Brain levels of the endocannabinoid 2-arachidonoylglycerol (2-AG) shape motivated behavior and nucleus accumbens (NAc) dopamine release. However, it is not clear whether mobilization of 2-AG specifically from midbrain dopamine neurons is necessary for dopaminergic responses to external stimuli predicting forthcoming reward. Here, we use a viral-genetic strategy to prevent the expression of the 2-AG-synthesizing enzyme diacylglycerol lipase α (DGLα) from ventral tegmental area (VTA) dopamine cells in adult mice. We find that DGLα deletion from VTA dopamine neurons prevents depolarization-induced suppression of excitation (DSE), a form of 2-AG-mediated synaptic plasticity, in dopamine neurons. DGLα deletion also decreases effortful, cue-driven reward-seeking but has no effect on non-cued or low-effort operant tasks and other behaviors. Moreover, dopamine recording in the NAc reveals that deletion of DGLα impairs the transfer of accumbal dopamine signaling from a reward to its earliest predictors. These results demonstrate that 2-AG mobilization from VTA dopamine neurons is a necessary step for the generation of dopamine-based predictive associations that are required to direct and energize reward-oriented behavior. Inhibiting 2-AG synthesis in ventral tegmental area dopamine neurons may impair cue-driven motivated behavior and dopamine release. Here the authors report this, showing the role of endocannabinoid signaling in dopamine-based predictive associations. It Is generally assumed that activation of the main substrate for 2-AG, the cannabinoid type-1 receptor (CB1R), is responsible for the disinhibition of dopamine neurons located in the ventral tegmental area (VTA)4. In contrast, anandamide binds CB1Rs with submaximal potency, thereby blunting accumbal dopamine cell excitability5. Intriguingly, dopamine neurons are among the few brain cell types with little to no CB1R protein expression8, a surprising phenomenon considering that CB1Rs are the most abundant G protein-coupled receptor in the brain9. A growing body of evidence suggests that CB1Rs  are strategically positioned throughout the mesocorticolimbic circuitry to fine-tune dopamine neuron excitability3. In the VTA, CB1Rs are found in GABAergic and glutamatergic terminals impinging upon dopamine neurons11. In their seminal work, Lupica and Riegel (2005)12 described that eCB release from dopamine cells preferentially activates CB1Rs localized in GABAergic terminals13, leading to the disinhibition of VTA dopamine cell bodies in a feed-forward loop resulting in a net increase of dopamine outflow in terminal regions14.The brain endocannabinoid (eCB) 2-arachidonoylglycerol (2-AG), detected at concentrations a thousand times higher than those of the other eCB anandamide15. It has been theorized, but never unambiguously demonstrated, that 2-AG mobilization from dopamine neurons is a necessary mechanism for the initiation and invigoration of reward-oriented behaviors triggered by conditioned stimuli17. The primary constraint in providing an explicit demonstration has stemmed from the lack of viral-genetic tools capable of selectively excising –in vivo– distinct molecular components of the eCB machinery deployed by VTA dopamine neurons or their afferent pathways. Advances in mouse mutant lines18 and viral-genetic recombinant constructs19 hold great potential to overcome the difficulties inherent to pharmacological approaches. 2-AG is synthesized ‘on demand’ following increases in Ca2+ influx, which activates the catalyzing enzyme diacylglycerol lipase α (DGLα)20. Therefore, a selective experimental strategy arises from the possibility of excising DGLα from midbrain neurons, allowing for the interrogation of 2-AG mobilization and its consequences on downstream dopamine signaling and behavioral functions.While notable progress has been made delineating the neurocircuitry and electrophysiological properties of the dopamine and eCB systems, less is known about the precise neuropsychological implications emerging from their interaction21. This has been extensively supported by evidence that VTA dopamine neuron firing and NAc dopamine release phasically increase in response to better-than-expected events22 and, as learning proceeds, the dopamine signal transfers –along with action initiation– to the earliest predictors of forthcoming reward access23. This encoding by dopamine signals stands as one of the most replicated phenomena in neuroscience24. However, despite its robustness, the underlying molecular mechanisms giving rise to these dynamic signaling changes remain largely unexplored. Pre-existing pharmacological evidence indicates that elevations of 2-AG tone25, or activation of CB1R2, boost execution of effort and dopaminergic encoding of reward-predictive cues. In light of this evidence, we hypothesize that mobilization of 2-AG is a requisite step for VTA dopamine neurons to progressively sculpt phasic dopamine release events to the earliest predictors of forthcoming reward. Subsequently, we postulate that midbrain dopamine neuron 2-AG mobilization promotes rapid cue-evoked reward seeking behaviors and effortful behavioral engagement.Mesolimbic dopamine neuron projections from the VTA to the nucleus accumbens (NAc) gate the selection and invigoration of appetitive behaviors triggered by outcome-predictive cuescre recombinase vector driven under the promoter of tyrosine hydroxylase (TH) –a dopamine neuron marker– was injected into the VTA of DGLαf/f adult mice, therefore preventing the ability of this neuronal population to mobilize 2-AG in behaving animals.Here, we sought to isolate the functional relevance of the 2-AG-synthesizing enzyme (DGLα) in VTA dopamine neurons in behaving mice using a viral-genetic approach with negligible effects on motor function and non-cued operant responding but striking implications for the acquisition and invigoration of conditioned reward seeking and accompanying dopamine signals. To achieve this, a cre recombinase under a tyrosine hydroxylase (TH) promoter in the VTA of wild-type (WT) and DGLαf/f mice  in the VTA of WT mice to confirm the specific expression of TH-driven cre recombinase in VTA dopamine neurons   breaking points  or rewards earned . Additional metrics were obtained following PR testing to eliminate non-specific confounds related to bodyweight  dopamine neurons in motivated behavior, we expressed ice Fig. . Figure ly) Fig. , male anly) Fig.  , into the VTA of male and female WT and DGLαf/f mice  displayed characteristic spontaneously-clustered lever pressing ‘bursts’. Should motor deficits arise from our manipulations, then it would be reasonable to expect significant changes in how lever presses clustered within each engagement event: i.e., reduced response density within each ‘burst’ (fewer presses per ‘burst)’. In contrast, changes in how lever-pressing behavior is hierarchically structured during the task  should be independent of potential motor impairments, but rather reflect the motivation of the animal to engage in reward-oriented behaviors. The distribution of inter-response intervals during the PR session revealed that the TH-cre viral injection, DGLαf/f genotype, or its combination (DGLαTH cKO) were not associated with slower rates of responding 34. Here, we examined whether conditional inducible deletion of DGLα from dopamine neurons resulted in the loss of DSE. Specifically, WT and DGLαf/f mice were bilaterally transduced with AAV2/10-TH-cre in addition to AAV5-DIO-Chr2-eYFP, hence allowing us to optogenetically depolarize dopamine neurons  is a hallmark form of 2-AG-mediated synaptic plasticityons Fig. . Single les Fig. . To furtTH cKO mice to simultaneously prevent 2-AG production and enable optogenetic intracranial self-stimulation (oICSS) of dopamine neurons35  to better resemble spontaneous NAc dopamine release events in response to rewarding stimuli. Next, WT and DGLαTH cKO animals were trained to lever-press for VTA laser self-stimulation throughout five FR1 and one variable time-out (VTO) oICSS sessions. These operant tasks were chosen to selectively deconvolve the role of outcome-predictive cues in the motivational phenotype associated with DGLα deletion from dopamine neurons. During FR1 reinforcement (30-min sessions), actuation of the laser-coupled lever resulted in non-cued optostimulation of VTA dopamine neurons , as well as the onset of proximal (lever extension) and distal predictors (compound light + tone cue). Representative FSCV color plots and in vivo NAc dopamine concentration traces can be seen in Supplementary Fig. ard Fig. . Secondlard Fig. , discardice Fig. .TH cKO mice significantly deviated from the patterns expected from the conceptual framework of dopamine signals, and those observed in WT mice. NAc phasic dopamine transients in response to the distal cue progressively increased across sessions in WT animals  in early versus late VTO sessions  as well as the lack of dynamic range observed for accumbal dopamine encoding of reward-predictive distal cues and rewards. In addition, these effects were accompanied by a notable decrease in effortful motivation. Importantly, experimentally induced dopamine cell-specific impairment of 2-AG mobilization was not linked to gross motor output alterations, basic associative learning deficits or reduced operant reward seeking in scenarios not requiring the exploitation of predictive cues or high effort. These studies support the critical role of 2-AG signaling by VTA dopamine neurons to sculpt accumbal release patterns consistent with encoding and appraisal of distal outcome-predictive cues.By modulating limbic-motor striatal regionsvia DGLα-synthesized 2-AG, providing a disinhibitory mechanism34. That is, when dopamine neurons burst at high-frequency, elevation in intracellular Ca2+ levels lead to DGLα catabolic activity43. Consequently, synthesis and retrograde release of 2-AG activates CB1R located on GABAergic terminals, thus procuring the suppression of GABA release30. This dopamine neuron disinhibition results in an increase in dopamine terminal release44. Thus, this empirical evidence supports a role for midbrain eCB signaling as a potential substrate sustaining the backpropagation of motivational information throughout the mesolimbic circuitry, closing the loop between top-down afferents to the VTA and bottom-up dopamine release45.Our data is consistent with the notion that VTA dopamine neurons communicate retrogradely with CB1R-expressing GABAergic afferences 46. Of note, the loss of DSE reported in DGLαTH cKO animals is not thought to directly participate in the behavioral and dopaminergic effects reported here. Blunting CB1R-induced inhibition of glutamatergic influences (DSE) should result in the facilitation of dopamine cell function and its related functions, in opposition to the phenotype here unveiled. Instead, we postulate that a loss of DSI was responsible for the observed effects, as it is consistent with a disinhibition of GABAergic input onto VTA dopamine neurons. Here, to demonstrate the effectiveness of our viral-genetic strategy, we used DSE, therefore assessing the effectiveness of eCB-mediated synaptic plasticity in recordings of adult VTA dopamine neurons47.In our experimental conditions, DGLα ablation negated the ability of VTA dopamine cells to backpropagate information about its depolarization events, which resulted in the absence of DSE. DSE/I is the most commonly observed form eCB-based plasticity that allows dopamine neurons to accommodate the influence of excitatory (DSE) or inhibitory (DSI) inputs to their burst firing events48. We thoroughly examined phenotypic differences to rule out the influence of extraneous effects of our viral-genetic model that could affect dopaminergic, behavioral or learning performance. Our data indicates that DGLα deletion from VTA dopamine cells did not change free-feeding, non-operant consumption of sucrose. It neither altered exploratory nor anxiety-like responses. Moreover, we conducted an in-task characterization of motor skills presumably involved in lever pressing, which would introduce a notable confound in the present findings. Nonetheless, our results discarded that down-regulation of 2-AG mobilization from dopamine cells impaired the motor execution of lever pressing, as we did not detect changes in how individual responses spontaneously clustered in high-frequency responding events (or lever pressings ‘bursts’). Instead, DGLαTH cKO engaged fewer times in reward-directed behaviors (number of ‘burst’ events), a metric independent of lever press execution. Next, we demonstrated that no gross alterations in dopamine release and basic learning functions were present in DGLαTH cKO mice. First, we proved that VTA dopamine neurons deprived of 2-AG synthesis can still sustain self-optostimulation and act as a primary reinforcer. Second, we revealed that DGLαTH cKO also exhibited dopamine predictive encoding in the VTO task, as lever extension  triggered normal NAc dopamine elevations known to precede action initiation toward appetitive stimuli50. The increased lever-evoked dopamine signal across sessions may be indicative of faster action initiation toward the lever , which was evident in both groups, albeit at different rates. However, this signal was not transferred to the antecedent cue following deletion of DGLa in VTA dopamine neurons. This finding also discards perceptual impairments related to the sensory characteristics of the specific compound light+sound distal cue here employed. We also observed that deletion of DGLα in VTA dopamine neurons increased reward-evoked NAc dopamine release during VTO. This is congruent with an underutilization of outcome-predictive cues preceding the reward’s availability. According to formal learning theories of dopamine signals, unpredicted rewards trigger greater release of NAc dopamine compared to otherwise predicted outcomes52. Interestingly, goal-seeking behavior in low-demand and non-cued scenarios (FR1) remained unaffected following genetic DGLα removal from VTA dopamine neurons. This finding supports that exploitation of cues becomes increasingly relevant as behavioral and temporal resources become scarce54, hence maximizing an organism’s evolutionary fitness to the environment when access to rewards is compromised. However, the question remains unresolved as to whether the mobilization of midbrain 2-AG is distinctly involved in both effortful motivation (PR) and cue-driven responding (VTO), or if these phenomena stem from a shared underlying cause. In our previous research utilizing an adapted PR schedule, we observed that the encoding of NAc dopamine in response to distal cues varies as a function of the anticipated effort needed to attain a reward25. Notably, cues indicating a high-effort trial trigger dopamine responses of reduced magnitude compared to cues indicating low effort25. Given the compromised dopaminergic encoding of distal cues exhibited by DGLαTH cKO mice, deficient dopamine encoding of such distal cues during PR responding  could explain the diminished execution of effort in absence of 2-AG mobilization. To ascertain the interdependence of these phenomena, future experiments should involve monitoring dopamine release through a PR chain schedule of reinforcement, wherein distal cues denote distinct effort requirements. Finally, no spurious effects of the DGLαf/f genotype were evident, as the disruption of effortful motivation and rapid conditioned responding was absent in DGLαf/fsham mice.eCBs are amongst the most ubiquitous signaling molecules in the brain and, consequently, a large volume of work has described its many functions in emotional regulation, motor function and memoryIn light of these findings, we propose that production of the endogenous CB1R ligand 2-AG by VTA dopamine neurons is a necessary mechanism for this cellular population to function as the canonical neural substrate of reward prediction during reward seeking.Guide for the Care and Use of Laboratory Animals. Ethical approval was granted by the Institutional Animal Use and Care Committee at the University of Maryland, Baltimore (IACUC protocol #00000054).All experimental procedures conformed to the National Institute of Health f/f on a C57BL6/J background mice . DGLαf/f mice were obtained from Vanderbilt University55. DGLαf/f mice were maintained by homozygote x homozygote breeding. Genotypes were determined by PCR of genomic tail DNA using the following primers (5′–3′): TGAGCCAGAGACATTTGCTG, CTGGTGAGGCCAAGTTTGTT and GGGACAGAAAACCACTTGGA. Animals were housed in a temperature- and humidity-controlled room  and maintained on a 12 h light/dark cycle (07:00–19:00 h). All experiments were conducted in the light cycle. The number of mice for each experiment are indicated in the respective figure legends. Sex of the animals is reported throughout the figures and its legends, with open circles representing female subjects and closed circles referring to male mice. Sex was added as an additional factor in all the parametric analyses performed. When no sex effect was detected, males and females were collapsed in the same group and analyses proceed without including sex as a biological variable. A significant sex difference was only observed for body weight but no interaction with our experimental manipulations was found .For all experiments, we used either wild-type C57BL6/J or DGLαMice underwent isoflurane anesthesia (5%) and transcardial perfusion with a 4% paraformaldehyde (PFA) solution in a 0.1 M sodium phosphate buffer (PB) at pH 7.4. Following perfusion, brains were post-fixed at 4 °C in PFA overnight. Brain sections, (40 µm thick) were obtained using a vibratome (Leica). For the immunohistochemistry of tyrosine TH DGLα, coronal sections were immersed in a PB solution containing 0.2% Triton X-100 (Sigma-Aldrich) and 3% normal donkey serum (Jackson 017-000-121) for a duration of 30 min. Subsequently, brain sections were incubated with primary antibodies  overnight. Secondary antibodies , were applied for 2 h, followed by staining with 4’,6-diamidino-2-phenylindole . Sections were then mounted on slides for visualization of TH, DGLα, and eYFP. Imaging was performed using a confocal microscope . Animals lacking viral expression in the target region were excluded from the data analysis.Cre, Th and Dagla mRNAs cellular distributions in the VTA were detected using the following RNAscope probes : Mm-Th-C2 , Mm-Dagla-C3  and Mm-CRE-C1 . The RNAscope mRNA assays were performed following the manufacturer’s protocols. Stained slides were covered with DAPI mounting medium  and scanned into digital images with an Olympus Fluoview confocal microscope at 20× magnification.RNAscope ISH was employed to ascertain the cell type-specific expression of Cre recombinase mRNA within dopamine TH-expressing cells. Following deep anesthesia induction, mice brains were promptly removed and frozen at −80 °C for coronal sectioning (14 μm thick) in a Leica microtome. The resulting brain slices were then mounted on glass slides (Fisher Scientific). Mounted samples were kept at −80 °C until ISH assays were conducted. f/f mice were transduced with AAV2/10-TH-cre in the VTA and left undisturbed in their home-cage for 3 weeks. On test day, mice were decapitated, and their brains were rapidly removed and transferred to an oxygenated (95% O2/5%CO2) ice-cold solution containing: 93 mM NMDG, 2.5 mM KCl, 1.2 mM NaH2PO4, 30 mM NaHCO3, 20 mM HEPES, 25 mM Glucose, 5.6 mM Ascorbic acid, 3 mM Sodium pyruvate, 10 mM MgCl2, 0.5 mM CaCl2. Horizontal slices containing VTA (220 µm) were transferred to a holding chamber filled with oxygenated solution containing: 109 mM NaCl, 4.5 mM KCl, 1.2 mM NaH2PO4, 35 mM NaHCO3, 20 mM HEPES, 11 mM Glucose, 0.4 mM Ascorbic acid, 1 mM MgCl2, 2.5 mM CaCl2. Slices were initially incubated at 35 °C for a duration of 10–12 min. Subsequently, they were transferred to room temperature, where they remained until the experiments began. The slices were then moved to a recording chamber and submerged in constantly-flowing (2 ml/min) oxygenated artificial cerebrospinal fluid (aCSF) (32–34 °C). The aCSF composition consisted of 3 mM KCl, 26 mM NaHCO3, 2.4 mM CaCl2, 126 mM NaCl, 11 mM Glucose, 1.2 mM NaH2PO4, and 1.5 mM MgCl2. Visualization of the slices was achieved using a differential interference contrast (DIC) optics upright microscope . We recorded lateral VTA neurons located anterior to the third cranial nerve and medial to the terminal nucleus of the accessory optic track.DGLα2, 10 mM Tris-phosphocreatine, 10 mM HEPES, 0.3 mM Na-GTP, 4 mM Mg-ATP, 0.1 mM EGTA. The pH of the internal solution was adjusted to 7.2, and the osmolarity was maintained at 290 mOsm. Picrotoxin (50 mM) was added to the aCSF for recordings to isolate excitatory transmission. EPSCs were evoked using a train of five-stimuli  delivered at 50 Hz every 30 s with bipolar tungsten stimulating electrodes with tip separation 100–200 µm. The amplitudes of EPSCs were calculated by taking a 1 ms window around the peak of the EPSC and comparing this with the 5 ms window immediately before the stimulation artifact. The depolarizing pulse used to evoke depolarization-induced suppression of excitation (DSE) was achieved by optostimulation of ChR2-expressing TH+ neurons . The magnitude of DSE was measured as percentage of the mean amplitude of consecutive EPSCs after depolarization (acquired between 10 and 50 s after the end of the pulse) relative to that of five EPSCs before the depolarization. Stimulation protocols were generated and signals acquired using the WinLTP program. In an additional experiment . Recording pipettes with a resistance of 3–5 MΩ were filled with an internal solution containing: 2 mM NaCl, 140 mM K-gluconate, 1.5 mM MgCl19 were injected bilaterally into the VTA  of DGLαf/f mice. In a separate set of animals . Mice were allowed 4 weeks to recover from surgery and allow viral expression.Mice were anesthetized with isoflurane in O2 . Then, to induce a conditional DGLα knockout in VTA dopamine neurons, 300 nl/side of AAV2/10-TH-iCre There were four main series of behavioral experiments, each one corresponding to each main figure. Mice underwent either sucrose PR Fig. , sucroseTH cKO mice underwent four PR sessions. Data from the last session is shown. (2) WT, DGLαf/fsham, and DGLαTH cKO mice, appearing in Fig. TH cKO animals  For the experiments reported in Fig.  For the s shown.  WT, DGLαals Fig.  were firals Fig. . 4) For ForTH cKIn all cases, mice were tested in operant chambers  housed within sound-attenuating enclosures. Each chamber was equipped with two retractable levers (located 2 cm above the floor), one LED stimulus light and a 2.5 kHz tone-generating speaker located above each lever (4.6 cm above the lever). A houselight and a white-noise speaker  were located on the opposite wall. For FR1, PR and VTO experiments, an external food magazine was placed within the box that delivered sucrose pellets  to a dispenser centrally located between the two levers. Two weeks after surgical procedures, mice were mildly food deprived (85–90% of starting body weight), receiving standard laboratory mouse chow daily in addition to food rewards earned during task performance.When FR1 training was required (see “Operant training schedules” subsection), mice were trained under FR1 schedule of reinforcement with a 10 s timeout in 30-min daily sessions. During FR1 training, animals had continued access to the active and inactive levers (except for the timeout period) and each active lever press was rewarded with a sucrose pellet. Pellet acquisition did not have any other consequences. Responses on the inactive lever were recorded but had no programmed consequences. For the experiment shown in Fig. (0.2 x reward number) - 5)’, after rounding to the nearest integer. The response ratio of the first sixteen trials was: 1, 2, 4, 6, 9, 12, 15, 20, 25, 32, 40, 50, 62, 77, 95 and 118. The last response requirement attained, also known as breakpoint, was recorded and used to infer the inherent motivation for the reward. At the beginning of each session, both active and inactive levers were extended, accompanied by the illumination of the cue light placed on top of the active lever and the house light. Actuation of the inactive lever had no consequences, but inactive responses were recorded. Upon response requirement completion, a pellet was delivered, and, for 10 s, levers retracted, lights were turned off and a 2.5 kHz tone was emitted. After the end of this 10 s time-out period, a new trial began. The PR session ended whenever no reward could be obtained within 20 min. Four PR sessions were conducted and data from the last session, –once the lever pressing behavior was acquired– is depicted.A progressive ratio schedule of appetitive reinforcement was used to estimate the effort mice were willing to expend for a sucrose pellet reward. On each successive trial, the response requirement (lever presses) needed to obtain a reward scaled near-logarithmically, as determined by the function ‘response requirement = . FSCV recordings were performed every session. For control purposes, a group of DGLαf/fsham mice were run along WT and DGLαTH cKO animals  ranging from 30–60 s (average 45 s). Then, mice were transferred to an operant VTO (30–60 s) operant schedule of reinforcement, similar to that used in prior workf/fsham mice were tested for anxiety-like behaviors using a procedure similar to one previously reported58. Briefly, The EPM test took place in a black maze that was elevated 30 cm off the ground and lit from above. Each mouse was placed in the center of the maze and allowed to explore for 5 min. The total time spent in the open arms were recorded using the EthoVisionXT 17 video-imaging system. The total number of entries  was also counted.WT, DGLαad libitum feeding conditions. WT and DGLαTH cKO  coupled to 62.5 mm core, 0.22 NA optical fiber (Thor Labs). Light output was ~10–20 mW at the tip of the ferrule. In all cases, laser stimulation consisted of 4 ms pulses during 1-s at 30 Hz. Presses on one lever produced immediate laser stimulation accompanied by a 1-s illumination of the cue light and activation of the 2.5 kHz tone placed above the lever, while presses on the other lever (inactive) or presses on the active lever during an ongoing stimulation (non-reinforced) were collected but had no programmed consequences. A separate group of WT mice  was applied (10 Hz) to carbon fiber microelectrodes aimed at the NAc of WT and cKO mice. Redox reactions around the carbon fiber tip were detected as fluctuations in faradaic current from which concentration changes of the electroactive analyte were derived. Thus, all voltammetric measurements are reported in current units (nA). Data was collected using LabView 2020. Using principal component regression (PCR), we statistically derived the dopaminergic component of the voltammetric measurements obtained during cue onset and the delivery of the reward (5-s window)59. Lever-evoked dopamine levels were calculated over the total time elapsed between lever extension and pellet delivery on a trial-by-trial basis. A visual representation of the time intervals used for quantification measurements is depicted in Supplementary Fig. As in prior studiest tests and Holm–Šídák post hoc test was used to correct for multiple comparisons when appropriate. For all parametric analyses, Welch’s corrections were applied to all the cases wherein violations of homoscedasticity were detected. If so, Welch’s-corrected analyses were followed by Dunnett’s post hoc test. When analyzing ordinal variables (rewards/breaking points on PR task), the non-parametric Mann–Whitney statistic was employed in unifactorial designs and the Kruskal Wallis test in multifactorial cases, followed by Dunn’s post hoc tests. Significance was set at p < 0.05 and all tests were two-tailed. Statistical analyses were performed in Prism 9.5 (GraphPad), Demon Analysis Software, Matlab 2020a and SigmaPlot 14.5. Statistical details and sample size for each experiment are presented in figure legends. No statistical methods were used to pre-determine sample sizes. Confirmation of viral expression and optic fiber/electrode implantation in each mouse was done through histology. Similar expression and/or localizations  ANOVA, or unpaired ns Figs.  were repFurther information on research design is available in the Supplementary InformationPeer Review FileReporting SummarySource Data


The rise of antimicrobial resistance (AMR) is a major global public health threat due to excessive and inappropriate use of antibiotics and is responsible for prolonged illness, longer hospital stays, and economic burden to society. This article aims to review the factors, role of antimicrobial stewardship, preventive strategies, and role of various organizations in combating AMR. Three major factors of AMR are inappropriate and excessive utilization of antibiotics, nonadherence to infection control measures, and the emergence of pathogens that are resistant to multiple drugs. Antimicrobial stewardship initiatives play a vital role in promoting judicious and targeted utilization of antimicrobials, thereby safeguarding their efficacy and mitigating the emergence of resistance. Implementing such programs optimizes patient outcomes by ensuring that individuals receive the most suitable therapeutic interventions. International organizations have a vital role to play in addressing AMR by promoting the responsible use of antimicrobials, developing new drugs, and improving surveillance systems. As AMR's impact grows, it is critical to take a collaborative and interdisciplinary approach to mitigate its consequences effectively. Antimicrobial resistance (AMR) is one of the top global public health issues, which is primarily caused by the misuse and overuse of antimicrobials. AMR is responsible for prolonged illness, longer hospital stays, and an economic burden to society. AMR is making antibiotics more and more ineffective, necessitating the urgent need for new antibacterials -3.AMR refers to the capacity of bacteria to proliferate and disseminate within the human body, even when exposed to medications intended to eradicate or impede their growth . The risCauses of AMRThree major factors of AMR are inappropriate and excessive utilization of antibiotics, nonadherence to infection control measures, and the emergence of pathogens that are resistant to multiple drugs. The widespread and indiscriminate use of antibiotics, spanning human and animal populations, imposes selective pressures that facilitate the development of bacteria with resistance capabilities.Misuse and Overuse of AntibioticsThe improper and excessive utilization of antibiotics represents a significant challenge in the realm of public health. In instances where antibiotics are used without genuine necessity or in an inappropriate manner, they can foster the propagation of AMR strains. Subsequently, these resistant strains can disseminate to other individuals, posing further risks ,9. The aFailure of Infection Control PracticesInfection control is a set of measures to prevent communicable diseases . MaintaiEmergence of Multiple-Drug-Resistant (MDR) PathogensMDR microorganisms have the capacity to withstand numerous categories of antimicrobial agents, thereby impeding effective treatment approaches ,18,19. PGlobal trends in AMRThe distribution of AMR exhibits geographical and regional variation, highlighting an inequitable burden across the globe. According to the World Health Organization (WHO), AMR affects approximately 75% of the population globally ; howeverStaphylococcus aureus (MRSA) and carbapenem-resistant Enterobacteriaceae (CRE). The dissemination of multidrug-resistant tuberculosis (MDR-TB) represents another prevailing occurrence. AMR instills mounting apprehension within the context of sexually transmitted infections, notably gonorrhea. Additionally, it progressively compromises the efficacy of novel antimicrobial agents, including antivirals and antifungals [AMR poses a considerable challenge in effectively combating numerous infectious diseases, rendering their treatment increasingly arduous . Certainifungals ,28. It iifungals .Mechanisms of AMRAMR emerges when microorganisms acquire the capacity to endure exposure to antimicrobial agents that are specifically formulated to eradicate them or impede their proliferation . Common Genetic MutationMicrobial entities possess the ability to acquire resistance against antimicrobial agents through genetic alterations, referred to as mutations, within their DNA sequences. These mutations can transpire spontaneously or may be provoked by the presence of antimicrobial substances .Genetic TransferMicroorganisms possess the capability to develop AMR via genetic transfer which facilitates the acquisition of resistance traits. This phenomenon can occur through diverse mechanisms, including horizontal gene transfer, which involves the direct transfer of genetic material between organisms. Additionally, resistance genes can be acquired through plasmids or transposons, which serve as carriers capable of conveying resistance genes from one organism to another ,33.Alteration of Drug Target, Drug Inactivation, and Efflux PumpsSome microorganisms can acquire resistance through alteration of the drug target, which denotes the precise location within the organism that the drug is intended to combat, thus rendering it less vulnerable to the effects of the drug attack ,34,35. BBiofilm FormationCertain microbial entities have the capacity to create biofilms, characterized by slender strata comprising microorganisms adhered to a surface and encompassed by an extracellular matrix. These biofilms possess the ability to shield against antimicrobial agents, resulting in heightened challenges in eradicating associated microorganisms -43.Role of antimicrobial stewardshipAntimicrobial stewardship encompasses the conscientious utilization of antimicrobial agents, including antibiotics, to mitigate the occurrence and dissemination of AMR ,45. The Antimicrobial stewardship can enhance patient outcomes by curbing the occurrence and dissemination of AMR and preserving the efficacy of currently available antimicrobial agents ,49. ThesAntimicrobial stewardship is of significant importance because of the adverse effects of the excessive and improper utilization of antimicrobials, which can lead to the emergence and proliferation of AMR. Antimicrobial stewardship initiatives play a vital role in promoting judicious and targeted utilization of antimicrobials, thereby safeguarding their efficacy and mitigating the emergence of resistance. By implementing such programs, patient outcomes can be optimized by ensuring that individuals receive the most suitable therapeutic interventions. Thus, the proper management of antimicrobials and balance between effective treatment and minimizing the development of resistance can be achieved -53. ThusThe key components involved in antimicrobial stewardship are education, surveillance, intervention, and evaluation Table .Strategies for preventing AMRTo tackle AMR, it is important to adopt an interdisciplinary approach that acknowledges the intricate and interrelated nature of the contributing factors. Numerous strategies can be implemented to prevent AMR Table .Social and economic impact of AMRAMR can have a massive social and economic impact. In terms of economics, AMR can lead to increased healthcare costs due to the need for more expensive or prolonged treatments, as well as the cost of managing outbreaks of resistant infections. AMR can also affect the agriculture industry, as antimicrobials are used to prevent and treat infections in animals . Also, lRole of international organizations in addressing AMRInternational organizations are uniquely positioned to address AMR, given that it is a global health threat that requires a collaborative and cross-sectoral approach. Several international organizations are working to address AMR.WHOThe WHO is the United Nations agency responsible for international public health. It has developed a Global Action Plan that outlines several strategies for addressing AMR, including improving surveillance, promoting the development of new antimicrobials, and improving access to clean water and sanitation .Food and Agriculture Organization of the United Nations (FAO)The FAO works to improve food security and nutrition, and has recognized the importance of addressing AMR in the livestock sector. It has developed guidelines for the responsible use of antimicrobials in animal agriculture and is working to improve surveillance and data collection on antimicrobial use in this sector ,77.World Organization for Animal Health (WOAH)The WOAH ) is an international organization that promotes the health and welfare of animals and works to prevent and control animal diseases. It has developed guidelines for the responsible use of antimicrobials in animal health and is working to improve surveillance and data collection on antimicrobial use in this sector ,79.The World BankThe World Bank is a financial institution that provides loans and grants to countries to support economic development and poverty reduction. It has recognized the economic consequences of AMR and has funded projects related to AMR, including efforts to improve surveillance and data collection and to promote the development of new antimicrobials ,80.Group of 20 (G20)G20 has recognized the importance of addressing AMR and has committed to taking action to address this issue ,82.Global Antimicrobial Resistance Partnership (GARP)GARP is a multi-sectoral partnership that brings together experts from a range of fields to address AMR ,84.International Council of Nurses (ICN)The ICN is a professional organization that represents nurses globally. It works to address AMR through the promotion of antimicrobial stewardship and the development of guidelines and recommendations for the appropriate use of antimicrobials in healthcare settings ,86.Global Antimicrobial Resistance Surveillance System (GLASS)GLASS is an initiative of the WHO that aims to strengthen global surveillance of AMR and improve the data available on the problem ,88.The roadmap for addressing AMR"A Roadmap to Tackle the Challenge of Antimicrobial Resistance - A Joint Meeting of Medical Societies in India" was convened as a symposium during the second annual conference of the Clinical Infectious Disease Society in Chennai, India, in 2012. This groundbreaking assembly marked the inaugural congregation of medical societies in India, united with the objective of devising a comprehensive strategy to combat the global challenge of AMR from an Indian standpoint. Distinguished representatives from numerous medical societies across India, eminent policymakers from both central and state governments, delegates from the WHO, National Accreditation Board of Hospitals, Medical Council of India, Drug Controller General of India, and Indian Council of Medical Research, alongside prominent personalities within the Indian medical domain, were in attendance, collectively steering the discourse towards an effective response against AMR .The WHO's Expert Committee on Selection and Use of Essential Medicines created the AWaRe classification system for antibiotics in 2017. It's an important tool for advancing antibiotic stewardship efforts worldwide. The classification system divides antibiotics into three categories: Access, Watch, and Reserve. This categorization takes into account the impact of various antibiotics and antibiotic classes on the development of AMR. It underscores the critical importance of using antibiotics judiciously and responsibly. The AWaRe classification is updated every two years. Its primary purpose is to monitor antibiotic consumption, establish benchmarks, and assess the outcomes of stewardship policies. The WHO 13th General Programme of Work from 2019 to 2023 outlines a country-level target. The target states that at least 60% of total antibiotic consumption should be attributed to antibiotics in the Access group. This effort aims to enhance the accessibility and appropriate use of antibiotics on a global scale .Overall, international organizations play a vital role in coordinating efforts to address AMR and in providing support for countries to implement strategies to reduce the emergence and spread of resistance.AMR is a growing health threat. It requires an interdisciplinary approach and collaborative efforts to recognize and combat AMR, which is a serious health and economic burden that affects all sectors. International organizations and countries need to support the development and implementation of policies to reduce the inappropriate use of antibiotics in both humans and animals. Greater investment in research and development for new antibiotics is needed.


First Person is a series of interviews with the first authors of a selection of papers published in Biology Open, helping researchers promote themselves alongside their papers. Lara Cantarero is first author on ‘ Lara CantareroDescribe your scientific journey and your current research focusI did my bachelor's degree in biology at the University of Barcelona. In 2010 I started my PhD at the Cancer Research Center of Salamanca (IBMCC-CIC), in Dr Lazo's group, where I characterized the molecular mechanism by which VRK1 kinase regulates the dynamics and stability of Cajal bodies. In 2016, I joined the Neurogenetics and Molecular Medicine group as a postdoctoral researcher, directed by Drs Palau and Hoenicka at the Sant Joan de Déu Research Institute (IRSJD). My research has been focused on the pathophysiology of Charcot-Marie-Tooth neuropathy, in cellular and murine models, patient fibroblasts, and induced neurons. In recent months, I have joined the Translational Diagnostic and Therapeutic Program (TDTP) of our laboratory for translational research in patient samples and the search for therapeutic targets. The TDTP objective is to use molecular and cellular biology to explore new treatments and clinical trials, through molecular characterization of patients, identification of cell phenotypes and biomarkers, drug screening for repositioning and personalized molecular therapies.Who or what inspired you to become a scientist?Cellular biology has fascinated me since childhood. Science is a way of thinking, and I think you are truly privileged if you ask the right question so that the answer comes back meaningful. And then, if nature tells you the answer, it's something magical!As the scientist Carl Sagan, whom I have always admired, wrote:Science is part and parcel humility. Scientists do not seek to impose their needs and wants on Nature, but instead humbly interrogate Nature and take seriously what they find. We are aware that revered scientists have been wrong. We understand human imperfection. We insist on independent and – to the extent possible – quantitative verification of proposed tenets of belief. We are constantly prodding, challenging, seeking contradictions or small, persistent residual errors, proposing alternative explanations, encouraging heresy. We give our highest rewards to those who convincingly disprove established beliefs.How would you explain the main finding of your paper?GDAP1 gene: the mitochondria–lysosome membrane contact sites, a microenvironment where many cellular processes are altered in patients.Charcot-Marie-Tooth neuropathy is a rare disease, although it is the most common hereditary neuropathy, affecting 2.8 million people worldwide. Currently, there is no effective treatment. It is for this reason that the study of the affected cellular processes is necessary to find out possible new therapeutic targets that help in the development of advanced therapies. In this work, we have delved into one of the cellular processes altered in Charcot-Marie-Tooth caused by mutations in the What are the potential implications of this finding for your field of research?GDAP1-related Charcot-Marie-Tooth, which can be very disabling. In addition, due to the very nature of GDAP1, an atypical GST-transferase, the results begin to shed light on the relevance of these proteins in the structure of the mitochondrial–lysosome contacts.I hope that these results will bring more functional knowledge to the field of Charcot-Marie-Tooth research. Specifically, in Which part of this research project was the most rewarding?GDAP1 mutations presents differences in clinical expression in relation to the Mendelian inheritance pattern: the recessive forms are very severe while the dominant forms are mild or moderate. The fact that we were able to show a correlation between the Mendelian pattern of inheritance and the different consequences on mitochondrial network morphology and mitochondria-lysosome contacts was remarkable. These results also suggest that, even for the same gene, the design of personalized therapies can vary to rescue the underlying defect at its source.Charcot-Marie-Tooth neuropathy due to What do you enjoy most about being an early-career researcher?Personally, I think it is a wonderful stage in my scientific career. After more than 10 years of dedicating myself to science, I have been learning a lot of techniques, acquiring great autonomy and, above all, starting to enjoy every question and answer achieved. You no longer have the rush and the time pressure of the doctoral thesis, but you can go driving a project, thinking about future experiments, and achieving major milestones. In addition, I love to be able to teach the younger researchers and students who come to the lab, to see their faces when they get their first western blot and to be able to transmit and spread them with a passion for science.What piece of advice would you give to the next generation of researchers?Starting a scientific career can be difficult at first, you will spend many hours in the lab. That's why I think it's very important for young scientists to be as passionate and inspired as possible. The life of a scientist is full of sacrifices, obstacles, experiments that do not work out and frustrations, however, all these difficulties are compensated by a fascinating side, when you confirm your hypothesis, when you get that experiment you have been struggling with for months, or when you identify the key cellular process that will lead to future therapies. I can only say that science gives you much more than it takes away. It is also crucial to have a great mentor, someone who orientates you, gives advice and helps you to grow.What's next for you?I have enjoyed doing science for more than 10 years. I still enjoy working at my bench, doing experiments, and writing papers and research projects. I hope I can continue to do this for a very long time. In the coming years, I will focus on the study and design of new personalized therapies in pediatric rare diseases, a research area where cell biology and clinical biology meet, in a round-trip path from the laboratory and the cell to the patient.


The rapid progress in the field of fluorescent probes and fluorescent sensing material extended this research area toward more complex molecular logic gates capable of carrying out a variety of sensing functions simultaneously. These molecules are able to calculate a composite result in which the analysis is not performed by a man but by the molecular device itself. Since the first report by de Silva of AND molecular logic gate, all possible logic gates have been achieved at the molecular level, and currently, utilization of more complicated molecular logic circuits is a major task in this field. Comparison between two digits is the simplest logic operation, which could be realized with the simplest logic circuit. That is why the right understanding of the applied principles during the implementation of molecular digital comparators could play a critical role in obtaining logic circuits that are more complicated. Herein, all possible ways for the construction of comparators on the molecular level were discussed, and recent achievements connected with these devices were presented. The great attention paid in the last thirty years to the design and fabrication of variety machinery and devices at the molecular level has led to significant progress, as a result of which this field has been expanded from intramolecular motion and molecular switches ,4,5,6,7 + (input 1) and benzo crown ether moiety for Na+ (input 2). In order to obtain a strong fluorescence signaling output from the anthracene fluorophore, the PET quenching processes needed to be suppressed from both receptors. In the presence of protons, the PET from the alkylamine receptor was interrupted, but fluorescence emission was not observed because of the PET quenching effect from the crown ether. The presence of sodium cations inhibits a PET process from the crown ether but not from the amino receptor, and the system again does not fluoresce. Simultaneous use of both H+ and Na+ interrupts both PET processes to the anthracene fluorophore, resulting in strong fluorescence. In binary, the resulting behavior satisfied AND logic gates  and TRUE (YES) are encoded in binary code as “0” and “1”, respectively. Usually, the semiconductor logic gates operate via two inputs and one output, which states they possess one of the two binary conditions “0” or “1” encoded using a low (0) or high (1) voltage. This process is achievable on the molecule level in many ways, but the most common ones are based on the optical switching properties of molecule chemosensors ,25, usinic gates .Since the first report by de Silva et al. , the fieComparison between two digits is the simplest logic operation, which could be realized with the simplest logic circuit. The right understanding of the applied principles during the implementation of molecular digital comparators could play a critical role in obtaining logic circuits that are more complicated. However, review papers in the literature are very rare. Generally, the reported reviews were focused on the design and fabrication of molecular logic circuits such as devices for addition and subtraction, key lock pads, multiplexers, demultiplexers, encoders, decoders, “write-read-erase-read” function ,59,60,61In general, there exist two basic types of digital comparators: identity comparators and magnitude comparators . The sim1  chemosensing architecture pyrrole in compound 6 leads to a bathochromic (red) shift. The presence of K+ and F− reduced their opposite individual effects and returned the absorption spectrum of 6 near the former state. Hence, the recording of the absorption spectrum of probe 6 at shorter wavelengths (650 nm) showed high binary output only in the presence of K+ as an input alone, which is well correlated with the INHIBIT logic gate. In addition, the monitoring at a long wavelength (715 nm) gives another IHIBIT gate in which only F− is able to generate high absorption output. Meanwhile, the desired molecular comparator XNOR logic gate was achievable by monitoring the initial maximal absorption at 680 nm. As can be seen from + is higher than F−. If the K+ input is less than F− then the output at 715 nm named “less than” is set to “1”. Finally, if the comparison between the both inputs gives equality the output “equal” at 680 nm is set to “1”.The binding of a positively charged KIn molecular logic architectures, the parallel action of three or more rationally selected logic gates is a real challenge. Fortunately, the magnitude digital comparator could be obtained using two gates instead of three. This is possible due to the specific connection between the three outputs, which always have a high value on one of them and a low value on the two others —after all, a comparison of two digits could give only one of the outputs “equal”, “greater than”, or “less than” at the same time. This connection allows the achievement of magnitude comparison only using two gates because if the output of two of the gates is low, then the third one necessarily must be high, and its measurement is not required. The one explained here was the most exploited logic in the design of magnitude comparators at the molecular level, and the majority of the reported systems operate on the parallel action of XNOR and one INHIBIT gate ,62,78,807 for selective recognition of Zn2+ cations and OH− anions  could serve as a magnitude digital comparator in this manner [2+ and OH− ions, compound 7 showed an absorption band centered at 442 nm and fluorescent emission at 615 nm. The addition of OH− deprotonated the phenolic electron-donating fragment in probe 7 and increased its electron-donating ability. Thus, the ICT efficiency increased, and the absorption band of 7 was red-shifted to 484 nm, which was accompanied by a strong fluorescence quenching. The complexation of Zn2+ with compound 7 bound its amino substituent, thus destabilizing the exited ICT process, which resulted in blue shifting to 415 nm and additional fluorescence quenching. The presence of Zn2+ and OH− simultaneously returned the absorption spectrum of probe 7 to the starting site and turned on the fluorescence emission at 615 nm. That is why the monitoring of the fluorescence at 615 nm in Boolean logic resulted in an XNOR gate. At the same time, based on the changes at 525 nm, where a high output was observed only in the presence of OH−, an INIHIBIT gate was constructed. The authors have used these two logic gates to construct a magnitude comparator at a molecular level. If both inputs are equal, then the fluorescence output at 615 nm is TRUE (“1”). If the OH− input is greater than Zn2+, then the absorbance output at 525 nm is TRUE (“1”). When both outputs are FALSE (“0”), then the input OH− is less than input Zn2+  shifting of the absorption spectrum from 510 nm to 567 nm is observed, which is due to protonation and increasing electron accepting ability of imido-imino substituent that enhanced the ICT nature of excited compound 8. This results in an XNOR logic gate at the molecular level using the fluorescence changes at 520 nm that could be used as equal output during comparison. Additionally, it was found that the addition of acid that provokes a high output at 567 nm generates an IHIBIT logic gate at a molecular level that could be canceled in the presence of OH−. This indicates that the H+ input is high than OH− output. Thus, the “off-on-off” fluorescence output of compound 8 possessing different absorption spectra of the both “off” states could be applied as digital magnitude comparator  and INHIBIT (input 2) gates could be constructed. Furthermore, the parallel performance of INHIBIT (input 1) and INHIBIT (input 2) in compound 9 allows digital comparison at a molecular level. When the H+ input is greater than OH− one, the fluorescence output is high. If the H+ input is less than OH− one, the absorption output is high. The low signaling values of both outputs indicate the equality of both inputs , then both outputs are high. If the input H+ is greater than OH− one, both outputs are low. If the input H+ is less than OH− one, then the fluorescence output is low, but the absorption output is high (The use of negative logic could be helpful during the construction of a molecular magnitude comparator. For instance, compound ON gates . The IMP is high .11 is an interesting example of a molecular magnitude comparator, which works only in the fluorescent mode [11 showed a strong yellow-green fluorescence at 511 nm. After the addition of acid as an input, due to the formation of a neutral molecule with reduced ICT efficiency, the fluorescence was blue-shifted to 482 nm. On the other hand, the addition of OH− resulted in a non-emissive dianion due to the presence of a PET quenching process from the formed phenolate anion. The presence of both acid and base cancels each other out, and the system returns to the starting yellow-green fluorescent form. The parallel monitoring of the fluorescence intensities at 511 nm and 482 nm results in obtaining XNOR and INHIBIT logic gates, respectively, whose performance is the above-mentioned classical requirement for the construction of a magnitude digital comparator at a molecular level, where the XNOR gate is “equal” output, while INHIBIT is “greater than”. Furthermore, the use of a paramagnetic quencher such as Cu2+ ion as a third chemical input disables both fluorescence outputs. Hence, based on probe 11, a magnitude digital comparator with disabled capability at a molecular level could be achieved (Probe ent mode . As a moachieved . The enaIn this tutorial review, all possible ways for the construction of digital comparators at the molecular level are discussed, and the recent achievements connected with these devices are presented. Comparison between two digits is the simplest arithmetic operation, and it could be realized with the simplest logic circuit. We hope the presented information will help young scientists to understand this field better and join it. The progress of molecular logic gates in the last thirty years is already impressive, but there are still plenty of outlets for the creative and innovative development of new concepts and applications for molecular computing of fluorescence probes. Improved recycling reset capabilities, reconfigurability, and integration of molecular logic gates on solid support will undoubtedly be an important theme. The higher-order logic circuits that could perform a comprehensive analysis of more than three targets are difficult to design at the molecular level. That is why such circuits need to be better designed and conceived. In addition, further intensive work on the functional integration of multichromophoric systems and interconnected cascades in supramolecular assemblies of multiple logic devices will be a great challenge.Further extension of the principles of logic to applications in biomolecular recognition or theranostics will be the ultimate goal. While there has been success in the direction of logic operations, computational algorithms, sensing, and communication, some of the more unsolved fundamental challenges refer to concepts of intracellular applications. This is an area that ‘‘silicon-based computing cannot reach’’ and an important direction that needs to be studied more deeply in the future. Some fluorescent probes are related to the pathophysiological conditions in specific diseases; thus, a strategy could be designed of molecular logic based on this relationship that could complete the intelligent analysis of multi-targets, giving a unique YES or NO output. For many diseases, it is difficult to obtain an accurate diagnosis using a single or limited number of fluorescence probes, and it is necessary to conduct a comprehensive analysis of different permutations and combinations of multiple targets. Fortunately, the molecular logic gates enable the highly sensitive detection of multiple types of targets and the production of multiple signaling outputs. This development of molecular and computer science makes it a promising tool for clinical use. Although complex molecular logic gates are still not readily available in the clinic, and they serve only as proof of concept, healthcare professionals could greatly benefit from their existence.As a whole, this exciting research area offers a great future working field that is limited only by the researcher’s imagination and inspiration.


Additive manufacturing (AM) can produce almost any product shape through layered stacking. The usability of continuous fiber-reinforced polymers (CFRP) fabricated by AM, however, is restricted owing to the limitations of no reinforcing fibers in the lay-up direction and weak interface bonding between the fibers and matrix. This study presents molecular dynamics in conjunction with experiments to explore how ultrasonic vibration enhances the performance of continuous carbon fiber-reinforced polylactic acid (CCFRPLA). Ultrasonic vibration improves the mobility of PLA matrix molecular chains by causing alternative fractures of chains, promoting crosslinking infiltration among polymer chains, and facilitating interactions between carbon fibers and the matrix. The increase in entanglement density and conformational changes enhanced the density of the PLA matrix and strengthened its anti-separation ability. In addition, ultrasonic vibration shortens the distance between the molecules of the fiber and matrix, improving the van der Waals force and thus promoting the interface binding energy between them, which ultimately achieves an overall improvement in the performance of CCFRPLA. The bending strength and interlaminar shear strength of the specimen treated with 20 W ultrasonic vibration reached 111.5 MPa and 10.16 MPa, respectively, 33.11% and 21.5% higher than those of the untreated specimen, consistent with the molecular dynamics simulations, and confirmed the effectiveness of ultrasonic vibration in improving the flexural and interlaminar properties of the CCFRPLA. Continuous fiber-reinforced polymers (CFRP) have been widely used in aerospace, rail transit, and marine equipment because of their excellent features, such as high specific strength and modulus, preferable compressive stability, and good design performance ,2,3. AddIn this study, an ultrasonic online vibration-assisted AM platform was designed to fabricate CCFRPLA composites, as shown in The fixed frequency of the ultrasonic transducer used in this study was 30 KHz, and the power was adjusted to within 30 W. The PLA  was chosen as the resin matrix material in this study owing to its excellent mechanical properties and environmental friendliness ,32, and Tensile and interlaminar shear tests were carried out in accordance with ISO 527:1997 and ISO 14130:1997, respectively, where the CCFRPLA specimen dimensions for the tensile test were 250 mm × 14 mm × 2 mm, and the fiber direction in the specimen was the same as the tensile direction. The specimen dimensions for the interlaminar shear test were 20 mm × 10 mm × 2 mm, and the direction of the carbon fiber in the CFRP was consistent with the long side of the specimen. In addition, the bending strength of specimens under different ultrasonic powers was studied, and the specimen for the three-point bending test was of a non-standard design with the dimensions 60 mm × 10 mm × 2 mm. The fibers were printed in the direction of 0°, corresponding to the longest side of the specimen. During testing, a vertical force was applied at the longitudinal midpoint at a loading rate of 2 mm/min. The displacements of the indenter and loads applied to the specimen during the experiment were recorded, and the results are shown in The experimental results illustrate that the introduction of ultrasonic vibration during the CFRP preparation process can significantly improve the mechanical properties of the composite. As the ultrasonic vibration power increased, the macroscopic tensile and bending properties of the CFRP specimens improved. When the ultrasonic power was 20 W, the tensile strength and bending strength of the specimens reached maximum values of 166.8 MPa and 111.5 MPa, respectively, which were 5.6% and 30.11% higher than those of the untreated part. However, when the ultrasonic power reached 30 W, the performance of the specimen was somewhat lower than the maximum value but still much stronger than that of the untreated specimen. This may be due to excessive ultrasonic power causing continuous fiber breakage, resulting in a decline in performance, and the interfacial shear strength coinciding with the macroscopic properties.The macroscopic properties of composite materials depend on the interfacial properties between the carbon fiber and the matrix, the conformation of the PLA molecular chains, and the microstructure of the matrix itself. The influence of ultrasonic vibration on the conformation of PLA molecular chains, internal microstructure, and properties, and the resulting impact on the macroscopic properties of PLA/CF composites, were difficult to obtain through the aforementioned macroscopic experiments. Molecular dynamics (MD) can be used to explore the effects of ultrasonic vibration on the conformation and activity of PLA molecular chains at the molecular scale ,34,35, aIn the ultrasonic vibration-assisted FFF manufacturing platform designed in this study, ultrasonic vibration acted directly on the PLA substrate through the nozzle, and the vibration effect was then transmitted to the carbon fiber through the PLA molecular chains. During the transmission process of ultrasonic vibration, an alternative fracture of the PLA matrix molecular chains may occur, leading to changes in the matrix motion activity, fiber/matrix interaction, and permeability. Therefore, before conducting the MD analysis, it is necessary to first determine the weight of the PLA matrix molecular chain. The effect of ultrasonic vibration on the molecular weight was determined through gel permeation chromatography (GPC), and the results are shown in As shown in 3, α = β = γ = 90°, and the density of PLA is 1.25 g/cm3. The parameters of the three MD calculation models were the same, except for the different degrees of polymerization of the PLA molecular chains.According to the GPC results and considering the effect of ultrasonic vibration on the molecular chains of PLA matrix, three different PLA molecular chains with polymerization degrees of 3 (PLA-3), 6 (PLA-6), and 10 (PLA-10) were constructed in this study, and the corresponding molecular dynamics models with these three molecular chain lengths were constructed in the commercial software Materials Studio, as shown in Before conducting MD calculations, a standard layer thickness surface in the (001) direction was cut based on the constructed PLA/CF model, and then a 20 Å vacuum layer was added on the surface as the activity space of the molecular chain after ultrasonic vibration, as shown in The interface of CFRP, as a key part of the composite, is vulnerable to failure under a load, and its structural performance plays a crucial role in the overall performance of the composite. Based on the MD model treated with ultrasonic vibration, the interface performance between the carbon fiber and the PLA matrix was explored at the molecular scale. The interface binding energy is an important indicator for characterizing the bonding strength . To explThe interaction energy between CF and matrix PLA is calculated by Equation (1) :(1)ΔE=EtAccording to Equation (1), the binding energy between the carbon fiber and matrix during the extraction process can be determined, and the maximum statistical value is taken as the interface binding energy between the fiber and matrix. In this study, the interfacial binding energy between the PLA matrix and fibers with three different degrees of polymerization under three different speed treatments was calculated b.As shown in The interfacial bonding force between the fiber and matrix is mainly composed of van der Waals and intermolecular electrostatic forces. Analysis of the change in interfacial binding energy components during the extraction process of CF revealed that the change in interfacial energy caused by the van der Waals force was consistent with the change in interfacial binding energy between the CF and PLA matrix, and the difference between the two was approximately 5 kcal/mol. However, the change in the interface binding energy caused by the electrostatic force was negligible, as shown in The interface binding energy between the CF and PLA matrix was mainly contributed by van der Waals forces. The van der Waals force between the CF and PLA matrix is mainly determined by the molecular spacing between the two. Subsequently, the effect of ultrasonic vibration on the distance between the CF and PLA molecules was investigated, and the results are shown in As shown in Changes in the conformation and activity of the PLA molecular chains caused by ultrasonic vibration can affect the entanglement infiltration inside the matrix, thereby altering the microstructure inside the matrix material and affecting its ability to withstand loads. According to the change in the PLA matrix energy during CF/PLA extraction, ultrasonic vibration not only improved the interface binding energy between the CF and PLA matrix, but also improved the deformation energy of the PLA matrix .As shown in As shown in Affected by the conformations of the PLA molecular chains with different degrees of polymerization, ultrasonic vibration showed different levels of impact on the activity and diffusivity of the PLA molecular chains. PLA-3, owing to its lower degree of polymerization, entanglement, and entanglement between molecular chains, is more susceptible to ultrasonic vibration, resulting in the greatest MSD values and diffusion coefficients, and the strongest interaction with CF and other molecular chains, which shared similarities with the SEM of the CFRP cross-section .As This study experimentally explored the effect of ultrasonic vibrations on the performance of 3D-printed CCFRPLA in conjunction with MD simulations. The experimental results showed that when the ultrasonic power was 20 W, the bending strength of the CFRP prepared by the ultrasonic-assisted FFF manufacturing system could reach 111.5 MPa and the interlaminar shear strength could achieve 10.16 MPa, which increased by 33.11% and 21.5%, respectively, compared with the untreated specimens. However, the tensile strength of the specimen did not increase significantly compared with the bending strength and interlaminar shear strength. At the same power, the tensile strength of the specimen was only 166.8 MPa, which was only 5.6% higher than that of the untreated specimen. When the ultrasonic power exceeded 20 W, the mechanical properties of the CCFRPLA deteriorated. This may be because excessive ultrasonic power damaged the main structure of the fibers and weakened the interface performance between fibers and matrix, leading to a partial fracture of the fibers and affecting their overall structural performance. In addition, we explored the enhancement mechanism of ultrasonic vibration on the molecular scale. Ultrasonic vibration can cause the alternative fracture of PLA molecular chains and form PLA chains of different lengths. In the MD analysis, ultrasonic vibration enhanced the activity of PLA molecular chains and changed their conformation, which promoted entanglement, infiltration, and interaction among matrix molecular chains and between the matrix and fibers. This increased the density of the PLA matrix and reduced the interface distance between the CF and PLA matrix, thus improving the interface binding energy between the CF and PLA matrix and the separation resistance of matrix layers and ultimately enhancing the bending strength and interlaminar shear strength of the CCFRPLA. However, the improvement of interfacial properties between the CF and PLA matrix, as well as between interlaminar layers, has a relatively small promoting effect on the overall tensile strength of CCFRPLA. The MD simulation results agree with the experimental results, illustrating the effectiveness of ultrasonic vibration in improving the mechanical properties of CCFRPLA.


In the field of pharmacochemistry, it is a time-consuming and expensive process for the new drug development. The existing drug design methods face a significant challenge in terms of generation efficiency and quality.In this paper, we proposed a novel molecular generation strategy and optimization based on A2C reinforcement learning. In molecular generation strategy, we adopted transformer-DNN to retain the scaffolds advantages, while accounting for the generated molecules’ similarity and internal diversity by dynamic parameter adjustment, further improving the overall quality of molecule generation. In molecular optimization, we introduced heterogeneous parallel supercomputing for large-scale molecular docking based on message passing interface communication technology to rapidly obtain bioactive information, thereby enhancing the efficiency of drug design. Experiments show that our model can generate high-quality molecules with multi-objective properties at a high generation efficiency, with effectiveness and novelty close to 100%. Moreover, we used our method to assist shandong university school of pharmacy to find several candidate drugs molecules of anti-PEDV.https://github.com/wq-sunshine/MomdTDSRL.git.The datasets involved in this method and the source code are freely available to academic users at  It is important to design effective and high-quality compounds, but the process is time-consuming and expensive for the new drug development. Molecular design  is usualMolecular structure determines the pharmacological properties and functions of compounds, therefore, molecular characterization is a fundamental problem in drug computing. In terms of intuitiveness, the 3D structural representation of drug molecules can provide spatial configuration information, facilitating the understanding of the molecule’s spatial properties and interactions . But theRegarding the design methods, deep learning techniques have found extensive application in molecular design. A series of methods based on recurrent neural network (RNN), variational autoencoder (VAE), generation adversary network (GAN) and reinforcement learning (RL) have realized the generation of candidate drugs for specific targets. RNN was a widely used neural network structure, which showed a strong processing ability in the drug molecular design . The VAEAiming at the above question, we propose a novel molecular generation strategy and optimization based on A2C reinforcement learning. The main contributions of our approach are the following:The generation strategy adopts transformer-DNN to retain the scaffolds advantages, while accounting for the generated molecules’ similarity and internal diversity by dynamic parameter adjustment, further improving the overall quality of molecule generation.In molecular optimization, heterogeneous parallel supercomputing is introduced for large-scale molecular docking based on message passing interface (MPI) communication technology to quickly obtain bioactivity information, thereby improving the efficiency of drug design.Experiments show that our model can generate high-quality molecules with multi-objective properties at a high generation efficiency, with effectiveness and novelty close to 100%.This paper presented a novel molecular generation strategy and optimization by combining transformer-DNN, heterogeneous parallel supercomputing and reinforcement learning. As shown in Molecular characterization is a fundamental problem in drug design. It directly affects the construction of a clear correlation between molecular structure and drug properties. Therefore, we proposed the molecular characterization based on balanced binary tree.We constructed a virtual fragment combinatorial library by fragmenting compounds. The molecules were partitioned into ring structures, side chains and linkers. To achieve this, single bonds extending from a ring atom were cleaved, and the corresponding attachment points were recorded for subsequent assembly steps.For the fragment combination library constructed previously, we calculated the similarity between fragments and then encoded fragments to achieve molecular fragment data preprocessing.Fragment similarity calculationsIn the method, through encoding all fragments as binary strings, similar fragments got similar encodings. Specifically, we employed the Tanimoto-MCS (TMCS)similarity for measuring the similarity, its formula was defined as follows:Here, Fragments encodingWhen constructing the tree, we calculated similarities among all fragments using All fragments were encoded into binary strings by constructing a balanced binary tree that was based on fragments similarity calculations. Afterwards, each fragment used this tree to generate binary strings. The fragments encodings were determined by the paths from the root to the leaves. Every branching to the left will add a one (“1”) to the end of the encoding and every branching to the right will add a zero (“0”).Molecular characterization is the foundation, so the molecular generation strategy is the core of drug design. Molecular generation is able to generate new compounds by performing fragment replacement on an initial set of lead compounds.In our method, the generation strategy adopted the transformer-DNN to identify one or more fragments for replacement. Firstly, the molecular sequence was preprocessed through molecular characterization to obtain fragment level embedding with the position information and scaffolds level embedding, which served as the input to the transformer-DNN. Then, using the transformer, the fragments importance was obtained by calculating the attention coefficient of each fragment. This process preserved the advantages of the molecular scaffolds and output a vectorized feature representation of a molecule with different fragment associations. Finally, the concatenated output was classified by the DNN, which estimated the probability distribution of which fragments to replace and with what fragments, as illustrated in hth head, three weight matrices The molecule’s multi-granularity information features were processed through a transformer layer. This transformer encoder comprised multi-head attention layers, position feedforward network (FFN), residual connection layers and normalization layers. The multi-head attention layer consisted of multiple attention heads, with each head performing linear transformations before calculating attention weights. Different trainable parameter sets were used for each attention head to capture distinct relationships between fragment sequences. This allowed learning of internal molecular structures through attention-weighted calculations. Specifically, the input of the multi-head attention layer consisted of three vector sequences: where H represented the number of heads, and Here, Then, the multi-head attention and the feedforward neural network were connected through residual connections and normalization. After the encoding transformation, we can obtain features for each molecular sequence:where Molecular generation strategy is the core, in that way molecular optimization is the key in the drug design. In this article, we added the evaluation of physical and chemical properties and molecular docking to the reward of reinforcement learning, the latter of which was used to add biological activity constraints. Considering the computational scale of molecular docking, we had adopted parallel supercomputing, greatly accelerating the speed of molecular generation.Preliminary generated molecules were screened for toxicity by checking for the presence of known toxic groups. In addition, molecular weight (MW), effectiveness (E), lipophilicity (LogP) and polar surface area (PSA) also affected the pharmacokinetic properties. Only those molecules satisfying the specified range of properties had excellent pharmacokinetic properties. The calculation formula was as follows:k represented multiple properties, l and u were the set range of molecular properties.where We used large-scale molecular docking to obtain docking scores, and then input them into a perceptron classifier for bioactivity prediction. Docking score in each epoch needed to perform docking calculations between the generated compounds in batches and specific target structures, which was obviously a time-consuming operation. To address this issue, we introduced MPI parallel computing based on the heterogeneous parallel supercomputing. In this process, the master process was responsible for reading protein target file data and analyzing target sequences. It intelligently distributed the data to computing nodes within the sub-communication domain based on the relationship between the number of protein targets and the number of worker processes. The worker processes handled the reading of ligand small molecule file data and broadcast it to all computing nodes, while also monitoring the overall program’s execution. The computing nodes were tasked with the actual molecular docking process, and once suitable docking conformations were found, they wrote the docking results to a file and sent a signal to the master process.Specifically, we first preprocessed ligand data and protein target files to create multiple ligand sets and specific target collections. a “target management node” was established to read all target files and docking pocket information in batches, which was then broadcasted to each computing node. Simultaneously, multiple “ligand management nodes” were assigned to read small molecule files and distributed them to computing nodes. Subsequently, molecular docking was performed between target files and small molecule files in the ligand sets, resulting in docking outcomes upon completion. In the molecular optimization, the bioactivity prediction was realized by constructing classifiers. Initially, the ic50 value was used as the benchmark, with active molecules as positive samples and inactive molecules as negative samples. The perceptron generated the activity information by virtually docking between the data of positive and negative samples and disease-related targets. From the docking results, we extracted the docking score, number of nonhydrogen atoms, and ratio of the first two. These features were then input into the perceptron to predict activity. In addition, a large-scale molecular docking was employed to quickly and accurately calculate the affinity score between small molecules and specific targets, and to predict the real activity of generated molecules, so as to further optimize the biological activity.In brief, the data obtained through molecular characterization was fed as the input to the model. The actor then adapted the generation strategy using transformer-DNN, with particular emphasis on molecular optimization being incorporated into the reward mechanism. Finally, critic calculated the value of the generation strategy adopted by the actor after receiving the reward. If yes, the actor’s action would be strengthened. If no, be blocked. Then, the current state was replaced by the new state. The actor was rewarded for each valid molecule generated. The reward was higher if it managed to produce molecules that met the expectations of multi-objective properties. The valid molecules generated were taken as the input for the next generation task. Through continuous iteration, the model accuracy was optimized to the maximum, which was marked by the stable proportion of high-quality molecules generated.We utilized two common datasets to evaluate model performance. The first one was the MOSES dataset : A distance measure that quantifies the chemical and biological similarity between two sets of compounds. As shown in G and R respectively.To assess our method, we generated 10 000 molecules by sampling and benchmarked the model using several different molecular generation methods based on the MOSES and GuacaMol datasets except activity, which including non-neural baselines [n-gram generative model and Hidden Markov Model (HMM)], JTN-VAE .We conducted molecular generation on the NOSES dataset and the GuacaMol dataset, sampling 10 000 molecules and counting various metrics for the generated molecules. For the specific Mpro target, we introduced an activity optimization strategy. Utilizing the dataset we had created, we successfully generated high-quality molecular compounds, offering promising candidates for anti-PEDV to the Shandong University School of Pharmacy. Furthermore, we also conducted a comprehensive evaluation, covering various critical indicators, including analyzing the property distribution, similarity, synthetic accessibility (SA), and drug-likeness, as well as the binding model of the generated molecule and its corresponding crystal structure, to ensure that these molecules had good potential and feasibility in anti-PEDV drug research.Molecular properties: We focused on optimizing four key properties, namely effectiveness, logP, PSA and MW. The percentage of generated molecules that met the target ranges for these properties was shown in In addition, we counted the properties distribution of MW, logP and PSA. Compared with the distribution in the initial set, the distribution of target properties generated in the last 10 epochs was shown in Molecular similarity: The similarity between newly molecules and the initial molecules remained stable at 0.8 under different Time parameters. As shown in Synthetic accessibility: SA score was used to assess the difficulty of compound synthesis. We analyzed the SA score distribution of the generated molecules in Binding model: We selected molecules with highest binding energy and ligand efficiency from the generated molecules and analyzed the binding model of the generated molecule and its crystal structure with SARS-CoV-2 Mpro of COVID-19. Then, we compared the binding energy and ligand efficiency of the generated molecules with their corresponding initial molecules. As shown in Drug-likeness: Drug-likeness (QED) scores were a quantitative estimate to evaluate the similarity between the generated compound molecules and known drugs. We randomly selected 10 molecules with better binding energy and ligand efficiency from the generated molecules and analyzed the change of QED score between the generated and the initial molecules. As shown in To validate the impact of properties optimization on generating high-quality molecules, we conducted a series of ablation experiments and analyzed validity, novelty, internal diversity as well as scaffold similarity of the generated molecules. As shown in Improving the efficiency and quality of molecule design is crucial in the process of the development of innovative drugs. The key is to balance the bioactivity and multi-physicochemical properties based on molecular scaffolds, which is difficult to achieve through opportunistic compound screening and artificial structure optimization. In this study, we proposed a novel molecular generation strategy and optimization based on A2C reinforcement learning. In molecular generation strategy, we adopted transformer-DNN to retain the scaffolds advantages, while accounting for the generated molecules’ similarity and internal diversity by dynamic parameter adjustment, further improving the overall quality of molecule generation. In molecular optimization, we introduced heterogeneous parallel supercomputing for large-scale molecular docking based on MPI communication technology to rapidly obtain bioactive information, thereby enhancing the efficiency of drug design. On this basis, future research can be extended to the characterization based on graph with 3D geometric information. In molecular generation strategies, it is possible to consider the introduction of diffusion models or their combination with larger models to optimize drug molecule design. For molecular optimization, the ADMET properties of molecules can also be considered in future research to facilitate the simplification of downstream molecular synthesis and drug measurement. Moreover, our method may provide important insights into the refinement of molecule generation and optimization for self-learning automatic molecular design.btad693_Supplementary_DataClick here for additional data file.