Magnesium-based bioresorbable stents (Mg-BRS) secure acute percutaneous coronary intervention (PCI) results without leaving, in the long term, a metallic caging effect. The purpose of this real-world study was to assess clinical and CCTA medium- and long-term follow-up of all our patients with implanted Mg-BRS. (2) Methods: The patency of 52 Mg-BRS implanted in 44 patients with de novo lesions (24 of which had acute coronary syndrome (ACS)) was evaluated by CCTA and compared to quantitative coronary angiography (QCA) post-implantation. (3) Results: ten events including four deaths occurred during a median follow-up of 48 months. CCTA was interpretable and in-stent measurements were successful at follow-up without being hindered by the stent strut’s “blooming effect”. Minimal in-stent diameters on CCTA were found to be 1.03 ± 0.60 mm smaller than the expected diameter after post-dilation on implantation ( Continuous techniques and scaffold advances have come a long way in coronary artery disease (CAD) treatment since Andreas Gruntzig’s pioneering work some 40 years ago ,2. The iThe appealing concept of a bioresorbable scaffold (BRS) aims to provide performance equivalent to existing DES by the delivery of a transient coronary device that would secure the acute results of percutaneous coronary intervention (PCI), preventing acute recoil and constrictive remodeling. Furthermore, the ideal BRS would elute an antiproliferative agent, avoiding neointimal hyperplasia before the biodegradation of its scaffold ,8. A morHowever, the daily clinical use of BRS nowadays remains limited secondary to safety concerns . The firOn the other hand, advances in coronary computed tomography angiography (CCTA) imaging have facilitated a more accurate interpretation of noninvasive coronary imaging . CCTA waWe sought to report short and long-term clinical and noninvasive imaging results of CAD patients treated at our institution with at least one Mg-BRS who were eventually scheduled to undergo a CCTA at any time post-implantation.This is a retrospective, monocentric, observational cross-sectional case series evaluating patients treated by at least one Mg-BRS. The Institutional Review Board (IRB) at the Centre Hospitalier Universitaire et Psychiatrique de Mons-Borinage (CHUPMB)—Belgium reviewed and approved the study protocol on 13 January 2022 (reference number HAP-2021-034). The board waived the requirement for written consent. Major adverse cardiovascular events (MACE) were defined as cardiovascular death, target vessel myocardial infarction (TV-MI), target vessel revascularization (TV-R) and clinically driven target lesion revascularization (CD-TLR). We retrospectively collected and assessed treated patients’ demographics and clinical information, including laboratory and procedural characteristics along with, when available, follow-up visits.The primary endpoint was to determine the diagnostic accuracy of CCTA for assessing in-scaffold luminal dimensions as compared to quantitative coronary angiography at the time of the procedure, as well as the feasibility to detect in-stent restenosis. The secondary endpoint was to report long-term clinical follow-up of all our patients treated by at least one DREAMS-2G scaffold.QCA analysis was performed at an independent core laboratory (UMONS CoeurLab) using a validated QCA software . The operator was blinded to CCTA results. Proximal and distal vessel diameter and surface area were determined as well as lesion and in-stent minimal surface area (MSA) and diameter (MLD). In-stent mean proximal to distal surface area was calculated as the average of the proximal and distal surface area at the stent edge /2).Assessment of stent patency was assessed by 240-slice Toshiba CCTA scans  using standard acquisition techniques, including 6 hours fasting, heart rate control using beta-blockers when indicated and tube settings depending on the patient’s body mass index. Images were reconstructed and analyzed by two independent physicians at 0.5 mm increments. After cross-sectional vessel reconstruction, implanted stents were identified using the two well-radiopaque stent markers. Automatic segmentation of the vessel lumen was performed for the measurements and manual correction was made when needed. The maximal and minimal lumen area (MLA), proximal and distal surface areas and diameters as well as the in-stent minimal and maximal diameters were determined for each scaffold. Mean proximal to distal surface area was calculated.2 and one-way ANOVA. The Mann–Whitney rank sum test, Kruskal–Wallis test and an independent t-test were used to compare continuous variables. A Wilcoxon signed-rank test was used to compare the median of differences. Agreement between measurement modalities was assessed by the Passing–Bablok and Bland–Altman methods. Long-term survival was represented by a Kaplan–Meier analysis. Statistical analyses were performed using SPSS software version 23.0  and MedCalc software version 20.125 .Categorical variables were described as frequency rates and percentages, and continuous variables were described as mean ± SD. Proportions for categorical variables were compared using Fisher’s exact test, χp < 0.05), males (p = 0.05) and smokers (p < 0.05). A majority of left anterior descending (LAD) arteries (63.5%) were treated. Mild calcifications were found in 19 lesions (37.3%) overall. Lesion classification, according to the ACC/AHA classification, was type A in 22 of the cases (42.3%). Pre-dilation was performed in all but three patients. The mean implanted stent diameter size was 3.15 ± 0.23 mm, inflated at a mean pressure of 14.9 ± 1.36 atm. Post-dilation was performed using a non-compliant balloon in all but three STEMI lesions (93.8%) at the discretion of the operator. The mean post-dilation balloon diameter was 3.4 ± 0.3 mm, inflated at a mean pressure of 18.5 ± 2.4 atm. This reflected the adherence of the operators to the predefined Mg-BVS implantation technique recommendations  months. Cardiac death, TV-MI, TV-R and CD-TLR rates were comparable among both ACS and elective groups: ten events (22.7%) were noted (four in the elective group and six in the ACS group). Three reported deaths (12.5%) were noted in the ACS group on days 3, 97 and 895 post-implantation. These were attributed to cardiac tamponade and two deaths of unknown origin, respectively (possible stent thrombosis following the ARC definition). In addition, one death was reported in the elective group attributed to COVID-19 infection complications 530 days post-implantation. Survival curves are shown in p < 0.05).Results of the quantitative coronary analysis (QCA) of the index procedures are given in 2 and the mean proximal to distal in-stent surface area was 8.55 ± 2.3 mm2 . Minimal in-stent diameters on CCTA were found to be 1.03 ± 0.60 mm smaller than the expected stent diameter after post-dilation after implantation (p < 0.05) using the post-dilation balloon diameters as reference.CCTA was performed in 36 out of the 44 patients (81.8%), out of which, 32 (88.9%) were interpretable and measured . Calcifi 2.3 mm2 . An in-sIn 32 scaffolds, the mean difference between QCA and CCTA in MLD and MSA were −0.08 (limits of agreement (LOA) −1.29 to 1.46) and −2.84 (LOA −2.3 to 8.0) mm, respectively. The mean minimal in-stent diameter by CCTA was 2.38 ± 0.56 mm while the post-PCI QCA minimal lumen diameter was 2.4 ± 0.5 mm, a non-significant statistical difference. Overall, there was no agreement between QCA and CCTA on a Passing–Bablok regression analysis .The appealing concept of BRS has received a lot of enthusiasm in the last two decades, with the advantage of a scaffold providing radial strength in the acute phase and dissolving after a period of time, hence reducing the long-term complications of DES ,29. It iThe main findings of this study are: first, Mg-BRS did not hinder in-stent lumen visualization measurements on CCTA. Second, Mg-BRS exhibited a trend for a higher rate of MACE in the setting of ACS although it did not reach a significant difference in our limited number of patients. Third, device-related thrombotic events were low in our cohort. Finally, intraluminal measurements using QCA and CCTA differed significantly, with a significant loss of in-stent diameter on CCTA.CCTA has gained much interest in the past years as a noninvasive method for the evaluation of patients with CAD and was adopted by the guideline-directed workup of patients suspected of having CAD . AdvanceTo the best of our knowledge, this is the largest cohort of patients implanted with a Mg-BRS evaluated by CCTA. The diagnostic accuracy of CCTA for the detection of bioresorbable scaffold obstruction and luminal dimensions has already been established using older-generation scaffolds . PreviouOur data are also in line with the MAGSTEMI, PRAGUE-19 and BEST-MAG studies concerning the safety of use of BRS in the setting of ACS all with a slightly higher rate of MACE ,43,44. AWe report a higher number of MACE than the recent 8.0% TLF published at 5 years follow-up in the BIOSOLVE II study . This isIn-scaffold luminal dimension evaluation has already been compared by QCA and CCTA, showing similar diagnostic accuracy . Our QCAWhereas dual antiplatelet therapy (DAPT) is recommended for at least 6 and 12 months post DES or BMS implantation in the elective and ACS setting, DAPT post-BRS implantation remains currently recommended ≥ 12 months independent of implantation setting due to a higher rate of late stent thrombosis . With anOur study highlights the safety of BRS implantation in a real-world simple, soft and mildly calcified de novo lesion, as recommended in BRS studies, allowing for a transient radial force securing acute PCI results and resorbing with time. Furthermore, our data underline the feasibility of CCTA follow-up of BRS, a non-invasive imaging tool, hence reducing the need for invasive coronary angiography for in-stent restenosis detection.This was a retrospective study on a relatively small cohort. The time frame of CCTA follow-up was short for assessing long-term outcomes. The observational nature of our study conveys its limitations. CCTA quality remains a paramount point to consider. Factors such as high heart rate, arrhythmias, calcifications and high body index will negatively impact CCTA interpretation.Our data demonstrate the feasibility and precision of CCTA for the follow-up of implanted Mg-BRS. Our results suggest good scaffold patency at 6 months, along with minimal lumen loss. Moreover, our data highlight the safety of Mg-BRS in the elective and ACS settings, with a slightly increased risk of MACE in the latter. In our cohort, one definite Mg-BRS thrombosis occurred despite optimal medical treatment, a rarely described event. Following an adequate selection of patients and implantation technique, Mg-BRS seems to be safe and efficacious in the long-term, with a word of caution for unstable patients. Further studies and improvements in CCTA imaging and Mg-BRS design might resolve some of these issues.


N-methyl-d-aspartate (NMDA), which is likely to be mediated indirectlythrough intermediary neuronal mechanisms rather than by a direct actionon dopamine terminals. On the basis of known modulatory processesin nucleus accumbens, the current experiments sought to test whetherthe effect of NMDA was mediated through cholinergic, GABA-ergic, ormetabotropic glutamatergic intermediate mechanisms. Fast-scan cyclicvoltammetry was used to measure electrically stimulated dopamine releasein nucleus accumbens of rat brain slices in vitro. Stimulated dopamine release was attenuated by NMDA, confirming previousfindings, but this attenuation was unaffected by either cholinergicor GABA-ergic antagonists. However, it was completely abolished bythe nonselective group I/II/III metabotropic glutamate receptor antagonistα-methyl-4-carboxyphenylglycine (MCPG) and by the selectivegroup II antagonist LY 341396. Therefore, group II metabotropic glutamatereceptors, but not acetylcholine or GABA receptors, mediate the attenuationof stimulated dopamine release caused by NMDA, probably by presynapticinhibition through receptors located extra-synaptically on dopamineterminals. This provides a plausible mechanism for the documentedrole of metabotropic glutamate receptor systems in restoring deficitsinduced by NMDA receptor antagonists, modeling schizophrenia, underliningthe potential for drugs affecting these receptors as therapeutic agentsin treating schizophrenia.Electrically stimulated dopamine release from the nucleusaccumbensis attenuated following application of  However, this finding is surprising for two key reasons.First, NMDA receptors are typically excitatory,15 and therefore it would be expected that activation of thesereceptors would increase dopamine release. Second, evidence suggeststhat NMDA receptors are not widely located on dopamine terminals inthis region  in rat brain slices on (refs  and 17)in vitro,on (refs ). Theref22 There are two classes of cholinergic receptor (AChR): nicotinicand muscarinic. The activation of nicotinic AChR by nicotine enhancesdopamine release in the NAc, which is disrupted by subchronic PCPpretreatment.23 It is established thatnicotinic AChRs are present on dopamine terminals in this region25 and that activation of these receptors leads to dopamine releasehere.27 Furthermore, activation of AChR with carbacholin freely moving rats enhances locomotor activity, a behavior primarilyunder the control of accumbal dopamine. Taken together, these studiessuggest that nicotinic AChR modulates dopamine release in the NAc.Acetylcholine containing interneuronsform only around 5% of thetotal number of neurones in the striatum, but their extensive arborizationmakes them exquisitely placed to provide powerful modulatory influenceon dopamine release through local actions on the mesolimbic terminalsin NAc.28 Furthermore,local application of muscarinic AChR antagonists into NAc disruptedreward-seeking,29 a behavior under thecontrol of mesolimbic dopamine. This was confirmed by additional experimentationwith FSCV suggesting further that muscarinic AChRs have some controlover dopamine release here.30 It is thereforeplausible that NMDA activates cholinergic interneurons, which in turninhibit the release of dopamine from mesolimbic terminals in NAc.The role of muscarinic AChR on dopamine release from the NAc iscomplex. Activation of the M5 subgroup of muscarinic AChR enhancesdopamine release in the midbrain, but M2 and M4 muscarinic AChRs inhibitdopamine neurones in the striatum.in vitro, group II (mGluRs2 and 3), and group III , and have been shownto be present in NAc.q-protein couples and mainly locatedpostsynaptically where they generally exert an excitatory action.38 Groups II and III, on the other hand, are Gi/o-proteincoupled and negatively regulate neurotransmitter release:40 while group III mGluRs are foundpre- and postsynaptically and on glial cells, group II mGluRs aremainly located on presynaptic terminals,40 including on dopamine terminals where theynegatively modulate dopamine release.37 Notably, presynaptic group II mGluRs are present both in the synapse,and outside the synapse, toward the axonal part, spatially removedfrom the transmitter release site41 where theyrespond to extra-synaptic “spillover” glutamate.42Group I mGluRsare Gin vivo indicated that activationof groups II and III but not group I mGluRs decreased dopamine releasein NAc,44 although there was some indication thatthe effect of group II agonists was biphasic, initially reducing dopaminerelease before increasing it again.43 Moreover,schizophrenia-like behavioral changes induced by ketamine in animalmodels are somewhat reversed following the application of a groupII agonist.45 Importantly, Yavas and Young14 showed that the broad spectrum mGluR antagonist,with little selectivity between groups I, II, and III, reduced theattenuation of electrically stimulated dopamine release by NMDA inNAc of rat brain slices in vitro, suggesting a keyrole of mGluRs in the attenuating action of NMDA. Therefore, thereis strong evidence for mGluR modulating dopamine release in NAc, whichcould mediate the NMDA-induced effects, of which mGluR-II’sare most likely, given their negative modulatory nature, inhibitingdopamine release, and their extra-synaptic localization.Microdialysis studies in vitro to ascertainwhich, if any, of these mechanisms mediate attenuation of electricallystimulated dopamine release by NMDA.Therefore,cholinergic, GABA-ergic, and mGluR systems all exertinhibitory control over local dopamine release in NAc. The experimentsused FSCV in rat brain slices in vitro, which represented a releasedconcentration of 0.204 ± 0.011 μM (n =112 slices). There were no significant differences in baseline releasebetween treatment groups. Similarly, there were no significant differencesin any of the parameters measured between slices taken from male andfemale animals (see Supporting Information), so the data for both sexes were pooled.Electrical stimulation evokeda consistent release of dopaminefrom brain slices 14In nondrug treatedslices the stimulated release remained stableacross the duration of the experiment comprising 14 or 18 stimulations,and in each experimental condition NMDA (30 μM) caused an attenuationof the stimulated dopamine release, to around 50% of baseline stimulationlevels, consistent with previous data.47 Although data from Yavas and Young14 suggested that DHβE may block the effectsof NMDA, interpretation was impeded by the fact that DHβE itselfincreases the stimulated release. For this reason an extended protocolwas used in the current experiments such that DHβE was appliedalone for 12 min (4 stimulations), to establish a new baseline releasein the presence of the antagonist, and then NMDA was added along withDHβE for a further 12 min (4 stimulations). DHβE alonecaused a rise in stimulated dopamine release to a maximum of approximately150% of baseline, consistent with previous findings.14Thenicotinic receptor antagonist, dihydro-β-erythroidine (DHβE)(1 μM), applied alone caused a substantial increase in stimulateddopamine release, as has been reported previously.When NMDA (30 μM) was added concomitantly withDHβE (1 μM), there was an attenuation of stimulated releasedown to near baseline levels, representing a reduction of approximately50% of baseline 1. This rF = 3.580; p = 0.0208) and of DHβE = 6.931; p = 0.0136) but not of NMDA  = 0.1849; p = 0.6705). The two-way interactionbetween stimulus and NMDA was significant  = 2.873; p = 0.0001) as was the stimulus ×DHβE  = 2.386; p = 0.0015), but neither the remaining two-way interaction  = 0.000 385 7; p = 0.9845)) or the three-way interaction  = 0.4751; p = 0.9636) was significant.Statistical analysis using a mixed-design three-wayANOVA showedmain effects of stimulus  showed that during theapplication of DHβE alone (stimulations S5–S8) the stimulatedrelease of dopamine was significantly augmented. In the DHβEalone condition, this augmentation was sustained through stimulationsS9–S12, during which DHβE alone continued to be applied,and through stimulations S13– S18, during the washout periodwhere the tissue was superfused with artificial cerebrospinal fluid(aCSF). NMDA alone applied during stimulations 9–12 causeda significant reduction in the stimulated release. In addition, whenNMDA was applied alongside DHβE, during stimulations S9–S12,there was a significant reduction in stimulated release, resemblingthe reduction seen when NMDA was applied alone during this period,although starting from an elevated baseline 1.The muscarinic AChR antagonist scopolamine(1 μM) alone hadno effect of stimulated dopamine release: therefore, the standard14 stimulus procedure was used rather than the extended 18 stimulusprotocol. Scopolamine had no effect on the attenuation of stimulateddopamine caused by NMDA.F = 6.713; p = 0.0150) but not of stimulus  = 1.227; p = 0.3050) or of scopolamine  = 0.2444; p = 0.6249). The stimulus× NMDA interaction was significant, but the other two two-wayinteractions  = 0.7865; p = 0.6747; NMDA × scopolamine, F = 0.0642; p = 0.8018) and thethree-way interaction  = 0.5006; p = 0.9239) were nonsignificant.Statistical analysis using a mixed-designthree-way ANOVA showeda main effect of NMDA  confirmed the significantattenuation of stimulated dopamine release by NMDA. Scopolamine appliedalone had no significant effect, nor did it have any significant effecton the attenuation of stimulated release caused by NMDA 2.The summary data 6 emphasi14 have shown that the GABA-A antagonistpicrotoxin failed to block the attenuation of stimulated release causedby NMDA.Previousstudies36 and therefore requiredthe extended (18 stimulus) protocol to establish a new baseline releasein the presence of the antagonist, before applying NMDA. In theseconditions, NMDA continued to reduce stimulated dopamine release,even in the presence of the GABA-B antagonist.We extended this by testing whether blockade of GABA-Breceptors affected the NMDA-induced attenuation. As with DHβE,the GABA-B antagonist CGP 54626 (1 μM) caused a marked increasein stimulated dopamine release when applied alone, consistent withprevious reports,F = 6.347; p = 0.0018), NMDA  = 8.674; p = 0.0060), and CGP 54626  =16.02; p = 0.0003). All two-way interactions andthe three-way interaction were also significant  = 9.064; p < 0.0001; stimulus× CGP 54626  = 7.404; p < 0.0001;  =6.188; p = 0.0183); three-way interaction  = 3.718; p < 0.0001)).Statistical analysisusing a mixed-design three-way ANOVA showedmain effects of stimulus,  showed a significant increasein stimulated dopamine release when CGP 54626 was applied alone duringstimulations S5–S8, although the onset was delayed and onlyreached statistical significance from stimulus S6. Where CGP 54626application continued alone over the next four stimulations (S9–12)the stimulated response continued to rise. As previously, NMDA appliedduring stimulations S9–S12 caused a significant attenuationon stimulated dopamine release, although the effect was delayed byaround 6 min, only showing up in stimulations S11–S14. WhenNMDA was applied in the presence of CGP 54626, the attenuation remainedintact, albeit that it started from a raised baseline and showed asignificant difference from CGP 54626 alone 3. As befThe broadspectrum  mGluR antagonist α-methyl-4-carboxyphenylglycine(MCPG: 100 μM) had no effect of stimulated dopamine releasewhen applied alone but completely abolished the attenuation of stimulatedrelease caused by NMDA (30 μM).F = 3.935; p = 0.0202) butnot of NMDA  = 3.169; p = 0.0859) or MCPG  = 0.6277; p = 0.4349). The two-way interaction between stimulus and NMDA  = 2.374; p = 0.0046) and thethree-way interaction  = 2.585; p = 0.0019) were significant, but the remaining two-wayinteractions were nonsignificant  = 1.264; p = 0.2323: NMDA × MCPG, F = 3.713; p = 0.0642). Post hocanalysis (Fisher’s LSD) confirmed the significant attenuationof stimulated dopamine release by NMDA during stimulations S8–S11.MCPG applied alone had no significant effect but completely reversedthe attenuation of stimulated release caused by NMDA . This also had no effect on stimulated dopaminewhen applied alone but abolished the attenuation of stimulated releasecaused by NMDA (30 μM).F = 3.048; p = 0.0454) but not of NMDA  =3.363; p = 0.0773) or LY 341495  = 1.219; p = 0.2789). The two-way interactionbetween stimulus and NMDA  = 2.414; p = 0.0039) and the three-way interaction  = 2.392; p = 0.0043) were significant,but the remaining two-way interactions were nonsignificant  = 1.573; p = 0.0906: NMDA × LY 341495, F = 3.139; p = 0.0873).Statistical analysis using a mixed-designthree-way ANOVA showeda main effect of stimulus showed that NMDA significantlyattenuated the stimulated dopamine release during stimulations S8–S11.LY 341495 alone had no effect on stimulated dopamine but completelyabolished the effect of NMDA 5The summary data 6 emphasi14 Neither the nicotinic AChR antagonist DHβE(1 μM) nor the muscarinic AChR antagonist scopolamine (1 μM),at concentrations previously shown to be effective at blocking cholinergicactions, affected the NMDA-evoked attenuation. Similarly, the GABA-Breceptor antagonist CGP 54626 (1 μM), at a concentration knownto block the GABA-B function,36 did notreverse the NMDA-evoked attenuation. However, the broad-spectrum (groupI/II/III) mGluR antagonist MCPG (100 μM) and the specific groupII mGluR antagonist LY 341495 (1 μM) completely reversed theeffect of NMDA.In summary, the results showed that repeated electricalstimulationat 3 min intervals evoked a reliable release of dopamine, which remainedstable over the duration of the experiment, comprising either 14 stimulations(39 min) or 18 stimulations (51 min). NMDA (30 μM) caused areliable attenuation of stimulated dopamine release to around 50%of baseline stimulation, consistent with previous studies.14 whichis thought to be mediated through an inhibitory intermediary ratherthan by a direct action on the dopamine terminals.It is known that dopamine release from mesolimbicneurones is undermodulatory control at the level of the terminals in NAc from manyneurotransmitters, including glutamate, GABA, and acetylcholine. Bymaking our recordings in brain slices, we can be confident that weare measuring local, intra-accumbens modulatory control, as long-loopnetwork connections are absent in slices. We previously showed thatNMDA (30 μM) caused an attenuation of electrically stimulateddopamine release in NAc slices,48 which are widely expressedin mesolimbic dopamine neurones in NAc.49 DHβE,when given alone, evoked an augmentation of the stimulated releaseof dopamine, similar to that seen previously,50 consistent with cholinergic inhibition of dopamine release throughlocal nicotinic receptor mediated mechanisms. However, DHβEdid not reduce the attenuation of stimulated dopamine release causedby NMDA: in slices superfused with DHβE, NMDA still caused anattenuation of the stimulated release of similar magnitude to thatseen in the absence of DHβE, albeit from an elevated baseline, and bo40 and, as such, seemed a likely candidateto mediate the mGluR action. Moreover, studiesusing the selective group II antagonist LY 341495 showed that blockinggroup II mGluR also abolished the attenuation of stimulated dopaminerelease caused by NMDA. Evidence suggests that group II mGluRs arelocated on dopamine terminals but are outside the synapse, towardthe axonal part of the terminal, and are activated by extra-synaptic,“spillover” glutamate.41 Moreover,NMDA receptors are also found extra-synaptically on glutamate terminals:these receptors are generally slower than their intrasynaptic counterpartsbut with higher affinity.54 They regulate neuronalmembrane potential, increasing excitability and further enhancingglutamate release from the terminal.56 The increasedglutamate release, in turn, leads to more spillover which activatesreceptors over a wider volume, including the group II mGluRs on surroundingdopamine terminals, leading to inhibition of dopamine release. Thisprovides a plausible mechanism through which NMDA can attenuate electricallystimulated dopamine release through group II mGluR-controlled signaling.It is pertinent to note that, in particular, high frequency stimulationincreases the spillover of glutamate:56 this is consistent with the findings from the current study, whichemployed high frequency (60 Hz) stimulation, and also with preliminarydata (Supporting Information) which showedno effect of NMDA on electrically stimulated dopamine release at lowfrequency stimulation.Group II mGluRs have a negative modulatory effect of transmitterrelease13 which leads to downstream effects on transmittersystems, including glutamate/dopamine dysregulation.5 Moreover, mGluR involvement in schizophrenia has been posited58 possibly through disrupted interactions between NMDA and mGluR.59 More specifically, group II mGluR agonists reversechanges in behaviors resembling schizophrenia in animal models ofschizophrenia,61 leading to these drugs being considered as potentialnovel antipsychotic drugs.63 The results shown hereprovide a plausible mechanism through which NMDA/dopamine dysregulationin schizophrenia may involve group II mGluR mechanisms, giving furtherimpetus for studying this class of drugs as potential therapeuticagents.63The glutamate theory of schizophreniaposits a core deficit isin NMDA receptor function,In conclusion, these experiments aimedto elucidate the mechanismthat mediates the attenuation of electrically stimulated dopaminerelease caused by NMDA in NAc. Three possible candidates for thismechanism were investigated: cholinergic, GABA-ergic, and mGluR. Thefindings showed that neither cholinergic nor GABA-ergic antagonistshave any effect on the change caused by NMDA but that mGluR antagonists,particularly group II, completely abolish the NMDA-evoked effect.This indicates that the attenuation of electrically stimulated dopaminerelease caused by NMDA in NAc is mediated through group II mGluR,but not cholinergic or GABAergic mechanisms, probably through a directinhibitory effect of these receptors located extra-synaptically ondopamine terminals.ad libitum. This projectreceived ethical approval from the University of Leicester EthicalCommittee (AWERB/2019/69).Male and female rats  were housed in independently ventilated, double-deckPlexiglas cages  in groups of four to six animals at the University of LeicesterPreclinical Research Facility. Animals were maintained under standardlaboratory conditions as temperature (21 ± 2 °C), humidity(55% ± 10%), and lighting  were held constant. Animals could access food  and water 36 Animals underwent cervical dislocation, and the brain was removedand placed into ice-cold aCSF, comprising (mM), NaCI (126.0), KCI(2.0), KH2PO4 (1.4), MgSO4 (2.0),NaHCO3 (26.0), CaCI2 (2.4), glucose (10.0).Coronal slices (400 μm) containing NAc were cut using a vibratingmicrotome . Each brainprovided three to four slices containing NAc: these were cut alongthe midline to provide two single hemisphere slices from each fullslice. With one hemislice used for each experimental condition, itallowed six to eight experimental conditions to be tested from eachbrain. Slices were then incubated in oxygenated (95% O2/5% CO2) aCSF at a temperature of 21 ± 2 °Cfor 60 min to recover from the trauma of slicing.The procedure was similar to that previouslydescribed.64 were placed into the NAc, with the recording electrodeapproximately 0.5 mm away from the stimulating electrode.For recording,slices were transferred to the recording chamber and superfused continuouslywith oxygenated aCSF . Slices equilibrated for 30min before a concentric bipolar tungsten stimulating electrode  and a carbon fiber recording electrode, custombuilt in the lab as described by Clark et al.,65 connected to a Chem-Clamp potentiostat with a 5 MΩ headstage. The current generated was recorded, anddopamine oxidation was measured on the forward scan at approximately+0.6 V, in the background subtracted signal (Demon Voltammetry software65).Atriangular waveform  was applied at a frequencyof 10 Hz using Demon voltammetry software,Slices were stimulated  at 3 min intervals, during a 15 s recording period, withthe stimulus onset 5 s after the start of the recording. For experiments1b and 3, 14 stimulus trains were administered at 3 min intervalswith the complete session lasting 39 min. In the no drug (control)condition, tissue was superfused with aCSF throughout. For drug conditions,slices were perfused with aCSF for the first 9 min  and then drug was applied in the superfusate for 12min (stimulations S5–S8). The drug was then removed, and slicessuperfused with aCSF again for the remaining 18 min of the experiment(stimulations S9–S14)For experiments 1a and 2, wherethe antagonist alone had an effectopposite to the NMDA effect, an extended 18-stimulation protocol wasutilized. Here, 18 stimulation trains were applied to the tissue at3 min intervals in a session lasting for a total of 51 min. As before,in the no drug (control) condition, aCSF was perfused throughout thesession for 51 min. In the NMDA alone condition, aCSF was perfusedfor 24 min, from stimulations S1–S8, before NMDA (30 μM)was applied in the superfusate for 12 min (stimulations S9–S12),after which the superfusate was returned to aCSF for 18 min (stimulationsS13–S18). Where antagonists were used, these were applied inthe superfusate for 24 min, after the initial 9 min baseline recordingperiod (stimulations S5–S12), before returning to aCSF for18 min (stimulations S13–S18). In experiments testing the effectof the antagonist on NMDA following 12 min of application of the antagonistalone (stimulations S5–S8), NMDA was applied concomitantlywith the antagonist for 12 min (stimulations S9–S12), beforethe final 18 min with aCSF alone (stimulations S13–S18).(a)Nicotinic Antagonist, DHβE. After 9 min baseline superfusionwith aCSF, the DHβE and NMDA + DHβE treatment groups receivedDHβE (1 μM) alone for 12 min (stimulations S5–S8),while the no drug and NMDA groups continued to receive aCSF. Duringthe next 12 min (stimulations S9–S12) either DHβE alone(DHβE group), NMDA (30 μM) alone (NMDA group), or a combinationof the two (NMDA + DHβE group) was applied in the superfusate.Finally, all groups were returned to aCSF superfusion for the remaining18 min of recording (stimulations S13–S18). The no drug conditionreceived aCSF throughout the full 51 min of recording.(b) MuscarinicAntagonist, Scopolamine. After 9 min baseline superfusion with aCSF,either NMDA (30 μM), scopolamine (1 μM), or the combinationof NMDA (30 μM) and scopolamine (1 μM) was applied inthe superfusate for 12 min (stimulations S5–S8), before returningto aCSF superfusion for 18 min.GABA-BAntagonist, CGP 54626. After 9 min baseline superfusion with aCSF,the CGP 54626 and NMDA + CGP 54626 treatment groups received CGP 54626(1 μM) alone for 12 min (stimulations S5–S8), while theno drug and NMDA groups continued to receive aCSF. During the next12 min (stimulations S9–S12) either CGP 54626 alone (CGP 54626group), NMDA (30 μM) alone (NMDA group), or a combination ofthe two (NMDA + CGP 54626 group) was applied in the superfusate. Finallyall groups were returned to aCSF superfusion for the remaining 18min of recording (stimulations S13–S18). The no drug conditionreceived aCSF throughout the full 51 min of recording.(a) GroupI, II and III mGluR antagonist, MCPG. After 9 min baseline superfusionwith aCSF, either NMDA (30 μM), MCPG (100 μM), or thecombination of both was applied in the superfusate for 12 min (stimulationsS5–S8), before returning to aCSF superfusion for 18 min.(b) Group II mGluR-II Antagonist, LY 341495. After 9 min baselinesuperfusion with aCSF, either NMDA (30 μM), LY 341495 (1 μM),or the combination of both was applied in the superfusate for 12 min(stimulations S5–S8), before returning to aCSF superfusionfor 18 min.65 recorded the Faradaic current following backgroundsubtraction of the signal. Dopamine (1 μM) calibration performedeach day before experiments allowed the calculation of peak dopaminerelease concentration following each stimulation.Demon Voltammetry and Analysis softwareFor all experiments,the first four stimulations (S1–S4) were used to calculatethe mean baseline of electrically stimulated dopamine release. Thendopamine release recorded at all 14 (18 in the extended protocol)stimulations was calculated as the percentage of this mean baseline.Time course data from replications are shown as mean ± SEM percentagesof this baseline stimulated dopamine release.Summary data werealso calculated for each portion of the recordingby calculating the mean release during NMDA application as a percentageof the mean baseline in the three stimulations immediately beforeNMDA application (S2–S4 or S6–S8 in the extended protocol).Due to the delay in onset of the NMDA response, the first two stimulationsafter application of NMDA were disregarded, and the mean of the nextthree stimulations (S7–S9 or S11–S13 in the extendedprotocol) was designated as the NMDA response.Statistical analysiswas carried out using GraphPad Prism v9.0.0.Alpha level for analysis was 0.05. Time course and summary data weresubjected to mixed-design three-way ANOVA (stimulus × NMDA ×antagonist), where stimulus was a repeated measure, and NMDA and antagonistwere between subject measures. Significant interactions were furtherinvestigated using Fisher’s LSD to ascertain differences betweenthe experimental conditions during each stimulation (planned comparisons).Violations in sphericity were compensated using Greenhouse–Geissercorrection.For depiction of the profile of stimulated release,the raw (currentvs time) data for stimulations S4 (baseline), S8 (drug), and S14 (washout)(or S4 (baseline), S8 (drug A), and S12 (drug B) and S18 (washout)for experiments using the extended protocol) were normalized to themaximum stimulation amplitude achieved in the baseline (S4) period.Pooled data (mean ± SEM) were then plotted for each condition.MCPG, LY 341496 and CGP 45626 weresupplied by Tocris : all other drugs and chemicalsfor aCSF were supplied by Sigma-Aldrich . All drugs weremade up as 10 mM stock solutions in water except for CGP 45626, whichwas made up in DMSO. Aliquots were frozen (−20 °C) untiluse. On the day of experiments, a drug aliquot was thawed and dilutedin aCSF to the appropriate working concentration. Drug concentrationswere derived from previous work in our lab (refs , and 3636 and un


Polycystic ovary syndrome (PCOS) is the most common cause of anovulatory infertility in women of childbearing age. Randomized controlled trials (RCTs) have reported that exenatide and metformin are effective in the treatment of PCOS. In this meta-analysis, we aimed to compare the effectiveness and safety of exenatide alone or in combination with metformin versus metformin in patients suffering from PCOS.RCTs of exenatide therapy were identified through a search of electronic databases in November 2022 and updated in October 2023. Eligible studies were identified independently by the reviewers. Outcomes were analysed with Revman 5.4.P = 0.006), sex hormone-binding globulin (SHBG) , and follicle-stimulating hormone (FSH) . The reductions in total testosterone (TT)  was more significant after treatment with exenatide than after treatment with metformin. In terms of safety, exenatide had a lower diarrhea rate  than metformin. In the other three studies, exenatide plus metformin was compared with metformin. Exenatide combined with metformin was more effective in improving SHBG , Matsuda index  and reducing free androgen index (FAI) , Weight  and WC . The incidence of side effects between exenatide plus metformin and metformin was not statistically significant.Nine RCTs among 214 studies on 1059 women with PCOS were included in the analysis, and among the nine RCTs, eight studies compared exenatide with metformin. Our meta-analysis demonstrated that exenatide was more effective than metformin in terms of pregnancy rate (RR 1.85  Exenatide alone or in combination with metformin is more effective than metformin for women with PCOS. Considering the evidence on effectiveness and safety, exenatide alone or in combination with metformin may be a better treatment approach than metformin for women with PCOS.https://inplasy.com/inplasy-protocols/ ID: 10.37766/inplasy2022.11.0055.INPLASY The online version contains supplementary material available at 10.1186/s12902-023-01497-x. Polycystic ovary syndrome (PCOS) is the most common endocrine disorder among women of childbearing age and is associated with high androgen levels, oligomenorrhea or amenorrhea, anovulation, and polycystic morphology of the ovaries, according to the Rotterdam criteria . AlthougClinical studies have demonstrated that metformin improves anovulation, menstrual disturbances, and hyperandrogenism in patients with PCOS , 12. MetExenatide, which belongs to the glucagon-like peptide-1 (GLP-1) receptor agonist family, has been used to treat diabetes and obesity chiefly owing to its prominent effects on insulin resistance, weight loss, and metabolic disorders , 15. SomOriginal studies published in English for this meta-analysis were sought from electronic databases . We searched for RCTs on humans in databases from their establishment date to October 2023 by utilizing a strategy combining MeSH words with free words. The utilized terms involved "Polycystic Ovary Syndrome" [MeSH], Ovary Syndrome, Polycystic [Title/Abstract], Syndrome, Polycystic Ovary [Title/Abstract], "Exenatide"[MeSH], Exendin-4 [Title/Abstract], Peptide, Ex4 [Title/Abstract], randomized controlled trial [Publication Type], and randomized [Title/Abstract]. The search history on PubMed is presented in Table SAll searched articles were independently screened by 2 authors according to the inclusion criteria, which were as follows: 1) participants: females diagnosed with PCOS on the basis of the Rotterdam criteria; 2) intervention(s): exenatide alone or plus metformin in the treatment of PCOS; 3) comparison(s): exenatide versus metformin; 4) outcomes: effectiveness estimated by pregnancy rate, sex hormone levels, change in body weight, and metabolic disorders and safety assessed by the incidence of side effects; 5) study type: human-based RCTs. The exclusion criteria were as follows: one-arm study with no control group, placebo-controlled trials, and studies with no results.Two authors (XS and SH) selected articles independently based on the inclusion and exclusion criteria and then reviewed the full texts to extract data from the eligible articles. In the case of disputes, a discussion meeting was organized by a third author (YM). Necessary information for outcomes included pregnancy rate, MFR, total testosterone (TT), follicle-stimulating hormone (FSH), luteinizing hormone (LH), dehydroepiandrosterone sulfate (DHEAS), sex hormone-binding globulin (SHBG), the free androgen index (FAI), homeostasis model assessment-insulin resistance (HOMA-IR), Matsuda index, fasting insulin (FINS), fasting blood glucose (FBG), weight, body mass index (BMI), waist circumference (WC), the waist-hip ratio (WHR), and side effects . Information on the trials included basic information , interventions , and study design. The MFR was calculated using the ratio of expected menses to observation weeks. Matsuda index is an indicator of insulin sensitivity calculated from the glucose tolerance test.The risk of bias for each RCT was evaluated by using the Cochrane Collaboration’s risk of bias assessment, which includes the aspects of sequence generation, allocation concealment, blinding, incomplete outcome data, selective outcome reporting and other bias. Studies were characterized as “low risk”, “unclear risk”, and “high risk”. Quality assessment of the RCTs was performed with the Grading of Recommendations and Evaluation (GRADE) system. Quality was defined as “high”, “moderate”, “low”, or “very low” according to the study design, risk of bias, indirectness, inconsistency, imprecision, and publication bias .2) test and presented as I-squared (I2) values. I2 < 50% and P > 0.1 indicate the existence of little heterogeneity among original studies. Analyses were employed by utilizing fixed-effects models. I2 ≥ 50% indicates high heterogeneity, and subgroup analysis or sensitivity analysis was performed. If no reason for the heterogeneity could be determined and the heterogeneity was within the relevant limits, a random-effects model could be utilized. P values < 0.05 were recognized to indicate significant differences.Meta-analysis plus forest plots were applied by utilizing Rev Man 5.4. Continuous variables were presented as the standardized mean difference (SMD) or mean difference (MD) with a 95% confidence interval (CI) on basis of the agreement of research units. For dichotomous data, the risk ratio (RR) with 95% CI for every original study was gathered for meta-analysis utilizing the Mantel/Haenszel model. Heterogeneity was appraised through the Chi-squared , and the fixed-effects model was used for examination. Exenatide had a higher pregnancy rate  than metformin . The random effects model of the meta-analysis indicated that exenatide is superior to metformin in TT  . This analysis indicated that the study "Elkind-Hirsch K 2008" greatly contributed to the observed heterogeneity. After excluding this study, heterogeneity testing was insignificant . The heterogeneity in the excluded study could possibly be attributed to its small sample size and the distinct population compared to the other three studies. The Meta-analysis results remained consistent after sensitivity analysis, suggesting the stability of the findings. ] P < 0.001)   and DHEAS  , a sensitivity analysis was performed. This analysis identified the study "Elkind-Hirsch K 2008" as a major contributor to the observed heterogeneity. Upon excluding this study, the heterogeneity testing was insignificant . The observed heterogeneity may be due to its small sample size and unique population compared to the other three studies. The Meta-analysis results remained consistent after conducting the sensitivity analysis, indicating the robustness of the findings ] P = 0.8)   Table .Meta-analysis demonstrated a greater reduction in HOMA-IR, FINS, weight, BMI, WC and WHR in the exenatide group than in the metformin group. The difference in FBG between the two groups was insignificant Fig S, Fig S2.2 = 0%, P = 0.64), and the fixed-effects model was used for analysis. Exenatide had a lower diarrhea rate than metformin  , and FAI    Fig. .P = 0.006) in the group of exenatide plus metformin  (Fig. WC was reported in 2 studies in the comparison between exenatide plus metformin and metformin containing 68 patients with PCOS. The fixed-effects model was applied for meta-analysis. Results revealed that exenatide was more effective in reducing WC (MD-5.6195%CI -8.4, -2.82] , -2.82 PThe incidence of diarrhea, nausea, vomiting, stomach pain, constipation, headache, and fatigue between two group was not statistically significant Fig.  Table 55.Fig. 6TPCOS is commonly associated with infertility, menstrual cycle disorders and abnormal sex hormone levels . In addiThe ability of exenatide to improve sex hormone disorders could be one of the reasons for this effect. It is well known that PCOS is the most common cause of anovulatory infertility . InitialThe second mechanism for the improvement of reproductive function is based on the improvement of IR and hyperinsulinaemia. PCOS is associated with a high prevalence of IR, hyperinsulinaemia, and high androgen levels , particuThe third mechanism for the improvement of reproductive function involves weight reduction , and thiThe present study has several strengths. Our study compared the efficacy and safety between exenatide alone or plus metformin and metformin in patients with PCOS from several perspectives. We not only focused on exenatide, but also summarized the effectiveness of exenatide plus metformin in PCOS patients, which previous meta-studies on GLP-1 receptor agonists have not done.Our research also has some limitations. To date, the clinical randomized controlled trials of exenatide in the treatment of PCOS have been short-term trials of 12–24 weeks, and there are no studies of longer duration. In addition, the quality of the included studies was low to moderate. Therefore, the long-term effect needs to be established by longer, larger, multicentre, and higher-quality clinical studies.This meta-analysis demonstrates that exenatide, either alone or in combination with metformin, exhibits superior performance over metformin in managing PCOS, while maintaining a comparable side effect profile. Exenatide, either alone or in combination with metformin, appears to be a safe and effective treatment choice for patients diagnosed with PCOS. Further studies with larger sample sizes and longer durations are warranted to investigate the combined treatment approach when obesity is linked to PCOS.Additional file 1. PRISMA 2009 Checklist.Additional file 2:Table S1. The search history on PubMed. Table S2. RCT Risk of Bias Assessment. Fig S1. Effects on HOMA-IR, FBG and FINS between exenatide and metformin. Fig S2. Effects on Weight, BMI, WC, WHR between exenatide and metformin. Fig S3. Sensitivity analysis of TT and FAI. Fig S4. subgroup analyses of FBG. Fig S5. Subgroup analyses of BMI. Fig S6. Putative mechanism of beneficial effects of exenatide.Additional file 3. Table for excluded literature in meta-analysis.


Antimicrobial resistance (AMR) is a major public health challenge, particularly in sub-Saharan Africa (SSA). This study aimed to investigate the evolution and predict the future outlook of AMR in SSA over a 12-year period. By analysing the trends and patterns of AMR, the study sought to enhance our understanding of this pressing issue in the region and provide valuable insights for effective interventions and control measures to mitigate the impact of AMR on public health in SSA.The study found that general medicine patients had the highest proportion of samples with AMR. Different types of samples showed varying levels of AMR. Across the studied locations, the highest resistance was consistently observed against ceftaroline (ranging from 68 to 84%), while the lowest resistance was consistently observed against ceftazidime avibactam, imipenem, meropenem, and meropenem vaborbactam (ranging from 92 to 93%). Notably, the predictive analysis showed a significant increasing trend in resistance to amoxicillin-clavulanate, cefepime, ceftazidime, ceftaroline, imipenem, meropenem, piperacillin-tazobactam, and aztreonam over time.These findings suggest the need for coordinated efforts and interventions to control and prevent the spread of AMR in SSA. Targeted surveillance based on local resistance patterns, sample types, and patient populations is crucial for effective monitoring and control of AMR. The study also highlights the urgent need for action, including judicious use of antibiotics and the development of alternative treatment options to combat the growing problem of AMR in SSA. Antimicrobial resistance (AMR) is a growing public health concern worldwide, and it is particularly acute in sub-Saharan Africa (SSA), where infections caused by resistant bacteria are prevalent and pose a significant threat to health systems. The increasing prevalence of AMR in SSA is driven by several factors, including poor antimicrobial stewardship and limited access to effective diagnostics and therapeutics –4.Klebsiella pneumoniae is a Gram-negative bacterium that is among the leading causes of healthcare-associated infections in SSA. Klebsiella pneumoniae can cause a range of infections, including pneumonia, urinary tract infections, bloodstream infections, and surgical site infections. However, its utility extends beyond its clinical significance, as it can serve as a proxy for other bacteria in studying AMR in SSA. One reason is that Klebsiella pneumoniae is commonly isolated from clinical specimens in the region, making it a useful indicator of the prevalence and patterns of AMR. Additionally, the bacterium has been associated with high rates of multidrug resistance, making it an ideal target for studying the evolution and spread of resistance in the region. Furthermore, Klebsiella pneumoniae has been found to share common resistance mechanisms with other Gram-negative bacteria, such as Escherichia coli, making it a potential proxy for studying resistance trends in other bacterial species [ species –12.Klebsiella pneumoniae as a proxy to provide valuable insights into the evolution and future outlook of AMR in SSA. By analysing phenotypic data for Klebsiella pneumoniae over a 12-year period, this research aimed to shed light on the patterns and potential trajectory of AMR in the region. The findings of this study have the potential to inform evidence-based interventions and policies to mitigate the impact of AMR on public health in SSA.Despite the growing threat of AMR in SSA, there is a scarcity of data on the evolution and future outlook of AMR in the region. This knowledge gap hampers the development of effective public health strategies and interventions to combat this escalating threat. Therefore, this study focused on In Nigeria, the majority of samples in 2014, 2018, and 2020 were from general medicine , general surgery , and general medicine , respectively. In South Africa, the majority of samples in 2014, 2018, and 2020 were from general medicine  , blood , and urine , respectively. In South Africa, the majority of isolates in 2014, 2018, and 2020 were from wounds , blood , and blood , respectively , while the lowest was to CEA, IMP, and MEM . In 2018, the highest resistance was to CER, while the lowest was to CEA, IMP, and MEM . In 2020, the highest resistance was to CER , while the lowest was to MEV . In South Africa, the highest resistance in 2014 was to CER , while the lowest was to CEA . In 2018, the highest resistance was to CER , while the lowest was to CEA . In 2020, the highest resistance was to CER , while the lowest was to MEV   represent the projected resistance trends up to 2026. The non-bold red lines represent the lower and upper confidence bounds.The bold blue lines represents resistance trends up to 2020. The bold red lines (in the middle) represent the projected resistance trends up to 2026. The non-bold red lines represent the lower and upper confidence bounds.Based on the results, the majority of samples were from general medicine in both Nigeria and South Africa. This distribution is in agreement with findings from other studies conducted in SSA, implying that targeted surveillance of AMR in general medicine patients could be an effective approach to monitoring and controlling AMR in the region. By focusing on the most common patient population, resources can be used more efficiently, and interventions can be more targeted to where they are most needed. Nonetheless, it is worth noting that similar distribution of patients across specialties has been observed in other studies, underlining the significance of collaboration between different medical specialties and a comprehensive approach to tackling AMR in healthcare settings –18.Based on the results, there is no consistent pattern in the types of samples from which isolates were most commonly obtained in both Nigeria and South Africa. This suggests that targeted surveillance of AMR should be based on the local epidemiology of infections and the sample types most commonly associated with AMR in each setting. For instance, if bloodstream infections are a significant problem in a particular hospital or region, then surveillance efforts should focus on blood isolates to monitor trends in AMR. Similarly, if urinary tract infections are more prevalent, then urine samples may be more useful for surveillance purposes. It is important to note that different sample types may have varying levels of AMR, and therefore, the selection of samples for surveillance should be guided by local data and expert opinion. Additionally, the use of standardized methods for specimen collection, transport, and processing is critical for ensuring the accuracy and comparability of surveillance data across different settings –25.Based on the results, there are significant differences in the resistance patterns of the isolates between Nigeria and South Africa. This suggests that targeted surveillance of AMR should be tailored to the local resistance patterns in a given setting, rather than a one-size-fits-all approach. For example, in Nigeria, CER had consistently high levels of resistance over the years, indicating the need for targeted surveillance and control measures for this antibiotic. In contrast, in South Africa, CER had varying levels of resistance over the years, and in 2020, the highest resistance was to a different antibiotic (MEV). This highlights the importance of regular surveillance to detect changes in resistance patterns and to guide appropriate interventions. It is also important to note that resistance patterns may vary not only between countries but also between hospitals, regions, and even different patient populations within the same hospital. Therefore, targeted surveillance efforts should be based on local data and guided by expert opinion to ensure that interventions are effective and appropriately targeted –28.The results indicate a statistically significant increased trend in resistance towards several antibiotics in both Nigeria and South Africa. This highlights the urgent need for targeted surveillance of AMR in these countries, particularly for the antibiotics showing an increased trend in resistance. By monitoring the prevalence of AMR in specific antibiotics, healthcare providers can tailor their interventions to ensure optimal use of antibiotics and minimize the development and spread of resistant organism , 30.It is important to note that the increase in resistance towards certain antibiotics may be attributed to various factors, such as antibiotic use patterns, prescribing practices, and patient demographics. Therefore, the implementation of effective targeted surveillance strategies should be coupled with other interventions, including antibiotic stewardship programs, infection control measures, and public awareness campaigns , 31–35.Furthermore, understanding the classes of antibiotics that are becoming more resistant can provide insight into the mechanisms driving the development of AMR. This can inform research and development efforts aimed at creating new antibiotics and identifying alternative treatment options –40. UltiKlebsiella pneumoniae and other relevant pathogens. Longitudinal analysis: The study analysed data over a 12-year period, which provides valuable information on temporal trends in AMR. However, a longer-term longitudinal analysis covering an extended period would offer a more comprehensive understanding of the evolutionary dynamics of resistance and the impact of interventions over time. Additionally, such a study would be better suited to capture the emergence and spread of novel resistance mechanisms. Multidisciplinary collaboration: Although this study focused on the analysis of phenotypic data, collaboration with experts from diverse fields such as molecular biology, epidemiology, and healthcare policy could provide additional perspectives and enrich the study’s findings. Integrating multidisciplinary approaches could enable a more comprehensive assessment of AMR and facilitate the translation of research findings into effective interventions and policies.Generalizability: While the results provide valuable insights into the situation in Nigeria and South Africa, caution should be exercised in generalizing the findings to other countries in SSA. The epidemiology of AMR can vary significantly across different regions and healthcare settings within SSA. Therefore, it is crucial to collect data from other countries in SSA to obtain a more comprehensive understanding of the patterns and drivers of AMR in the region. Data scope: This study focused on analysing data collected by the ATLAS programme (PFIZER) from Nigeria and South Africa over a specific period. While these countries were chosen due to the availability of complete data, it is important to acknowledge that they may not represent the entire spectrum of AMR dynamics within SSA. Including data from a larger number of countries within the region would contribute to a more representative assessment of AMR trends and enable better generalisation of the findings. Methodological considerations: The retrospective nature of the study and the use of phenotypic data analysis have inherent limitations. Molecular characterisation of resistance mechanisms and genomic analyses were not performed, which could provide additional insights into the genetic basis of resistance and the spread of resistant strains. Further studies could incorporate advanced molecular techniques to enhance the understanding of the mechanisms driving AMR in Overall, this study highlights the urgent need for action to address the growing problem of AMR in SSA, and the importance of a multifaceted approach that includes targeted surveillance, judicious use of antibiotics, and the development of alternative treatment options. The findings underscore the significance of tailoring interventions based on local resistance patterns, sample types, and patient populations. By implementing evidence-based strategies informed by comprehensive surveillance data, healthcare systems can effectively mitigate the impact of AMR and safeguard public health in SSA.This study retrospectively analysed data collected by the ATLAS Programme (Pfizer). This programme’s aim is to do AMR surveillance across 80 countries, including 6 countries in SSA . This study targeted datasets for the 6 countries for the years 2014 to 2020. However, only 2 countries (Nigeria and South Africa) had complete data for these years hence were considered in the analysis. Only speciality, source, antimicrobial susceptibility profiles were extracted from the datasets. Antibiotic data targeted was for aztreonam (AZT), a monobactam; amoxycillin clavulanate (AMC) and piperacillin tazobactam (PTZ) both penicillin-β-lactam inhibitor combinations; ceftazidime (CAZ), a 3rd generation cephalosporin; ceftazidime avibactam (CEA), a 3rd generation cephalosporin-β-lactam inhibitor combination; cefepime (CEP), a 4th generation cephalosporin; ceftaroline (CER), a 5th generation cephalosporin; imipenem (IMP) and meropenem (MEM) both carbapenems; meropenem vaborbactam (MEV), a carbapenem-β-lactam inhibitor combination; and colistin (CT), a polypeptide. Prediction was done up to the year 2026.


By comparison of the behaviour of differently fluorinated molecules, experimentally and computationally, we can identify properties of molecules with regard to interactions and molecular packing motifs that are required for an effective utilisation of the observed effect.Since molecular materials often decompose upon exposure to radiation, lithographic patterning techniques established for inorganic materials are usually not applicable for the fabrication of organic nanostructures. Instead, molecular self-organisation must be utilised to achieve bottom-up growth of desired structures. Here, we demonstrate control over the mesoscopic shape of 2D molecular nanosheets without affecting their nanoscopic molecular packing motif, using molecules that do not form lateral covalent bonds. We show that anisotropic attractive Coulomb forces between partially fluorinated pentacenes lead to the growth of distinctly elongated nanosheets and that the direction of elongation differs between nanosheets that were grown and ones that were fabricated by partial desorption of a complete molecular monolayer. Using kinetic Monte Carlo simulations, we show that lateral intermolecular interactions alone are sufficient to rationalise the different kinetics of structure formation during nanosheet growth and desorption, without inclusion of interactions between the molecules and the supporting MoS Structuring organic films is of scientific and technological interest. Here, the authors use partially fluorinated organic molecules exhibiting strong intermolecular interactions to form extended 2D molecular nanosheets and control their shape through growth and desorption kinetics. In contrast to conventional inorganic electronics that are continuously approaching physical limits as device miniaturisation progresses4, structuring of organic films is still in its infancy. One major reason for this is the radiation sensitivity of organic materials, hampering the application of well-established lithographic techniques for top-down structuring6. Instead, bottom-up methods are required10, such as molecular self-assembly which provides precise control of molecular packing and scalability for mass fabrication11. This is commonly achieved through covalent molecule-substrate bonds or covalent intermolecular linkage, often catalysed by metallic substrates, allowing for the fabrication of supramolecular nanostructures such as porous networks or nanoribbons17. Naturally, such structures are rigid and cannot be reshaped after covalent bonds are formed.Molecular materials are a focus of scientific and technological interest due to the possibility to flexibly tailor their electronic properties. This enables diverse applications ranging from biochemical nanotechnology to organic thin-film electronics18 or the organisation of proteinaceous surface layers19 that rely on non-covalent intermolecular interactions only, proving that such interactions can drive self-assembly of molecules into complex structures. Interestingly, biologic self-assembly processes often require non-equilibrium operation to adopt non-minimum energetic states20. Non-equilibrium processes could similarly be used to achieve new means of structural control in artificial molecular nanostructures, for instance through tempering of already assembled vdW structures21.An alternative and so far hardly studied approach is to rely on non-covalent van der Waals (vdW) interactions. Despite their relative weakness, nature has countless examples of complex self-assembly processes, such as DNA association2), which is a popular representative of the class of two-dimensional (2D) materials22. MoS2 allows an easy preparation of perfectly flat, long-range ordered surfaces by exfoliation of single crystals23. Previous studies have shown that MoS2 exhibits an exceptionally weak molecule-substrate interaction potential, in the absolute strength as well as in its spatial corrugation, for the closely related molecules pentacene and perfluoropentacene27. As discussed in more detail in the ‘Methods’ section, this experimental evidence for a negligible corrugation of the molecule-substrate interaction potential shows that structure formation of molecular layers is not influenced by the supporting MoS2 substrate and consequently only determined by intermolecular interactions. Nonetheless, MoS2 is sufficiently conductive for scanning tunnelling microscopy (STM) measurements, in contrast to similarly inert substrates such as hexagonal boron nitride.In this study, we show that control of the mesoscopic shape of molecular assemblies that preserves molecular packing order can be achieved, which is a step towards true bottom-up patterning through self-assembly. We use regioselective fluorination of parental pentacenes to tune intermolecular interactions and correlate the kinetics of structure formation upon ad- and desorption with these interactions. As support for our molecular nanosheets, we chose molybdenum disulphide 28, a pentacene derivative that is fluorinated unilaterally adjacent the molecular L-axis  crystal, prepared by deposition of a nominal film thickness of 3.0 Å as measured by a quartz crystals microbalance. The molecules adsorb in a coplanar orientation on the substrate surface as confirmed by X-ray absorption measurements  showed notable desorption of the first molecular monolayer at temperatures above 400 K25. For the similar L-F6PEN, we have tested different annealing temperatures between 390 K and 420 K, finding no deviation from the preferred nanosheet elongation in direction 〈The first molecule chosen for our study is 1,2,10,11,12,14-hexafluoropentacene . To distinguish between the different edges of a nanosheet for attachment and desorption processes, we denote them according to the molecular side that is exposed at the respective nanosheet edge as illustrated in Fig. 6PEN molecules. Here, blue and red areas correspond to attractive and repulsive interactions, respectively. The white area in the centre of the AEM, sometimes referred to as excluded area , in dependence of the 2D sheet dimensions (cf. ‘Methods’). The results are shown in Fig. math row and the nbth column, counted from the bottom left corner, corresponds to p(LE) for an island of ma × nb molecules, as exemplarily illustrated in the left panel for a 3 × 2 island. As one would expect, the geometrical aspect ratio of the nanosheet has a notable influence on p(LE), which tends to be larger for 〈p(LE) is always larger than 50%. Therefore, attachment of additional molecules to a nanosheet always favours 〈-extended growth, as p(LE) mostly exceeds 50%, and any growth of a sheet in direction 〈p(LE), thus further promoting growth in direction 〈To validate that this anisotropy of the attachment energy favours attachment of admolecules at the LEs and hence the formation of 〈6PEN island consisting of ma = 20 by nb = 50 molecules. A map of the average desorption sequence of molecules from this nanosheet, determined from 50 individual simulations of desorption of such a nanosheet, is shown in Fig. In addition to the kinetics of growth, we have also simulated the kinetics of desorption (cf. ‘Methods’). Supplementary Movie 6PEN, C20N2H6F6)30. This molecule is fluorinated above the median molecular M-axis and exhibits a distinctly bipolar electrostatic potential along the L-axis  M-F6PEN on MoS2. Here, as for adsorption, molecular islands are preferentially elongated in direction 〈6PEN nanosheets. By contrast, heating of a complete monolayer of M-F6PEN to 410 K for 1 min to induce partial desorption yields islands with no clear shape preference . Though strongest attraction is found along the LEs, there are weaker but longer-ranged attractive interactions along the SEs. The longer range of those attractive interaction might promote attachment at the SEs despite overall weaker attraction. Indeed, in agreement with the experimental results, Monte Carlo simulations of the single-molecule attachment process to nanosheets  is always below 50% and quite homogeneous. In a simple picture, the alternating attractive and repulsive cusps at the LEs  is almost independent of the nanosheet shape in the case of M-F6PEN, but shows a distinct dependence on the nanosheet shape for the L-F6PEN. Simulations of the dismantling process during desorption of an M-F6PEN nanosheet , have the weakest bond to the nanosheet and therefore tend to desorb first, leading to the formation of islands elongated in the 〈To examine the influence of the molecular shape on the growth kinetics upon ad- and desorption, we have extended our computational analysis to the partially fluorinated perylene derivative 1,2,3,10,11,12-hexafluoroperylene  Molecules must interact attractively to promote the formation of stable nanosheets. (ii) Interactions must be anisotropic, which also requires shape anisotropy of the molecule. If interactions are isotropically attractive, no preferential direction for lateral bonding of molecules can be observed. (iii) Molecules must prefer a packing motif that allows the formation of edges with a homogeneous termination. These edges are crucial for opposite nanosheet extensions upon growth and partial desorption, as they are both the preferred edges for ad- and desorption as discussed above. (iv) Interactions must be sufficiently strong to prohibit detachment of molecules from the nanosheet without desorption. If molecules could detach from nanosheets during thermal treatment and reattach elsewhere, it is likely that no shape difference between nanosheets that were grown and ones that were created by partial desorption would occur.6PEN), a packing motif with uniform molecular orientation will occur, allowing the formation of uniformly terminated nanosheet edges.In summary, we rationalise the kinetically controlled formation of 2D molecular nanosheets composed of partially fluorinated polycyclic aromatic hydrocarbons with bipolar electrostatic potentials using high-resolution STM data and simulations of ad- and desorption processes. We find that packing motifs that allow the formation of uniformly, i.e., only hydrogen- or fluorine-terminated edges, promote attachment of molecules to and detachment of molecules from these edges upon ad- and desorption, respectively. This provides means of control over the shape of nanosheets that are elongated in one direction upon growth of the sheets and in the other upon desorption. A key property of the molecules used in this work is the pattern of fluorination and their shape anisotropy. In case of distinctly elongated molecules with partial fluorination along one long side only , this is not a material niche. In particular with regard to the interesting potential of organic/2D material hybrid systems for future device architectures6PEN and M-F6PEN films were grown by means of organic molecular beam deposition under ultrahigh vacuum (UHV) conditions from resistively heated Knudsen cells at room temperature with typical deposition rates of 2 Å/min as determined by quartz crystal microbalances. Molecular materials with an initial purity of approximately 98% were carefully degassed before deposition to increase material purity. MoS2 crystals were grown by means of chemical vapour transport from stoichiometric amounts of Mo and S using Br2 as source for the transport agent MoBr4 formed in situ. The reaction was performed in an evacuated quartz glass ampoule with a temperature gradient from 1300 K to 1220 K for 20 days23. The MoS2 crystals were exfoliated under ambient conditions prior to evacuation and annealed at 650 K for 15 min prior to the deposition of organic material. STM measurements were carried out in UHV (base pressure <10−10 mbar) using an Omicron VT STM in constant current mode with etched tungsten tips at sample temperatures of 110 K. Complementary work function measurements were conducted in situ in the same UHV system by means of a Kelvin probe setup . As a reference for the contact potential, we used highly oriented pyrolytic graphite  with a work function of 4.4 eV33. NEXAFS measurements in partial electron yield mode (retarding field: 150 V) were performed at the HE-SGM beamline of the electron storage ring BESSY II in Berlin (Germany). Details on the experimental setup and the data analysis can be found in ref. 34.L-FAttachment and desorption processes were simulated using self-programmed code. The source code is available free of charge (cf. ‘Code availability’) with a detailed documentation. Here, we briefly describe the underlying model and assumptions for the simulations performed throughout this work.22H14, PEN) and perfluoropentacene , two closely related molecules to the L-F6PEN and M-F6PEN used in this work. In ref. 25, it is shown by means of temperature-programmed desorption (TPD) and STM that the repulsively interacting acenes are highly mobile on the MoS2(001) surface, even at cryogenic temperatures at which well-ordered films of the same molecules are observable on metal surfaces. In ref. 24, a point-on-line epitaxy is reported for multilayer films of PFP grown on different transition metal dichalcogenides, including MoS2. The observed structure shows that growth of the molecular film is indeed not dictated by molecule-substrate interactions in the first layer of the film, but only by intermolecular interactions. Finally, ref. 27 shows that PEN deposited on 2D flakes of MoS2 on a Au(111) substrate adsorbs preferentially on the Au(111) substrate, showing that molecules can diffuse from the MoS2 flake to the Au(111) substrate and that the adsorption energy for PEN on Au(111) is significantly larger than that for PEN on MoS2, which is supported by DFT computations and experimental data in refs. 35. Together, these studies show that the molecule-substrate interaction potential and its spatial corrugation must be negligibly small, as it appears that all structure formation is purely dictated by intermolecular interactions. For these reasons, we have neglected any corrugation of the molecule-substrate interaction potential and represent the molecule-substrate interaction through a constant term that is only relevant for TPD simulations. Such simulations with the same assumption were already performed for PEN and PFP adsorbed on MoS2 in ref. 25, successfully replicating experimental data.One fundamental assumption underlying all molecular dynamics simulations is that the corrugation of the molecule-substrate interaction potential is negligible. This assumption might appear crude first, but it is based on experimental evidence reported for pentacene  surface, and grow in a single layer, molecular diffusion is restricted to two dimensions. Intermolecular interactions were modelled as described by Kröger et al.29, considering electrostatic interactions between the intramolecular charge distributions, attractive dispersion forces, and short-ranged Pauli repulsion. In absence of any interface dipole moments, as supported by work function measurements  with the B3LYP functional and aug-CC-pvTZ basis set39. All interaction parameters used in this work can be found in Supplementary Note Since we need to appropriately model electrostatic interactions in the near field, we cannot rely on simple far-field approximations of dipole-dipole interactions but need a finer subdivision of the intramolecular charge distributions. Therefore, all intermolecular interactions were treated in an atomistic model, in which molecules are modelled as rigid structures consisting of, in the cases of L-F6PEN, optimisation yields a = 16.78 Å, b = 7.33 Å, and γ = 70.81° with an angle of 80° between the molecular L-axis and b. For M-F6PEN, we use a = 16.4 Å, b = 15.0 Å, and γ = 90° with an angle of 90° between the molecular L-axis and b and two molecules per unit cell. For the uniformly oriented L-F6PER structure, we find a = 12.06 Å, b = 9.14 Å, and γ = 65.6° with an angle of 84.4° between the molecular L-axis and b. For the alternating L-F6PER structure, we find a unit cell with a = 23.58 Å, b = 9.24 Å, and γ = 90° with an angle of 90° between the molecular L-axis and b and two molecules per unit cell.Based on the above-described intermolecular interactions, interaction potential maps were calculated by positioning a single molecule or a rigid molecular nanosheet with fixed orientation and location in the centre of the coordinate system and then calculating the interaction energy of a probe particle of fixed orientation with the centre molecule or island as a function of the relative probe displacement. For calculations of the outer molecular electrostatic potential Fig. , an elecma × nb molecules. A single additional molecule was placed at a random location on a ring around the nanosheet centre with a radius of thrice the width or height of the nanosheet, whichever is larger. The admolecule was then allowed to move for a fixed number of 100 steps along the force vector acting at its current location. The step size is coupled to the admolecule-nanosheet distance so that it decreases as the molecule approaches the nanosheet. After completion of the movement steps, the location of the admolecule is categorised as follows: If its distance to the closest molecule exceeds 1.2 times the diagonal of the unit cell of the structure, it is considered to not have attached. Else, the closest edge is determined to determine whether the admolecule has attached to an LE or an SE. This simulation was performed 1000 times per nanosheet size (ma × nb) and repeated for varying nanosheet sizes, allowing for the calculation of an LE or SE attachment probability, p(LE) or p(SE) = 1 – p(LE), respectively, as a function of the nanosheet LE and SE extension.Molecular attachment at existing nanosheets was modelled by creation of a rigid molecular nanosheet consisting of 40. This algorithm was already successfully used to simulate TPD of PEN and PFP from MoS2 substrates25. Therefore, a surface is populated with To analyse desorption of molecules from nanosheets, TPD was simulated based on an algorithm described by Meng and Weinberg in ref. 35. The prefactor 19 s−1 for a crystalline monolayer of PEN35. 2(001) surface. Since optoelectronic properties such as energy levels and frontier orbitals of partially fluorinated pentacenes usually lie between those of PEN and PFP41, and 2 and PFP adsorbed on MoS2 with 122 ± 5 kJ mol−1 and 131 ± 4 kJ mol−1, respectively, we have chosen the experimental data for PEN of 122 kJ mol−1. Note that, since not quantitative conclusions are drawn from the TPD simulations, the exact values for 6PER.Desorption was simulated based on the reaction rate constant The TPD algorithm proceeds as follows: The reaction desorption rate constant 25. Therefore, between individual desorption events, molecules were allowed to diffuse for a predetermined time period. Initially, all molecules were given Boltzmann-distributed random initial (angular-) velocities that were increased according to the temperature increase between desorption events. Periodic boundary conditions were included to allow for smooth diffusion in the confined sample surface area. Due to high computational demand of diffusion simulations that is further increased by the periodic boundary conditions, diffusion time was limited to 1 ps at low coverage and reduced proportional to the surface coverage at higher coverages. TPD of PEN/MoS2 was simulated for varying initial surface coverages (cf. Supplementary Note 25, thus proving that the interaction potential used in this work are reasonably accurate.For validation, TPD simulations were also carried out for PEN. Here, due to repulsive intermolecular interactions, molecular diffusion must be includedFurther information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Movie 1Reporting Summary


Because of the current unmet medical needs, new drugs are urgently required. In this context, the activities related to drug discovery and development play a key role, allowing the delivery of new drugs to the market, and thus available at the pharmacy shelf . Traditiin vivo, and, very importantly, are compatible with several analytical strategies for high throughput drug screening and evaluations, including mass spectrometry (MS) and high-performance liquid chromatography (HPLC), two well-established methods employed in drug discovery and development ’s dataset was used instead of public databases for molecular generation, allowing to overcome the above limitation. DEL data-based molecular generation could represent a powerful tool allowing not only the improvement of drug discovery but also the enhancement of structural optimization technique.Molecular generation through machine learning has speeded the structural optimization of drugs, especially in the case of targets for which a large amount of bioactivity data has been reported, while is, unfortunately, often powerless when considering new targets . The secPrivitera et al., describing the coupling of microfluidics to HPLC to study the protective activity exerted by carnosine on human microglia challenged with lipopolysaccharides (LPS) and ATP, an in vitro model mimicking the molecular alterations observed in depressed patients . Microglia represent a common model of LPS-induced activation to identify novel pharmacological targets for depression and Alzheimer’s disease, and numerous studies have linked the impairment of energy metabolism to the onset of depressive episodes  for analytical evaluation and synthetic preparation of pharmaceuticals. EESI-MS allows the effective ionization of molecules of interest, even if at low concentration, in complex mixtures, with high sensitivity, selectivity, and responding speed, with no need of any sample pretreatment. In addition to that, this technique makes it possible at high-energy molecular species to undergo specific reactions leading to advanced functionalization. As highlighted by the authors, EESI-MS could be further developed in several aspects, including in vivo analysis and reliability, thus providing a unique platform for wide applications in numerous disciplines such as chemistry, biology, and the life sciences (The review by sciences .in vitro and in vivo preclinical studies with in silico ones is of utmost importance to increase the findings related to drug discovery and development and to translate them to clinical practice.In conclusion, all the articles published covered different and current aspects related to the proposed Research Topic, considering the importance of microfluidics and/or mass spectrometry in drug discovery and development processes. The combination of


Klinefelter syndrome (KS) is the most common sex chromosome disorder and genetic cause of infertility in males. A highly variable phenotype contributes to the fact that a large proportion of cases are never diagnosed. Typical hallmarks in adults include small testes and azoospermia which may prompt biochemical evaluation that typically shows extremely high follicle-stimulating hormone and low/undetectable inhibin B serum concentrations. However, in prepubertal KS individuals, biochemical parameters are largely overlapping those of prepubertal controls. We aimed to characterize clinical profiles of prepubertal boys with KS in relation to controls and to develop a novel biochemical classification model to identify KS before puberty.Retrospective, longitudinal data from 15 prepubertal boys with KS and data from 1475 controls were used to calculate age- and sex-adjusted standard deviation scores (SDS) for height and serum concentrations of reproductive hormones and used to infer a decision tree classification model for KS. Individual reproductive hormones were low but within reference ranges and did not discriminate KS from controls. Clinical and biochemical profiles including age- and sex-adjusted SDS from multiple reference curves provided input data to train a ‘random forest’ machine learning (ML) model for the detection of KS. Applied to unseen data, the ML model achieved a classification accuracy of 78% . Supervised ML applied to clinically relevant variables enabled computational classification of control and KS profiles. The application of age- and sex-adjusted SDS provided robust predictions irrespective of age. Specialized ML models applied to combined reproductive hormone concentrations may be useful diagnostic tools to improve the identification of prepubertal boys with KS. Klinefelter syndrome (KS) is the most common sex chromosome disorder affecting approximately 1:660 males , 3. HoweThe hypothalamo–pituitary–gonadal (HPG) hormone axis shows varying activities during postnatal life in healthy subjects; high activity is observed in minipuberty , followeTraditional references in pediatric hormone concentrations are stratified in distinct age intervals, but this crude approach may be sub-optimal for diagnosing specific endocrine conditions. Recent adoption of continuous hormone references using the well-established ‘LMS’ growth curve algorithm provides a clinically useful framework to standardize age- and sex-dependent variance and obtain standard deviation scores (SDS) , 7, 8, 9Supervised machine learning (ML) is an artificial intelligence method that is used for optimizing the interpretation of complex or large datasets. During recent years, ML has increasingly been implemented in the field of medicine as a tool for optimizing diagnostics , 13, 14.The aim of this study was to characterize the combined reproductive endocrine profiles of prepubertal boys with KS in relation to those of controls and to develop a novel classification model to predict new cases of KS in the pediatric population. Hereby, we intend to increase the likelihood of detecting, diagnosing, and providing healthcare for KS patients at a much younger age.n = 1475) aged 5.8–20.9 years were retrieved from the COPENHAGEN Puberty Study conducted in 2006–2008, as previously described (n = 729) as defined by TV <4 mL were aged 5.8–13.4 years.Longitudinal, retrospective clinical and biochemical data from a total of 35 visits were obtained from 15 untreated prepubertal boys with 47,XXY KS aged 6.3–13.4 years. Data from routine clinical visits were obtained from patient records from a tertiary center at The Department of Growth and Reproduction, Rigshospitalet, Copenhagen, Denmark. The Klinefelter diagnosis was established prenatally in ten boys and due to psychosocial disorders or learning difficulties in four boys. The reason for diagnosis was unknown in one boy. All boys with KS were prepubertal as defined by testicular volume (TV) < 4 mL, Tanner genital stage G1, and Tanner pubic hair stage PH1. All three puberty measures were used since boys with KS may have entered puberty despite having a TV below 4 mL. Corresponding clinical and biochemical profiles for controls  and anti-Müllerian hormone (AMH) changed during the study period as previously described .Serum concentrations of total testosterone, dehydroepiandrosterone sulfate (DHEAS), 17-hydroxyprogesterone (17-OHP), androstenedione (4A), SHBG, follicle-stimulating hormone (FSH), luteinizing hormone (LH), AMH, and inhibin B were measured. Total testosterone, DHEAS, 17-OHP, and 4A were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS) with inter-assay coefficients of variation (CVs) ranging from 1.4 to 2.5%, as previously described  with an inter-assay CV below 5% for both and a limit of detection (LoD) of 0.06 and 0.05 IU/L for FSH and LH, respectively. Until September 2014, serum SHBG was measured by time-resolved immunofluoresence assay   with a CV below 6% and an LoD of 0.23 nmol/L, while SHBG from September 2014 and onward was measured by a chemiluminescence immunoassay   with a CV below 5% and an LoD of 0.35 nmol/L. All Access2-derived SHBG results were factored to corresponding AutoDELFIA SHBG results after internal method comparison. An enzyme-linked immunosorbent assay (ELISA) was used to determine inhibin B . The LoD and inter-assay CV for inhibin B were 3 pg/mL and 11%, respectively. AMH was measured by AMH-ELISA (Immunotech) until 2015  and by chemiluminescence immunoassay  from 2015 and onwards . Based on an internal validation, the results of the two methods were comparable. The LoD and inter-assay CV for AMH were 0.14 pmol/L and less than 2.5%, respectively.https://cran.r-project.org/web/packages/gamlss/gamlss.pdf). The LMS method summarizes local skewness (L), mean (M), and coefficient of variation (S) and is universally applied to establish anthropometric growth charts (n = 64).Reference curves for body height and serum concentrations of LH, FSH, inhibin B, AMH, total testosterone, SHBG, A4, 17-OHP, and DHEAS were extrapolated from observations of the controls in the COPENHAGEN Puberty Study and modeled using the ‘LMS’ method provided in the Generalized Additive Model for Location, Scale, and Shape (gamlss) package in R  and prepubertal controls  curves using the ‘pROC’ package in R.The ‘randomForest’ and ‘caret’ packages in R were used to randomly split the dataset rows into training (75%) and testing (25%) data frames and generate a random forest prediction model. The proportion of prepubertal boys with KS  function ‘prcomp’ in R was applied to the SDS profiles of prepubertal controls and boys with KS in the dataset, as previously demonstrated . The ‘ggAccess to and management of data from patient files from boys with KS was approved by the Danish Patient Safety Authority (no. 3-3013-1376/1/), the Team for Medical Records Research, Centre for Health, the Capital Region of Denmark (R-22031906), and the Danish Data Protection Agency (no. 2015-235 (I-Suite no. 04204) and P-2022-364).The COPENHAGEN Puberty Study was approved by the local ethical committee (KF 01 282214 and V200.1996/90) and was conducted in accordance with the Second Helsinki Declaration. All participants and their parents signed informed consents.Body height and serum concentrations of LH, FSH, inhibin B, AMH, total testosterone, SHBG, A4, 17-OHP, and DHEAS in prepubertal boys with KS according to chronological age are depicted in Body height was equally distributed through the reference range, and each boy with longitudinal measurements followed his own curve .Age- and sex-adjusted SDS for body height and serum concentrations of LH, FSH, inhibin B, AMH, total testosterone, SHBG, A4, 17-OHP, and DHEAS were calculated for both prepubertal controls and for prepubertal boys with KS . When coIn order to evaluate the feasibility of detecting the KS profile by means of biochemical and clinical data, predictive values of all current variables including body height as well as serum concentrations of LH, FSH, inhibin B, AMH, total testosterone, SHBG, A4, 17-OHP, and DHEAS were assessed by ROC analyses. The ability of each variable to distinguish prepubertal boys with KS from controls is listed as ROC, area under the curve (AUC), and in terms of sensitivity and specificity . As evidPCA was applied to age- and sex-adjusted SDS of prepubertal control boys and prepubertal boys with KS to explore unbiased systematic differences with regard to serum concentrations of LH, FSH, inhibin B, AMH, total testosterone, SHBG, A4, 17-OHP and DHEAS, and body height. Dimensionality reduction by PCA was used to explore potential clusters in the total dataset variance. Variance clusters are detectable by PCA when two or more phenotypes differ systematically across several feature variables, and we hypothesized that this would apply to the current dichotomy of KS and controls. PCs 1 and 2 explained 24.4 and 17.3% of the total dataset variance, respectively. The resulting biplot, visualizing individual observations of KS patients and controls in context of the total dataset variance, largely indicated overlapping endocrine profile clustering (i.e. systematic differences) between prepubertal KS patients and prepubertal controls, and neither PC1 nor PC2 coordinates differed significantly between the two groups .Figure The predictive values of the parameters presented in P < 0.05). We found no difference regarding total testosterone, inhibin B, A4, and body height . Dimensional reduction of the prepubertal phenotypes was resolved by PCA and revealed some degree of clustering)  in the KS endocrine profiles compared to that of controls. Leveraging all the available feature variables above to define the prepubertal KS phenotype, we trained a decision tree-based ML model to differentiate between KS patients and age-matched controls. When applying the ML model to predict (i.e. binary classification of control or KS status) unseen data, the obtained ML model achieved a prediction accuracy of 78%  and Cohen's kappa of 0.65 , thus outperforming the predictive value of any singular variable .This study was supported by