was mixed with rZetnfb for the first immunization, and incomplete Freund's adjuvant  was mixed with rZetnfb for the last immunization. Five SD rats of SPF grade were inoculated with the mixed substance through multiple subcutaneous injections to prepare polyclonal antibody. A part of SD rats collected before immunization was used as a negative control in the experiments. All the serum samples of SD rats were collected after the immunization. The titer of the serum polyclonal antibody was determined by enzyme-linked immunosorbent assay (ELISA) according to the method described previously . The spe2 and 95% air at 28°C. V. vulnificus FJ03-X2 cells in the logarithmic growth phase were diluted with (TSB) medium at a density of 1.0 × 106 CFU/ml. One group of cells without V. vulnificus inoculation was used as a negative control. The other three groups were inoculated with V. vulnificus for 12 h at three multiplicity of infection (MOI) of 1:1, 1:10 and 1:100, respectively. Optical density (OD) was measured at 490 nm (OD490) with a full-wavelength microplate reader . The percent cytotoxicity in each plate was calculated through the level of lactate dehydrogenase (LDH) using the standard procedure from CytoTox96 non-radioactive cytotoxicity assay . A low control (LC) and a high control (HC) were set up, and the percentage of cytotoxicity was calculated according to following equation:The ZF4 cells were plated in 6-well culture plates and divided into 4 groups with 3 wells per group. These were incubated overnight in Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DME/F-12) in an incubator with 5% CO5 CFU/g, 5.61 × 104 CFU/g and 1.4 × 104 CFU/g V. vulnificus, respectively. The negative control group was injected with 10 μl of sterilized phosphate-buffered saline (PBS). The zebrafish status and survival rate were observed and recorded once every 3 h until 24 h. The LD50 of V. vulnificus infection in zebrafish was determined according to the recorded survival rate and the concentration of bacteria for challenging zebrafish.A total of forty zebrafish of similar size (Tail weigh of 0.22 g ± 0.02 g) and good growth condition were divided into 4 groups. According to the weight of the zebrafish, three groups were injected intraperitoneally with 1.4 × 10V. vulnificus [LD50 =4.39 × 104 (CFU/g) per fish]. The control group was injected with 10 μl of sterilized PBS. Kidney, muscle and intestine samples were collected from three fish of each group at different time points  for RT-PCR detection. Kidney samples from healthy and infected zebrafish at different time points  were collected for western blotting. The activity and survival rate were recorded.The experimental group was injected intraperitoneally with 10 μl V. vulnificus at different time points . Intestinal, muscle and kidney samples were collected from control and experimental groups. The TNF inhibitor AG-126 was purchased from Sigma-Aldrich . RNA extraction was conducted by using Trizol Reagent  according to the manufacturer's protocol. qRT-PCR was performed using TB GreenTM Premix Ex TaqTM II reagent Kit  with the Step Two TM Real-Time PCR System . Primers specific for Zetnf-b and β-actin are shown in Zetnf-b was calculated using the relative quantification analysis module of the 2Ct−ΔΔ method , and the protein concentration was detected by BCA protein content detection kit . Samples with equal amounts of protein were subjected to SDS-PAGE and transferred to polyvinylidine difluoride membrane . After blocking, the membrane was incubated with the primary polyclonal antibody of Zetnf-b , which was prepared as descripted in 2.4 and rabbit polyclonal anti-β-actin  at 4°C for 12 h. After washing, the membrane was incubated with goat-anti-mouse  and Goat Anti-mouse Rabbit  secondary antibodies for 2 h at 28°C. Detection was performed using an ECL hemiluminescence kit . After the film was scanned, the gray value of each protein band was measured, and data were presented as a ratio of the value to that for-actin. Zetnf-b expression in the kidney of mock-infected zebrafish was set to 1.0 and marked as 0 h, and the expression of Zetnf-b in infected zebrafish at 2–24 hpi was compared to the expression at 0 h.t test analysis. The marker * represents P < 0.05, which is considered to be statistically significant, and ** represents P < 0.01, which is considered to be significantly different.Experiments were performed in triplicate and the results were expressed as mean ± standard error (SE). All data were analyzed using Statistical Package for the Social Sciences (SPSS) for Windows (Version 16.0). The difference between the control group and the experimental group was tested by independent sample V. vulnificus infection were elongated, and these were in a healthy cell growth state with high fusion and adherence. Cells in the first group (MOI = 0.01) shrank significantly. Most of the cells in the third group (MOI = 1) lost their adhesion, and the cell structure was broken  was cloned  and extracellular protease (ECPase), can cause cell injury  was significantly up-regulated after stimulating by V. pelagius , IL-6, IL-8, and IL-10 . Sepsis nfection . In vivopelagius . In zebrdeclined . Moreoveuillarum . Our stuV. vulnificus infection, Zetnf-b polyclonal antibody or TNF inhibitor AG-126 was pre-injected to zebrafish. We found that pre-injection with Zetnf-b polyclonal antibody significantly increased the survival rate of V. vulnificus infected zebrafish. V. vulnificus infection in zebrafish. In addition, it confirmed that pre-injection of Zetnf-b polyclonal antibody aimed to inhibit TNF release or TNF activity, thereby reducing the inflammatory cytokine storm caused by bacterial infection and decreasing fish mortality, which is an effective attempt to treat bacterial infection in zebrafish.However, the overwhelming production of inflammatory cytokines can lead to organ dysfunction and eventually death. To confirm the function of cytokine Zetnf-b in fighting against Zetnf-b plays an important role in inflammatory storms induced by V. vulnificus infection. Pre-treatment of Zetnf-b polyclonal antibody and TNF inhibitor AG126 can help zebrafish to resist bacterial infections and improve the survival of zebrafish. This study provides a useful reference for the study of the biological characteristics of TNF, especially TNF-b, in important aquaculture fish and presents the TNF antibody therapy for the prevention and treatment of the V. vulnificus in the aquaculture industry.In conclusion, our study demonstrates that NM_001024447.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number can be found below: accession no.: V. vulnificus infection model in ZF4 cells and zebrafish, and also prepared the TNFb polyclonal antibody. HC was responsible for ZF4 cell and V. vulnificus culture. LZ and LK performed the experiment of pre-injecting the TNFb polyclonal antibody in zebrafish before V. vulnificus infection. CL reviewed the manuscript and provided critical suggestions. All authors discussed the results and approved the final manuscript for publication.XC and CL were responsible for the conception of the study. SL performed the cloning, expression, protein purification of zebrafish TNFb, performed data interpretation and wrote the manuscript. CJ performed the This work was funded by the Fujian public welfare research project (2019R1027-5). Meanwhile, this work was supported by the National Nature Science Foundation of China , Innovation Team Projects of Fujian Academy of Agricultural Science (STIT2017-03), Special Fund for Public-interest Scientific Institutions of Science and Technology Plan Projects of Fujian Province (2018R1019-7), and Provincial special support for high-level talents Double Hundred Plan special project.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.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.


Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). For the characterization of the nanofiber sheets incorporated with the drug, several techniques were used, such as Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), and scanning electron microscopy (SEM). Our results showed that the nanofiber sheets containing ceftriaxone had potential inhibitory activity against E. coli and S. aureus as they had inhibition zones of approximately 20–25 mm on Mueller-Hinton-agar-containing plates. In conclusion, our nanofiber sheets fabricated with ceftriaxone have potential inhibitory effects against bacteria and can be used as a dressing to treat wounds in hospitals and for other biomedical applications.Nanotechnology provides solutions by combining the fields of textiles and medicine to prevent infectious microbial spread. Our study aimed to evaluate the antimicrobial activity of nanofiber sheets incorporated with a well-known antibiotic, ceftriaxone. It is a third-generation antibiotic that belongs to the cephalosporin group. Different percentages  of ceftriaxone were incorporated with a polymer such as polyvinyl alcohol (PVA) via electrospinning to fabricate nanofiber sheets. The Kirby-Bauer method was used to evaluate the antimicrobial susceptibility of the nanofiber sheets using  P. aeruginous bacteria and a 50% reduction in S. aureus bacteria [E. coli and S. aureus bacteria. They discovered that CuO-ZnO-loaded electrospun nanofibers with a concentration of 450 g/mL significantly inhibited E. coli bacteria and S. aureus growth [The appearance and extent of lethal diseases caused by viruses and bacteria are a serious challenge of public health and safety, despite the medical system improving over the 20th and 21st centuries ,4. ElectS. aureus bacteria [Various investigations have been carried out on the production of antibacterial nanofibers using electrospun nanofibers loaded with various medicines. Ajmal et al. created electrospun poly (lactic-co-glycolic acid)-alginate nanofibers and subsequently loaded the ciprofloxacin drug with these electrospun nanofibers. They achieved this by utilizing the broth microdilution method and determining ciprofloxacin’s MIC value against bacteria .Escherichia coli and Staphylococcus aureus, and it has been reported with various materials such as zinc oxide, silver nanoparticles, copper salt, and chitosan nanoparticles [E. coli and S. aureus bacteria and showed prominent results.Ceftriaxone is a commonly used, third-generation active antibiotic agent against many Gram-positive and Gram-negative pathogens, such as articles ,25,26,27articles . Due to articles ,30. TherCellulose acetate (CA) was purchased from Sigma Aldrich  (Mw 30 K). Polyvinyl alcohol (PVA) polymer  was purchased from Sigma Aldrich . Ceftriaxone was obtained from Macter International Limited, Karachi.Cellulose acetate (CA) solution of 18% was prepared in a solvent mixture of acetone and DMF with a ratio 2:1 and was kept on magnetic stirring for 24 h. After complete dissolution of CA polymer, different concentrations of ceftriaxone  were added. Then, stirring continued for 30 min to form a homogeneous solution. The prepared solution was electrospun by filling it into a plastic syringe that was attached to a feed pump, setting the solution feed rate at 1 mL/h, setting the tip-to-collector distance at 12 cm, and supplying a 12.5 kV power for the formation of ceftriaxone-loaded CA nanofibers.Similarly, 10% PVA solution was prepared by mild stirring at room temperature. After the complete dissolution of PVA polymer, different concentrations of ceftriaxone  were added. Then, stirring continued for 30 min to form a homogeneous solution. The prepared solution was electrospun at 16 kV applied voltage, feed rate was 1 mL/h, and the distance between syringe tip to collector was set at 10 cm. The prepared ceftriaxone-loaded PVA nanofiber membranes were dried for 24 h at room temperature.Escherichia coli and Staphylococcus aureus were cultured in nutrient broth at 37 °C for 24 h. The number of viable bacterial cells was adjusted from 3 × 105 cfu/mL to 4 × 105 cfu/mL by serial dilution (four times) with 0.03 mol/L PBS. After the desired bacterial growth was reached, 0.03 g of nanofibers was placed into 65 mL of 0.3 mM PBS culture solution and 5 mL suspension of prepared bacterial solution. The flask was then agitated on a rotary shaker for 18 h at 150 rpm at 37.8 °C. The solution was diluted 10 times with 0.3 mM PBS, and this was repeated four times for a serial dilution. Finally, bacterial suspensions of various concentrations (1 mL each) were placed on an agar plate. The number of colonies grown on the agar plate was calculated by Equation (1) after 24 h of incubation at 37.8 °C:The disc diffusion method was used to test the antibacterial activity against Staphylococcus aureus and Escherichia coli bacteria. The solutions were evaluated using a dilution method after E.coli and S. aureus pathogens. Each nanofiber sample of 6 mm diameter was mounted on Mueller-Hinton agar plates spread with bacterial colonies. Afterward, the Petri dishes were incubated for 48 h at 37 °C. After the incubation, the inhibition zones were measured and compared.The Kirby Bauer method (disc diffusion method) was used to determine the antibacterial activity of PVA and CA nanofibers against −1 at 25 °C. The crystalline and amorphous regions of the prepared nanofibers were observed by X-ray diffraction .The physical morphology of neat CA, neat PVA, CA/CEF, and PVA/CEF nanofibers was assessed by scanning electron microscopy . The chemical structure and changes were examined using IRPrestige-21 from Shimadzu  at ATR-FTIR mode at adsorption wavelengths between 500 and 4000 cmPrepared nanofibers were characterized by SEM; the SEM images are shown in −1, which corresponded to OH stretching, C-H stretch vibrations, C=O carbonyl stretching, and C-O-H stretching, respectively, in the PVA polymer [−1, which corresponded to C=C stretching in the ceftriaxone drug [−1, indicating the presence of C=O, C-CH3, and C-O-C groups, respectively [The nanofibers were characterized by FTIR to analyze their chemical structure and compatibility with ceftriaxone. As shown in  polymer . After lone drug ,27,31. Tectively . HoweverThe XRD spectra of pure CA, pure PVA, ceftriaxone-loaded PVA (PVA/CEF), and ceftriaxone-loaded CA (CA/CEF) nanofibers were analyzed to assess the crystallinity of the nanofibers. The XRD spectra of pure PVA nanofibers, shown in Staphylococcus aureus (Gram-positive) microorganisms was evaluated. As shown in Staphylococcus aureus. When the amount of the ceftriaxone drug was increased from 5% to 20%, both CA and PVA nanofibers created an inhibition zone that had an approximate diameter of 25 mm against Staphylococcus aureus. Furthermore, the nanofibers containing 5% to 20% ceftriaxone created the same inhibition zone. It was concluded that a small amount (5%) of ceftriaxone in nanofibers was sufficient to create an inhibition zone. Both nanofibers were further evaluated for E. coli microorganisms, and the results are presented in E. coli. In the case of E. coli, the inhibition zone increased with an increasing amount of loaded drug. We found that 15% loaded drug was sufficient to create an inhibition zone against E. coli.The antimicrobial susceptibility of neat CA, neat PVA, and ceftriaxone-loaded nanofibers to E. coli and S. aureus, respectively. CA/CEF nanofibers created inhibition zones of 20 and 25 mm against E. coli and S. aureus, respectively. The produced nanofiber composites maybe used in antibacterial applications for different medical purposes.The nanofiber composites of PVA/CEF and CA/CEF were successfully prepared by the electrospinning technique, which was followed by an antibacterial assessment. The ceftriaxone drug was loaded into nanofiber sat different concentrations . The produced nanofibers were smooth and beaded and had a diameter of 220 nm. The nanofibers showed a good inhibition zone against pathogens. PVA/CEF nanofibers showed inhibition zones of 20 and 25 mm against


The rapid diffusion of the Covid-19 worldwide has accelerated the need for companies to address the sustainability issue at different levels, as nowadays the attention of stakeholders with respect to this theme has grown considerably. As a result, companies had to set up CSR communication strategies to build and strengthen their legitimacy and reputation. Among the communication channels to convey messages of firms’ CSR initiatives, social media are becoming increasingly important and, particularly, Twitter is the social media platform where more CSR-related content is generated. By adopting the theoretical lens of constitutive communication of organization, the aim of this paper is to investigate with a textual approach how the CSR communication in the energy sector has evolved in the post Covid-19 scenario. Specifically, our attention will be focus on: (1) the exploratory analysis based on the hashtags; (2) the identification of CSR communication topics and (3) the proposal of topics network in order to discover subgroups of topics. Findings of this research show that the CSR communication on Twitter has undergone changes compared to the pre Covid-19 era. Particularly, we identified 11 CSR related-topics which, as the proposed topic network demonstrates, are interconnected. On the one hand, our results corroborate previous research regarding some CSR-related issues; on the other hand, we identified some topics such as safety, people and work which have exploded in Twitter conversations in the post Covid-19 scenario. Finally, this study provides managerial implications for professionals dealing with CSR communication, digital communication and social media marketing activities. Since Bowen, in 1953, defined Corporate Social Responsibility (CSR) as a set of obligations of business men to pursue policies, make decisions and follow desirable courses of action in terms of the objectives and values of our society, this concept has evolved profoundly. As early as 1999, Carroll defined CSR as an essential part of business languages and practices, consistent with what the public expected from the business community of that time. Today, business development calls for an understanding of the relevance of implementing CSR with the aim to achieve legitimacy among consumers , according to which organizations maintain themselves through communicative practices  and textual network analysis using a two mode network approach  topic is very broad and has considered different theoretical and methodological perspectives. Particularly, among the most recognized authors in the marketing literature, Kotler and Lee  define CSR as “a commitment to improve community well-being through discretionary business practices and contributions of corporate resources”.Marketing and corporate communication scholars agree that CSR must be recognized as a key element of the strategic approach to communication  a wide reach of stakeholders in reduced times and costs and (2) the possibility of receiving immediate feedbacks by generating positive dialogues in order to establish a better relationship with the target. Contrariwise, the challenges of the CSR communication on social media concern (1) the management of any negative users’ comments on these virtual platforms, including Twitter, Facebook, YouTube, and (2) to reduce the skepticism of stakeholders  separated by spaces and punctuation marks. All other alphanumeric characters and symbols have been eliminated. Next, we reduced the characters of all terms to lowercase, a so-called normalization operation. We have eliminated stopwords, words useful to compose a complete sentence but which, taken alone, do not give any information (e.g. prepositions and articles). These tokens are called empty words or stopwords because they do not give any additional information. From this point begins the processing phase of the textual data as a lexical feature through an approach to lemmatization called Part of Speech. In this step we introduce an innovative approach, related to an automatization of this phase of data pre-processing which consist in an annotation of language elements in the texts with a language-agnostic tool, the R package UDPipe , we select a random sample representing the word distribution of the topic Z, this distribution is φ, from φ we choose the word w. LDA has shown to work generally better than other models due to its potential to easily generalize new documents. In this model the data set acts as training data for the distribution of Dirichlet for topic-document distributions. The number of topics to extract from the corpus with this model is a parameter to be defined a priori. We need a methodological solution that allows us an automatic extraction of the topics. There are various methods for evaluating the model with the optimal number of topics .The measure proposed by the authors corresponds to the sum of the calculated values of the Kullback–Leibler divergence on two stochastic matrices M1 and M2:Our methodological strategy is based on KPI selected from three areas of sustainability: social, environmental and economic. We preferred to analyze the communication of companies on Twitter, taking advantage of the mobilization on social media regarding environmental issues : degree, betweenness and closeness centrality. The degree of a node expresses the receptivity and popularity, the betweenness is the importance of the actor in terms of probability to be alone all the possible paths that connect the actors of the network and the closeness use the geodesic distance to identify the prestige of an actor, as the one that can most easily reach all the other players in the network. The topic networks in Fig. Topic 3, topic 10 and topic 13 are the nodes with main degree, so they have the highest number of links. These themes are recurring topics in the communication, and have a shared vocabulary. The closeness measure denotes most important nodes, these are topic 1, topic 3 and topic 8. The betweenness helps to identify two sub-networks represented in network in Fig. 5, one links topic 1, topic 6, topic 7 and topic 8, this is the network of themes related to the dimension of sustainability and the other links topic 3, topic 4 and topic 10, this is the network related to the content communication.The overall aim of this research is to investigate how the CSR communication of companies evolved in the post-pandemic scenario. Specifically, we conducted an exploratory hashtag analysis (RQ1), identified the relevant CSR topics of the energy companies communication on Twitter (RQ2) and proposed a topic network to highlight how these topics interrelate (RQ3). In these different research steps, we have taken into consideration the theoretical lens of CCO according to which communication covers a formative role with respect to practices within organizations Cooren, .Generally, the contribution of this paper is twofold: on the one hand, we identify new dimensions of CSR communication emerged due to the Covid-19 pandemic and which, therefore, previous studies had not intercepted; on the other hand, through the analysis of the interrelation of the topics visible in the topic network, a systemic view of CSR communication on Twitter during the pandemic is provided. Moreover, although other studies which have analyzed the issue of CSR communication on digital platforms exist , confirming that the topic of the Environment (Topic 6) is key in the discussions of CSR on Twitter. Specifically, from the proposed topic network emerged that the themes of the environment, the link with the territory and the country represent a set of topics addressed by energy companies today. Also, the topic of the Education, which we have identified as Topic 12, supports the recent research by Saxton et al. , which sRegarding the new CSR dimension emerged from this study, we have noticed that the communication of CSR on Twitter has been strongly affected by the impact of the pandemic as issues such as “Safety” (Topic 6), with a focus on resources, people, work, which are emerging more and more clearly in the communication of companies on this digital platform. In fact, although Etter  in his pThese thematic clusters emerged from the analysis of communication flows on Twitter we conducted confirm that in a constructivist perspective communication becomes fundamental to dictate the agenda of the CSR activities that will be implemented within the organizations. Overall, research is showing that the topic of sustainability is key after the pandemic  in relation to sustainability recurs more and more often in CSR dialogues on Twitter—for this reason, companies that decide to communicate their CSR initiatives on this platform should leverage on aspects concerning sustainable business innovation.Finally, compared to the pre-Covid-19 era, the issue of the education, which was already widely communicated in the CSR communication strategies on social media (Saxton et al., This exploratory study aims to investigate how the CSR communication evolved on Twitter in the post Covid-19 era and, as any research, it presents some limitations. First, this study was conducted considering a limited sample of 5 companies belonging to the same territorial context, namely Italy, and the same industry, i.e. the energy sector. Secondly, the research was conducted on only one social network, i.e. Twitter, and, consequently, does not consider the dynamics of the CSR communication evolution on other social media in the post Covid-19 days.Thus, future research could therefore extend this perspective by investigating how CSR communication is evolving nowadays on other social media platforms and highlighting any differences both in terms of topics and interaction with users. For example, future studies could focus on the engagement and identify which types of content and which CSR topics generate the most engagement degree with Twitter and other social media users.


Cholangiocarcinoma (CCA) is an aggressive and lethal malignancy with limited therapeutic options. Despite recent advances in diagnostic imaging for CCA, the early diagnosis of CCA and evaluation of tumor invasion into the bile duct and its surrounding tissues remain challenging. Most patients with CCA are diagnosed at an advanced stage, at which treatment options are limited. Molecular imaging is a promising diagnostic method for noninvasive imaging of biological events at the cellular and molecular level in vivo. Molecular imaging plays a key role in the early diagnosis, staging, and treatment-related evaluation and management of cancer. This review will describe different methods for molecular imaging of CCA, including nuclear medicine, magnetic resonance imaging, optical imaging, and multimodal imaging. The main challenges and future directions in this field are also discussed. Molecular imaging has higher sensitivity and specificity than conventional imaging for the diagnosis of cholangiocarcinoma.Different molecular imaging methods have specific advantages for cholangiocarcinoma diagnosis.Molecular imaging has great potential for finding targets, probe synthesis, and clinical applications in cholangiocarcinoma.Cholangiocarcinoma (CCA) is a primary malignant tumor that occurs in intrahepatic and extrahepatic bile duct epithelial cells with high invasiveness and heterogeneity , 2. In >CCA is diagnosed via a combination of clinical symptoms, imaging manifestations, biochemical features, and histological examinations, and imaging plays a crucial role. US is the first and more common choice for screening CCA because it is inexpensive and simple to perform. However, US is difficult to assess the range of tumor invasive and is not efficient for detecting small invasive CCAs. Moreover, the accuracy of US varies according to tumor type, equipment quality, and operator experience , 13. CT Molecular imaging is an emerging discipline at the intersection of molecular biology and traditional medical imaging. It uses imaging methods to display specific molecules at the tissue, cellular, and subcellular levels. It can assess the changes at the molecular level in vivo, and perform qualitative and quantitative imaging studies on the biological behaviors of molecules. Molecular imaging provides a noninvasive, timely, and cost-effective method to study the fundamental behavior of organisms, thereby improving our understanding of diseases , 27. Com18F-FDG), a glucose analog that can be selectively absorbed by cells characterized by high glucose metabolism. In addition, there are some PET/CT tracers used in preclinical studies, such as those targeting COX-2, VEGF and CXCR4.Nuclear medicine imaging is an imaging technology that shows physiological and pathological activity by detecting the metabolic processes of tracers with radionuclide. PET/CT is one of the earliest functional metabolic imaging methods used in the clinic. It has certain advantages for tumor diagnosis, differential diagnosis, and tumor monitoring. PET/CT tracers are synthesized by covalent connections of isotopes . The tra18F-FDG-PET in primary CCA is 90% or higher iodooctyl fenbufen amide ([123I]IOFA) on CCA in a similar way. The results showed that a lower level and homogeneous pattern of [123I]IOFA uptake were observed in the liver of normal rats. However, in the liver of rats with CCA, higher [123I]IOFA radioactivity absorption and heterogeneous patterns were regarded as hot spots of tumor lesions. An increase in COX-2 expression was detected by immunostaining in the bile ducts of CCA rats, but not in normal rats. Thus, the SPECT reagent [123I]IOFA has imaging potential for CCA with overexpression of COX-2 , and found that the uptake level of radioactive tracer in CCA patients was the highest -2-deoxy-d-glucose (2-NBDG) is a fluorescent tracer that enters living mammalian cells via GLUT in a time-, concentration-, and temperature-dependent manner. The fluorescence intensity of cells expressing GLUT increases significantly after exposure to 2-NBDG, and the cells can be distinguished more clearly . Th. Th77]. CCA is a highly aggressive and heterogeneous malignancy with a dismal prognosis. Despite extensive research, current treatment strategies remain limited and ineffective in most cases. Medical imaging is an important method for the diagnosis and treatment of patients, which is a prerequisite for accurate medicine. The purpose of molecular imaging is to improve the sensitivity and specificity of traditional image detection. The molecular imaging study of CCA is still in the early stages and mostly limited to animal experiments and preclinical research. Thus, there are many difficulties and challenges ahead. The selection of a reliable target to distinguish CCA from other tumors or normal tissues is essential for molecular imaging of CCA. Accordingly, there are currently few mature probes used in CCA molecular imaging. On the one hand, developing or discover probe complexes for CCA targeting is expensive and time-consuming. On the other hand, the safety, specificity, and sensitivity of molecular probes used in the human body need to be further verified. For these reasons, the development of molecular probe complexes for specific monitoring of tumor cell growth and death has become the focus of CCA molecular imaging research. Currently, there is an activatable probe known as “smart probe” that can change the molecular conformation independently according to the microenvironment , thus affecting the strength of imaging signals. This is the most advanced tumor imaging probe, and the improved signal-to-noise ratio makes it a potentially effective probe for the molecular imaging of CCA .This review focuses on four different molecular imaging modalities. The current advantages and limitations of each method should be considered when designing future studies involving molecular imaging. PET and SPECT imaging are the earliest molecular imaging modalities used in clinical practice; they are sensitive for the detection of CCA and play a vital role in tumor grading and recurrence monitoring. However, nuclear medicine has poor spatial resolution; therefore, PET and SPECT should be combined with CT or MRI for multimodal imaging in practical applications. MRI has several advantages such as high spatial resolution, excellent soft tissue contrast, and no radiation exposure. Since MRI uses various sequences for imaging, it can be used in CCA to assess the extent of tumor invasion, providing differential diagnosis and prognostic information. Although MRI is a useful imaging method, it has low sensitivity and a long acquisition time. In MR molecular imaging, the unique characteristics of molecular imaging such as high sensitivity are combined to make MRI one of the best molecular imaging modalities. Optical imaging has been widely used in many cell and animal studies in the past few decades. Despite several advantages such as high sensitivity, no radiation, and low cost, the clinical application of optical imaging remains uncertain because of limitations such as light attenuation with increased tissue depth. Endoscopic technology and intraoperative real-time dynamic detection are the main directions for the application of optical imaging to the clinical diagnosis and treatment of CCA. Finally, the multimodal nanomolecular imaging platform can integrate the advantages of these imaging modalities and compensate for the corresponding shortcomings. These techniques increase the diversity of CCA molecular imaging, thus greatly improving the diagnostic accuracy and prognosis of CCA patients.There are still many new opportunities in CCA molecular imaging research. Molecular imaging research of CCA, which is part of personalized medicine, is moving in the direction of miniaturization of targeted materials, nanoscale imaging media, and multi-modal imaging methods. The translation of animal and preclinical trials to clinical application is the next focus of molecular imaging. In the future, the use of molecular imaging for early screening will greatly benefit patients with CCA.


Colorectal cancer (CRC) is one of the leading causes of cancer related death in the western world. Its successful treatment requires early detection and removal of precursor lesions as well as individualized treatment of advanced disease. During recent years, molecular imaging techniques have shown promising results to improve current clinical practice. For instance, molecular endoscopy resulted in higher detection rates of precursors in comparison to conventional endoscopy in preclinical and clinical studies. Molecular confocal endomicroscopy allowed a further classification of suspect lesions as well as the prediction and monitoring of the therapeutic response. In this review, we summarize recent achievements for molecular imaging of CRC in preclinical studies, initial clinical trials and the remaining challenges for future translation into clinical practice. Colorectal cancer (CRC) is among the leading causes of cancer-related mortality in the Western world. For instance, CRC has been estimated to result in more than 50.000 deaths in both men and women in the United States in 2021, making it the 3rd most common cause of tumor-related death in both genders , 5. Therin vivo, molecular imaging approaches can provide important information in realtime and therefore improve decision making during diagnostic procedures. In the field of cancer diagnostics and therapy, molecular imaging has become an important research topic in recent years , Kirsten rat sarcoma virus (KRAS), and tumor protein 53 (TP53). Until today, these genes are considered as major tumor suppressor genes (APC and TP53) or oncogenes (KRAS) of CRC development, although mutations in other genes have subsequently been discovered within the adenoma-carcinoma sequence model. Because of these findings, initial studies aiming at the detection of CRC precursors with optical molecular imaging targeted molecular pathways that were associated with mutations occurring early in the adenoma-carcinoma sequence. As the genes mutated within the adenoma-carcinoma sequence are mostly intracellular and therefore not well suited for molecular endoscopy approaches, markers have been identified that are associated with a dysregulation of pathways of the adenoma-carcinoma sequence. For instance, Marten et al. developed an activatable probe for the proteinase cathepsin B for the detection of adenomas, as early occurring mutations in APC could induce the activation of proteolytic enzymes . Therefore, endoscopic molecular imaging of serrated polyps might need other targets in comparison to polyps of the adenoma-carcinoma sequence. For instance, Joshi et al. developed a peptide that showed specific binding to human colorectal cancer cells with the V600E mutation of d polyps . If thesin vivo microscopic evaluation of tissues including CRC. Both types of devices together could enable a two-step approach: widefield molecular endoscopy could be used as a red-flag technique for the detection of suspicious lesions, which could then further be characterized by microscopic molecular imaging  were developed to enable an  imaging . The fir imaging , 13. Typ imaging , 29. Thi imaging . This or imaging . These i imaging .In contrast to widefield fluorescence endoscopes, CLE devices were built to deliver microscopic information about suspect lesions during endoscopy. Initially, two different systems have been developed and used in preclinical as well as clinical trials . In one The first studies that evaluated the potential of molecular imaging to identify precursor lesions of CRC were mainly focused on showing an upregulation of the molecular target and the possibility to visualize this upregulation through the detection of fluorescence. For instance, in the previously mentioned study by Marten et al., it was shown that Cathepsin B, the target of the used activatable protease sensing NIR probe used in this study, is upregulated in adenomatous polyps of the APCMin/+ mouse model of CRC . FurtherIn this regard, Schwegmann et al. used a NIR matrix metalloproteinase specific probe for the endoscopic detection of colon tumors induced through the administration of azoxymethane and dextrane sodium sulfate (AOM+DSS) in mice . They evin vivo  in several studies , 50. As Nearly 20 years ago, initial studies proposed molecular imaging approaches as new diagnostic modalities for the early detection of colorectal cancer. Since then, efforts in the development of molecular imaging probes, fluorescence endoscopes and their usage resulted in initial clinical trials that propose that molecular endoscopy might be superior for the detection of CRC precursors during screening colonoscopy. Data from preclinical studies further show a usage for the prediction of therapy or therapeutic monitoring. However, as new insights are gained on the genetic heterogeneity of CRC and efforts for personalized therapy are constantly progressing, there is still a huge demand on further research about the full potential of molecular endoscopy in the clinical management of CRC. Recently developed multispectral systems that allow the simultaneous detection of various targets at the same time pose important steps for future applications. However, the identification of appropriate molecular targets of CRC development and biomarkers for a response to therapy is still the bottleneck for molecular imaging approaches.MW and MN performed literature research, planned and wrote the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.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.


Neisseria gonorrhoeae genomics. In particular, we explore how genomics continues to advance our understanding of this complex pathogen.The sexually transmitted infection (STI) gonorrhoea remains a major global public health concern. The World Health Organization (WHO) estimates that 87 million new cases in individuals who were 15 to 49 years of age occurred in 2016. The growing number of gonorrhoea cases is concerning given the rise in gonococci developing antimicrobial resistance (AMR). Therefore, a global action plan is needed to facilitate surveillance. Indeed, the WHO has made surveillance leading to the elimination of STIs (including gonorrhoea) a global health priority. The availability of whole genome sequence data offers new opportunities to combat gonorrhoea. This can be through (i) enhanced surveillance of the global prevalence of AMR, (ii) improved understanding of the population biology of the gonococcus, and (iii) opportunities to mine sequence data in the search for vaccine candidates. Here, we review the current status in  Neisseria gonorrhoeae, the gonococcus, is an obligate human pathogen that causes the sexually transmitted infection gonorrhoea1. Gonococcal infection often results in severe complications, ranging from disseminated infection to salpingitis or pelvic inflammatory disease, and gonococcal infections are found to be asymptomatic in over 50% of women and associated with a significant cause of infertility2. Therefore, prompt diagnosis and treatment are essential for a positive clinical outcome. However, the gonococcus has developed resistance against all available classes of antimicrobials and options for the effective treatment of this infection are becoming increasingly limited3. This is particularly concerning as gonorrhoea, after Chlamydia trachomatis, is the second most prevalent sexually transmitted infection and is globally associated with high levels of morbidity and economic cost4.N. gonorrhoeae and C. trachomatis, which present with similar clinical symptoms5. However, treatment has become complicated by the rapidly changing antimicrobial susceptibility patterns of N. gonorrhoeae, raising concerns about the development of untreatable gonorrhoea. Internationally, the prevalence of gonococci resistant to most antimicrobials recommended for treatment is high. For example, the recent occurrence of failures to treat gonorrhoea with the extended-spectrum cephalosporins (ESCs) cefixime and ceftriaxone and, the emergence of gonococci exhibiting high-level resistance to all other available therapeutic antimicrobials8, have caused great concern and highlight the need for a targeted approach to limit the spread of infection and prevent transmission globally.Infections are diagnosed by microbiological culture (with Gram stain in men with urethritis) or nucleic acid amplification tests. In settings with limited laboratory capacity, diagnosis is often based on clinical symptoms alone, followed by syndromic management. This commonly includes the use of doxycycline, an antibiotic that targets both N. gonorrhoeae WGS data have become available, allowing an increasingly forensic examination of this bacterium. The knowledge gained from these analyses strengthens our capacity to detect antimicrobial resistance (AMR) while improving our understanding of the resistance phenotype and how it evolves. This knowledge also allows the population biology of the gonococcus to be resolved, facilitating global surveillance. Finally, WGS data provide opportunities for vaccine development through the characterisation of antigenic variability. Here, we review progress made in the field of gonococcal genomics, examining how WGS data have increased our knowledge of this pathogen. In particular, we focus on advances made in the detection of AMR, the genetic determinants conferring this phenotype, and the tools developed to identify AMR. We assess progress made in understanding the population biology of this pathogen and discuss the current status in vaccine development and how WGS can support this activity. To conclude, we review the challenges that remain and discuss the approaches needed to overcome them.Understanding the causative agents of disease is paramount to curing or preventing disease, and the availability of whole genome sequence (WGS) data offers novel opportunities to do this. In particular, a large amount of 9. The effectiveness of antibiotics to treat gonorrhoea is becoming limited by the global emergence and spread of gonococci resistant to antimicrobials, combined with increasing rates of gonorrhoea. The total number of confirmed cases reported in 2018 in 28 European Union/European Economic Area (EU/EEA) member states was 100,673 , representing a 12% increase in the total number of reported cases compared with 201711. In the UK alone, 61,775 confirmed cases were reported in 2018, representing a 26% increase compared with 201710. In the US and its territories, 556,413 cases of gonorrhoea were reported in 2017, representing a rate of 168.9 cases per 100,000 population and a 19% increase compared with 201612. In Australia, the number of confirmed cases rose from 23,875 in 2016 to 28,364 in 2017 with a rate of 118 per 100,000 population13. This was associated with a 63% increase in notification rates between 2012 and 2016. In China, 133,156 confirmed cases were reported in 2018, representing a rate of 10.06 cases per 100,000 population14. Similarly, a rate of 88.6 cases per 100,000 population was identified in 2015 in Japan15. These data are consistent with a global trend of increasing rates of gonorrhoea. However, there are differences in the reporting of incidence and prevalence data among countries, even where effective surveillance systems are in place. In addition, different jurisdictions have their own surveillance methodologies and diagnostic tests such that global estimates have to be interpreted with caution3. So it is challenging to assess effectively global rates of gonorrhoea prevalence.In the absence of vaccines, antimicrobial therapy is critical to cure infection and limit its spread16. The gonococcus employs multiple genetic mechanisms to overcome the effects of antimicrobial agents and is extremely adept in developing AMR  from commensal Neisseria species43; (ii) non-synonymous mutations in the ponA gene, which encodes penicillin binding protein 127; (iii) the presence of mutations in the mtrR repressor gene or its promotor sequence, resulting in overexpression of the MtrCDE efflux pump system and secretion of antimicrobial agents38; and (iv) mutations of the outer membrane channel porin PorB variant PIB, resulting in a decreased influx of penicillin, through reduced permeability of the porin36. In addition, mutations in rpoB, encoding RNA polymerase subunit B, and rpoD, encoding the RNA polymerase sigma factor, have been identified  that enhance the capacity of the protein to export antimicrobial agents40.To monitor resistance, gonococci are classified as either multidrug-resistant (MDR-GC) or extensively drug-resistant (XDR-GC): MDR-GC is defined as a gonococcus with decreased susceptibility to one currently recommended therapy  plus resistance to at least two other antimicrobials, whereas XDR-GC is a gonococcus with decreased susceptibility to two currently recommended therapies  plus resistance to at least two other antimicrobials71; (ii) the Pathogenwatch AMR prediction module, PAARSNP, which can be used through the Pathogenwatch portal and which uses resistance databases developed in-house73; and (iii) ABRICATE74. Performance of these in silico AMR prediction tools is dependent on the availability of accurate and curated AMR databases such as ResFinder75 and CARD76 and those reviewed by Hendriksen et al.77. Concordance between phenotype and genotype is stronger for some genetic determinants than others. For example, ciprofloxacin resistance can be readily determined from sequence data, whereas predicting beta-lactam–resistant genotypes is more challenging, particularly as this is often an additive effect resulting from multiple mutations. As a result, several studies have encountered difficulties when using WGS to predict such AMR phenotypes79.Genomic technologies therefore offer the tantalising prospect of enhancing surveillance of AMR with several bioinformatic tools developed to predict AMR from WGS data. These include (i) Antimicrobial Resistance Identification By Assembly (ARIBA), which identifies AMR genes and variants from paired sequencing reads through the use of a combined mapping/alignment and targeted local assembly approach80. More recently, a method called ‘genomic neighbour typing’, which allows the AMR phenotype of a bacterial sample to be predicted through the identification of its closest relatives in a database of genomes which have corresponding AMR metadata, has been described. This method was tested in both Streptococcus pneumoniae and N. gonorrhoeae datasets, and preliminary results indicate that this approach may work well for the pneumococcus given the greater sequence diversity observed in its core genome, which allows specific signature sequences to be more readily detected and matched to individual lineages. However, the lower diversity found in the gonococcal core genome results in the identification of multiple related neighbours which confounds obtained signals81.Other approaches have employed multivariate linear regression models to identify genetic predictors of minimum inhibition concentrations (MICs). These models predict MICs for five gonorrhoea antimicrobials that exhibit different genetic mechanisms of resistance: cefixime, penicillin, azithromycin, ciprofloxacin and tetracyclineNeisseria gonorrhoeae sequence typing for antimicrobial resistance), which indexes variability found in nucleotide sequence fragments from seven genes associated with AMR 82. Using an online, publicly available database (https://ngstar.canada.ca), users can submit new alleles for curation, allowing variation in genes conferring resistance to be defined and tracked. New allelic profiles are assigned NG STAR sequence types (STs), which are associated with corresponding isolate records. Since its publication, this tool has been used in several international studies assessing AMR, thus providing a tool with which to track the worldwide dissemination of resistant clones. All described genes associated with AMR in the gonococcus, both chromosomally and plasmid-mediated, have also been defined in the pubMLST.org/neisseria database with mutations annotated in allelic variants83. This database includes the NG STAR, multi-locus sequence typing (MLST) and N. gonorrhoeae multi-antigen sequence typing (NG MAST v2.0) schemes84. As a result, WGSs deposited in PubMLST are annotated in all of these schemes, allowing AMR to be evaluated in combination with conventional typing schemes and providing a publicly available resource for the analysis of gonococcal WGSs. Furthermore, WGSs can be directly queried for any of these schemes without the need for WGSs to be deposited. An additional publicly available tool, Gen2Epi, facilitates gonococcal WGS assembly followed by automatic retrieval of molecular epidemiological information, including NG STAR, MLST and NG MAST and AMR genotypes85.An alternative approach for tracking AMR in the gonococcus is through the use of a multilocus sequence typing scheme, NG STAR 91. A number of these ccs, ‘the hyperinvasive meningococci’, are responsible for the majority of invasive disease cases worldwide and are stable over time and global spread92. The levels of linkage disequilibrium observed in meningococci result in the non-random association of MLST allelic profiles, producing discrete persistent meningococcal lineages, some of which exhibit an invasive phenotype93. Studies of the genetic diversity of gonococcal housekeeping genes have shown that, in addition to diversification arising from randomly distributed point mutations, these genes are frequently subject to HGT95. As a result, some gonococci, though possessing the same seven locus MLST STs, may have different ancestry in the majority of their loci and consequently MLST STs cannot be used to reliably evaluate gonococcal populations96. In addition, combinations of MLST alleles in gonococci are unlikely to be associated with transmission fitness or stabilising selection in contrast to the meningococcus94.Several molecular typing tools have been developed to identify gonococcal isolates, including (i) MLST, which indexes the diversity found at seven housekeeping gene fragments, and (ii) the NG MAST scheme, where nucleotide sequence fragments of the outer membrane proteins PorB and TbpB are used to define NG MAST STs95. Genomic population studies have shown otherwise97. Hierarchical Bayesian analyses  uses nucleotide polymorphism–based alignments to generate clusters of related isolates after accounting for HGT identified by tools such as Gubbins99. With this method, gonococcal genome data from several studies have been found to consist of between six and 12 BAPS clusters, some of which associated with AMR phenotypes100. This is consistent with a semi-clonal population structure 101. The presence of distinct gonococcal lineages was also evident in studies of the gonococcal core genome. For example, more than 1,600 genes were identified as ‘core’ in WGS data from over 4,000 gonococci: these genes have been defined in the PubMLST database (https://pubmlst.org/neisseria) and form part of the N. gonorrhoeae cgMLST version 1.0 scheme84. Core genome STs are assigned for each isolate and, through single-linkage clustering and the use of increasing allelic difference thresholds, isolates can be grouped into related core genome groups  pilE gene, which encodes the major pilus subunit (pilin), provides a silent catalogue of sequences that can be reassorted into an expression locus to generate new pilE variants111. N. gonorrhoeae is also highly competent for genetic transformation, a process facilitated by a type IV secretion system which is present in over 90% of gonococci and which, in a contact-independent manner, secretes single-stranded DNA into the environment, providing a source of DNA for HGT112.Immune evasion is also facilitated by the ability of gonococci to exhibit extensive antigenic diversity in surface-exposed antigens, a consequence of mutation, HGT, and the modulation of expression through phase variationN. gonorrhoeae antigens, some of which are promising vaccine candidates based on their antigenic conservation, distribution in gonococcal populations, and immunogenicity, have been characterised  proteomics to identify the relative abundance, post-translational modifications and protein–protein interactions of those vaccine candidates; (ii) genomics to assess the genetic diversity, prevalence and distribution in gonococcal populations; and (iii) animal models to examine in vivo host responses and the potential for an immune response to be elicited.In spite of these challenges, several cterised . Severalelopment 113. HoweN. gonorrhoeae population. In this respect, outer membrane vesicle (OMV) vaccines may be suitable since they contain many of the gonococcal surface antigens in their natural conformation116. Support for the use of OMV as vaccines comes with the epidemiological evidence provided by a retrospective case control study undertaken in New Zealand, which suggested that individuals vaccinated with the OMV vaccine MeNZB were less likely to contract gonorrhoea117. The MeNZB vaccine had been specifically designed in response to a serogroup B N. meningitidis epidemic in New Zealand, and although vaccine efficacy towards gonorrhoea was estimated to be 31%, ensuing studies have shown that cross-reactive anti-gonococcal antibodies are induced by MeNZB OMV proteins, providing hope that a gonococcal OMV vaccine is achievable118.It is likely that vaccines containing a ‘cocktail’ of antigens will be required in order to generate broad protection against the in silico appraisal of vaccine candidates to be undertaken prior to more costly and time-consuming vaccine development approaches. In particular, the gene-by-gene characterisation of the gonococcal core genome available on PubMLST allows the diversity of putative vaccine candidates to be catalogued across global gonococcal populations spanning decades120.A vaccine targeting gonococci is now a possibility with the availability of WGS allowing an 96. However, it is conceivable that a much longer association, potentially spanning millennia and consistent with descriptions in ancients texts, has existed121.Gonorrhoea has been long considered to be an ancient human disease on the basis of clinical descriptions, but recent research has suggested that the emergence of gonorrhoea may date back to as recently as the sixteenth century, calculated through the comparison of genome sequence data and through inference of the time to most recent common ancestor122. The coexistence of gonococci with humans has led to the development of multiple mechanisms of genetic and antigenic variation, allowing adaptation, persistence and evasion of both environmental and host challenges. In addition, it is most likely that human migration has widened the gonococcal gene pool with increased diversity as a result145. In more recent times, antimicrobial use has further shaped the gonococcal population, leading to the expansion of AMR lineages96.In any case, the coexistence of humans and gonococci has had, and will continue to have, a profound effect on both populations. In humans, effects include reduced birth rates resulting from infertility caused by ascending gonococcal infections, in addition to increased mortality rates through coinfection with HIVN. gonorrhoeae isolates circulating globally will remain uncharacterised at the WGS level for the near future. Our current understanding will be based on a subset of the gonococcal population and at the time of writing this was strongly biased towards isolates with an AMR phenotype. To further our ability to cure or prevent gonorrhoea, larger and more representative datasets need to be assembled and sequenced. Sequencing technology has made major advances in the last decade and what seems impossible now may be achievable in the future. Until then, it is likely that the gonococcus will continue to elude us.Genomics plays an increasingly important role in combatting gonococcal disease, allowing the definition and characterisation of complex characters, including the genetic elements associated with AMR, persistence and antigenic diversity. However, at the time of writing, only a very small proportion of gonococci causing infection were analysed by WGS. For example, 56,259 infections were diagnosed in England in 2018 and it would be both expensive and impractical to sequence or culture all of these in one year using current sequencing platforms. Therefore, a large proportion of the Neisseria gonorrhoeae multi-antigen sequence typing; NG STAR, Neisseria gonorrhoeae sequence typing for antimicrobial resistance; OMV, outer membrane vesicle; ST, sequence type; XDR-GC, extensively drug-resistant gonococci; WGS, whole genome sequencingAMR, antimicrobial resistance; BAPS, Bayesian Analysis of Population Structure; cc, clonal complex; ESC, extended-spectrum cephalosporin; EU/EEA, European Union/European Economic Area; HGT, horizontal gene transfer; MDR-GC, multidrug-resistant gonococci; MIC, minimum inhibition concentration; MLST, multi-locus sequence typing; NG MAST,