Can a quality improvement antibiotic stewardship intervention implemented through external and internal facilitation decrease urine culture orders and antibiotic use for asymptomatic bacteriuria (ASB)?In this quality improvement study of 11 299 patients in acute medical and long-term care units at 4 Veterans Affairs facilities, case-based teaching on applying an evidence-based algorithm to distinguish urinary tract infection and ASB was associated with fewer urine culture orders, fewer days of antibiotics, and shorter length of antibiotic therapy. The 3 clinical outcomes did not improve at 4 comparison sites.These findings suggest that individualized antibiotic stewardship for ASB can be implemented effectively at a distance via external and internal facilitation. This quality improvement study explores external and internal facilitation as an implementation strategy for antibiotic stewardship in both acute and long-term care among patients with asymptomatic bacteriuria. Antibiotic stewardship for asymptomatic bacteriuria (ASB) is an important quality improvement target. Understanding how to implement successful antibiotic stewardship interventions is limited.To evaluate the effectiveness of a quality improvement stewardship intervention on reducing unnecessary urine cultures and antibiotic use in patients with ASB.This interrupted time series quality improvement study was performed at the acute inpatient medical and long-term care units of 4 intervention sites and 4 comparison sites in the Veterans Affairs (VA) health care system from October 1, 2017, through April 30, 2020. Participants included the clinicians who order or collect urine cultures and who order, dispense, or administer antibiotics. Clinical outcomes were measured in all patients in a study unit during the study period. Data were analyzed from July 6, 2020, to May 24, 2021.Case-based teaching on how to apply an evidence-based algorithm to distinguish urinary tract infection and ASB. The intervention was implemented through external facilitation by a centralized coordinating center, with a site champion at each intervention site serving as an internal facilitator.Urine culture orders and days of antibiotic therapy (DOT) and length of antibiotic therapy in days (LOT) associated with urine cultures, standardized by 1000 bed-days, were obtained from the VA’s Corporate Data Warehouse.P = .56). However, difference-in-differences analysis comparing intervention with comparison sites found a significant reduction in the number of urine cultures ordered by 3.24 urine cultures per 1000 bed-days (P = .003). In the segmented regression analyses, the relative percentage decrease of DOT in the postintervention period at the intervention sites was 21.7% (P = .007), from 46.1  to 37.0  per 1000 bed-days. The relative percentage decrease of LOT in the postintervention period at the intervention sites was 21.0% (P = .001), from 36.7  to 29.6  per 1000 bed-days.Of 11 299 patients included, 10 703 (94.7%) were men, with a mean (SD) age of 72.6 (11.8) years. The decrease in urine cultures before and after the intervention was not significant in intervention sites per segmented regression analysis (−0.04 ; The findings of this quality improvement study suggest that an individualized intervention for antibiotic stewardship for ASB was associated with a decrease in urine cultures and antibiotic use when implemented at multiple sites via external and internal facilitation. The electronic health record database–derived outcome measures and centralized facilitation approach are both suitable for dissemination. This intervention package, the Kicking CAUTI (catheter-associated urinary tract infection) Campaign, consists of a validated guideline-based algorithm plus individualized audit and feedback.12 The Less is More for ASB project was designed to evaluate whether the Kicking CAUTI intervention could be scaled up and delivered at a distance to 4 geographically distant VA medical centers with 4 comparison sites. Although we retained the Kicking CAUTI name, the intervention’s scope was expanded to address all cases of suspected UTI.A urine culture is often the first step in a chain of events leading to unnecessary antibiotic use, longer hospital stays, and other harms.15 Our centralized coordinating center served as the external facilitator, or the agency responsible for promoting and supporting positive change.16 The external facilitators  supported each intervention site’s champion, who became an internal facilitator for uptake of the intervention . The site champion or internal facilitator at each site was a local leader in infectious diseases, well-positioned to deliver training on how to use Kicking CAUTI components and promote organizational culture change. In the Less is More for ASB project, we hypothesized that our intervention would lead to the following outcomes: (1) the rate of urine culture orders would decrease over the course of the intervention period in intervention sites and would not decrease in the comparison sites; and (2) antimicrobial use related to urine cultures would decrease in the intervention sites and would not decrease in the comparison sites.A novel aspect of the Less is More for ASB project was the exploration of external and internal facilitation as an implementation strategy for antibiotic stewardship in both acute and long-term care.SQUIRE 2.0) reporting guidelines were referenced as a framework for quality improvement methods, data analysis, and reporting. Although this project was considered quality improvement from the perspective of each implementation site, the central facilitation team had access to individual health records at each site. Therefore, we sought and received approval by the institutional review board at each site. The need for informed consent was waived because the goal of the intervention was to implement standard of care based upon published guidelines.We used an interrupted time series design with 4 intervention sites and 4 comparison sites for this quality improvement study. This project engaged 4 geographically distant VA facilities in the intervention; each was paired with a control site in the same geographic region. Our intervention and comparison sites were similar in terms of geographic location, number of beds overall, intensive care unit beds, types of wards, academic affiliation, and teaching status, but they were specifically chosen to be in different VA Service Networks to avoid cross-contamination. Standards for Quality Improvement Reporting Excellence  for advice and encouragement. The internal facilitators received standardized teaching cases that embedded the algorithm in an interactive decision tree, and they were encouraged to deliver 2 to 4 teaching cases per month in various settings  of their own choice.17 The Kicking CAUTI algorithm steps clinicians through 2 questions they should ask themselves before ordering a urine culture or starting antibiotics for suspected UTI  an evidence-based algorithm that distilled the guidelines into a streamlined clinical pathway and (2) case-based education to train clinicians to use the algorithm.We intended for each site to spend at least 12 months in the intervention phase. We staggered the start of the intervention across sites to focus external facilitation on 1 site at a time . The intWe included all patients admitted to or residing in an acute care medicine ward or a community living center long-term care unit (VA nursing home) during the study period. Patient data were extracted from the VA Corporate Data Warehouse housed on the VA Informatics and Computing Infrastructure. Baseline was from October 1, 2017, until the start of the intervention in each site or its matched control; intervention was from start date through April 30, 2020 .19 For example, if a patient is prescribed 2 antibiotics to take together for 5 days, the DOT is 10 days and the LOT is 5 days. By using both metrics, we understand the number of antibiotics used and the true lengths of therapy. Our pocket card decision algorithm  were ASB . After the intervention, the percentage of ASB cases treated with antibiotics fell from 113 of 480 (23.5%) to 43 of 280 .To validate that our antibiotic use measures reflected antibiotics prescribed in reaction to a suspected UTI or to the results of a urine culture, we performed a medical record review of 1261 cases of positive urine cultures in intervention sites—726 in the baseline period and 535 in the intervention period. Each culture was classified as ASB or UTI per standardized algorithm19 both measured for antibiotic regimens started temporally in relationship to a urine culture.The primary outcome (hypothesis 1) was the total number of urine cultures ordered by inpatient or long-term care clinicians (urine cultures ordered before admission in the emergency department or outpatient clinics were excluded). Our outcomes for antimicrobial use (hypothesis 2) were metrics of inpatient antimicrobial consumption: DOT and LOT,17 We performed descriptive statistics for patients’ demographic characteristics  and site of care  stratified across the baseline and intervention periods and the intervention and comparison sites. Race and ethnicity are recorded in each patient’s electronic health record, and this information was included to support the generalizability of the study.Data were analyzed from July 6, 2020, to May 24, 2021. The sample size calculation for this study was reported previously.P < .05 indicated statistical significance. Analyses were conducted with STATA statistical software, version 17 (StataCorp LLC) and SAS statistical software, version 9.4 .Data for each outcome variable (urine cultures and urine culture–related DOT and LOT per 1000 bed-days) were aggregated monthly by facility level, and the period was divided into baseline and intervention segments. Autoregressive integrative moving average interrupted time series techniques were used to examine change in slope or level owing to the intervention. The details of the segmented linear regression models are available in eMethods 2 in the Of 11 299 patients included, 10 703 (94.7%) were men, 524 (4.6%) were women, 71 (0.6%) did not disclose, and 1 (0.01%) was a transgender woman, The mean (SD) age was 72.6 (11.8) years. Demographic characteristics in the baseline and intervention periods in the intervention and comparison sites are presented in 1) are negative and significant for both intervention and comparison sites , which indicates that the rate of urine culture ordering decreased in the intervention and comparison sites during the baseline period. The coefficient estimate for the change in regression slope (β3) in the intervention sites is not significant , indicating that the rate of urine culture ordering remained unchanged after the intervention (continued to decrease at the same rate). The relative percentage decrease of urine cultures at the intervention sites after the intervention was 6.9%. In contrast, the rate of urine culture ordering increased during the intervention period in the comparison sites, as indicated by significant and positive coefficient estimate  for the change in regression slope during the intervention period compared with the preintervention period (β3). The relative percentage increase of urine cultures at the comparison sites in the postintervention period was 28.8%.The overall mean number of urine cultures ordered was 15.1  per 1000 bed-days in the baseline period and 11.9  per 1000 bed-days in the intervention period at the intervention sites. The overall mean number of urine cultures was 14.0  per 1000 bed-days in the baseline period and 12.6  per 1000 bed-days in the intervention period at the comparison sites . We dispP = .003) in the intervention sites compared with comparison sites.Because the baseline trends of urine culture ordering were similar for the intervention and comparison sites (decreasing baseline slopes in 1) was not significant in the intervention sites , indicating that the rate of urine culture–related DOT remained unchanged during the baseline period (1) in the comparison sites was negative , which shows that the rate of DOT was decreasing. The coefficient estimate for the change in regression slope (β3) at the intervention sites is significant and negative , which indicates that the rate of urine culture–related DOT decreased after the implementation of the intervention (3) at the comparison sites is significant and positive , indicating that the rate of urine culture–related DOT increased in the intervention period at the comparison sites compared with the preintervention period. The relative percentage increase of DOT at the comparison sites in the post-intervention period was 35.1%. We did not perform DID analysis for DOT, because the assumption of parallel trends in the baseline period was not met  in the baseline period and 37.0  in the intervention period at the intervention sites. The overall means for urine culture–related DOT were 46.2  in the baseline period and 40.5  in the intervention period at the comparison sites. The time series analysis for DOT is presented in e period . In contrvention . The rel1) was not significant in the intervention sites , indicating that the rate of urine culture–related LOT remained unchanged (1) in the comparison sites was negative , which shows that the rate of LOT was decreasing. The coefficient estimate for the change in regression slope (β3) at the intervention sites was significant and negative , which indicates that the rate of urine culture–related LOT decreased after the implementation of the intervention (P = .001). In contrast, the coefficient estimate for the change in regression slope (β3) at the comparison sites was significant and positive , indicating that the rate of urine culture–related LOT increased in the intervention period at the comparison sites compared with the preintervention period. The relative percentage increase of DOT at the comparison sites in the postintervention period was 37.3%. We did not perform DID analysis for LOT, because the assumption of parallel trends in the baseline period was not met  in the baseline period and 29.6  in the intervention period at the intervention sites. The overall means for urine culture–related LOT were 36.8  in the baseline period and 32.8  in the intervention period at the comparison sites. We displayed the time series analysis for LOT as a graph C and sumnchanged . In contrvention . The rel2 term in the model, thereby excluding the immediate level change after the intervention  in all 3 clinical outcomes: urine cultures, DOT, and LOT. More than half of treated patients received more than 1 antibiotic, which underlines the importance of including both DOT and LOT metrics to target both the number of antibiotics used and the true LOT being used.21 Patients admitted for COVID-19 infection often receive unnecessary antibiotics and intensive care unit admission,23 both of which could contribute to the observed increases in urine cultures, DOT, and LOT at the comparison sites. Increasing awareness of antibiotic resistance among uropathogens could also have driven increased antibiotic use. In addition, these increases also may suggest the natural tendency in medicine of “to do more, treat more” if not checked by a focused stewardship program.24 Likewise, a prepandemic literature evaluation of antibiotic use in the US did not find a decline over time.25In contrast, we observed a significant increase in urine cultures, DOT, and LOT in the comparison sites, which is consistent with recent national reports of increased health care–associated infections (including catheter-associated urinary tract infections) during the pandemic.16 is a viable implementation strategy for implementing stewardship26 at a distance. The study team in the coordinating center provided the external facilitation through coaching calls and by troubleshooting logistical challenges. The local champions delivered the interactive, case-based education in small group settings, which encouraged the engagement of frontline clinicians. A modest investment of resources in identifying and training a local internal facilitator may well be worth the significant reduction in infection surveillance and unnecessary antibiotic treatment of ASB. Also, our metrics were derived from a data warehouse, in real time, thus streamlining this project for clinical implementation without medical record reviews.1The success of this project implies that our external and internal facilitation27 decreased antibiotic prescribing for ASB in nursing homes through a multifaceted intervention that included external facilitation (coaching). Chambers et al28 conducted a virtual learning collaborative in 45 nursing homes, using diagnostic stewardship, which reduced urine culturing and antibiotic use. On a smaller scale, Doernberg et al29 demonstrated the value of having an infectious disease pharmacist make weekly visits to 3 community-based long-term care facilities, leading to a 26% decrease in antibiotic prescriptions for UTI. Acute care stewardship interventions decreased treatment of ASB, one with internal medicine residents30 and the other focused on hospitalist physicians.31 To our knowledge, the present study is the first to address both acute and long-term care together in the same ASB stewardship intervention in an integrated VA health care system.Comparison of our findings with those of other stewardship interventions for ASB is challenging, because the measurements used across projects vary considerably. Nace et al32 in which 68% of urine cultures ordered on inpatients lacked a clinical indication. The differences in our outcomes were relatively small on an absolute basis. When comparing within the intervention sites using statistical comparison of time trends before and after the intervention, we did not find a significant difference in urine cultures before or after the intervention, but the DID analysis comparing intervention with comparison sites showed a significant decrease in urine cultures in the intervention sites after the intervention. Urine culture orders are a blunt measure but are suitable to automation for use outside the scope of a research project. The same limitations apply to DOT and LOT; we know that some antibiotic use in temporal association with urine cultures is appropriate. We excluded antibiotics that were either not relevant to treating UTI or for which therapy was not started in the same time frame as a urine culture. We had hoped to measure sustainability after each site completed a full 12 months in the intervention phase. However, the pandemic placed such time demands on our site champions that we ended data collection in May 2020. Our implementation strategy is likely generalizable to the other VA facilities that have infectious diseases services, an antibiotic stewardship program, and an academic affiliation, but we cannot assess the applicability to health care centers that do not have these characteristics.Our study has certain limitations, some of which are intrinsic to the use of database measures, others of which are related to the COVID-19 pandemic. Our primary outcome was total urine cultures ordered; this metric does not account for whether the cultures are appropriate or not. Some patients will have legitimate reasons for urine culture. We suspect that many of the urine cultures were inappropriate, extrapolating from the study by Leis et alThe findings of this quality improvement study suggest that external and internal facilitation can be used to implement the evidence-based Kicking CAUTI intervention. The Kicking CAUTI approach to stewardship resulted in reductions in orders for urine cultures and antibiotic use while being implemented at a distance. In contrast, sites that were not participating in the invention had an increase in urine cultures and antibiotic use temporally associated with urine cultures. Our use of database-derived metrics and a centralized facilitation approach are suitable for further dissemination to affect a greater number of patients and clinicians.


Caenorhabditis elegans. Here, we describe the development of a synergistic pipeline of molecular genetic, imaging, and computational technologies designed to allow high-throughput, precise quantitation with cellular resolution of reporters of gene expression in intact whole tissues with complex cellular constitutions such as the brain. We have deployed the approach to determine with exactitude the number of functional neurons and glia in the entire intact larval Drosophila CNS, revealing fewer neurons and more glial cells than previously predicted. We also discover an unexpected divergence between the sexes at this juvenile developmental stage, with the female CNS having significantly more neurons than that of males. Topological analysis of our data establishes that this sexual dimorphism extends to deeper features of CNS organisation. We additionally extended our analysis to quantitate the expression of voltage-gated potassium channel family genes throughout the CNS and uncover substantial differences in abundance. Our methodology enables robust and accurate quantification of the number and positioning of cells within intact organs, facilitating sophisticated analysis of cellular identity, diversity, and gene expression characteristics.Establishing with precision the quantity and identity of the cell types of the brain is a prerequisite for a detailed compendium of gene and protein expression in the central nervous system (CNS). Currently, however, strict quantitation of cell numbers has been achieved only for the nervous system of  Caenorhabditis elegans   , the exa56 glia) .Drosophila melanogaster, which in scale and behavioural repertoire has been viewed as intermediate between nematodes and rodents , remain unknown, though several approximations have been suggested. For the larval CNS, a range of 10,000–15,000 active neurons has been proposed  to summarise the CNS in terms of multiscale topological structures. Utilisation of these topological summaries in a support vector machine also supports that sexual dimorphism extends to deeper features of CNS organisation. Finally, we applied our pipeline to quantitate the whole CNS expression frequency of the Drosophila family of voltage-gated potassium channels, which revealed divergent channel expression frequencies throughout the CNS. We envision that our method can be employed to allow precise quantitation of gene expression characteristics of the constituent cells of the brain, and potentially other intact whole organs, in a format suitable for sophisticated downstream analysis.Here, we have sought to develop a synergistic molecular genetic, imaging, and computational pipeline designed de novo to allow automated neuron, glia, or other gene expression features to be precisely quantitated with cellular resolution in an intact whole CNS. Central to the approach are high signal-to-noise gene expression reporters that produce a punctate, nucleus-localised output, facilitating downstream automated computational measurements and analysis. Exploiting multiple genetic reagents designed to selectively identify only functional neurons with active synaptic protein expression, we identify substantially fewer neurons than most previous estimates in the Drosophila larval CNS function, we sought to develop a pipeline utilising genetic reporters designed to expediate automated neuron and glia quantitation from three-dimensional intact organ images. While membrane-associated reporters are generally employed to label Drosophila neurons  when homozygous, the line was employed in heterozygotes to capture Brp protein expression with high fidelity. To complement this line, we used the Trojan/MiMIC technique   is a critical presynaptic active zone component widely used to label analyses . We emplsequence  to efficechnique , to genetagmin 1 , the fastagmin 1 . Lastly,obrevin) , which eobrevin) , is usedobrevin) . All thrlav-GAL4 , which wlav-GAL4 . To charlav-GAL4 . Howeverlav-GAL4 , all thrlav-GAL4 . We alsolav-GAL4 . We founlav-GAL4 , suggestlav-GAL4 . Lastly,lav-GAL4  using inbrp, Syt1 and nSyb GAL4 lines to generate point cloud mathematical objects and compared them to point clouds of glial nuclei labelled by the repo-GAL4 line. We then plotted and compared the distributions of inter-nuclei distances in these lines. Using this measurement, we found that the inter-nuclei distance of glial cell nuclei exhibited a unimodal distribution  neurons, Syt1-GAL4 had 10,097  neurons, and nSyb-GAL4 had 9971  neurons . Averaging across the lines, we found that the female third instar larval CNS had 10,312 ±5.03%, n=16, neurons .We next counted the number of nuclei labelled by these neuronal and glial lines, again beginning with females . We foun neurons . We test neurons . To ensurepo-GAL4 line  neurons, Syt1-GAL4 labelled 9012  neurons, and nSyb-GAL4 labelled 9286  neurons in male larvae , while the number of neurons labelled by nSyb-GAL4 was not significantly different from Syt1-GAL4, similar to what we had found in females. Averaging across the lines, we found that the male third instar larval CNS had 9396 ±5.59%, n=15 neurons, significantly fewer than those of females . This difference was also consistent within individual genotypes with brp-GAL4 labelling , Syt1-GAL4 labelling  and nSyb-GAL4 labelling  less neurons in males than in females.We next carried out a similar analysis on the CNS of male larvae . We foune larvae . In malerepo-GAL4 in males  sex of the animal.Drosophila CNS at the larval stage.The 5000 splits were used to produce 5000 correlations with the true sex and 5000 correlations with a randomly assigned sex. The distribution of these correlations , exhibitDrosophila studies defined the founding members (Sh) (Kv1 family), Shab (Shab) (Kv 2 family), Shaw (Shaw) (Kv3 family), and Shal  (Kv4 family)   genes us family) . As the urements . We founurements , consistSh-GAL4 Shal-GAL4 Shab-GAL4 Shaw-GAL4 Sh and Shal were expressed in large numbers of neurons 8204 ±5.67%, n=10 and 8261 ±3.1%, n=5 respectively, though significantly less  than the average number of all male neurons  and Shaw  were expressed in smaller numbers of neurons (BDSC#39171) (repo-GAL4 (BDSC#7415) (repo-QF2 (BDSC#66477) (Shaw-GAL4 (BDSC#60325) (Ddc-GAL4(BDSC#7009) (TH-GAL4(BDSC#8848) (Trh-GAL4(BDSC#38389) (brp-GAL4 (this manuscript), UAS_H2A::GFP-T2A-mKok::Caax (this manuscript). All lines were raised on standard media at 25°C, 50% RH.The following stocks were employed - y[1] w[*]; Mi{y[+mDint2]=MIC}Syt1[MI02197] (BDSC#35973) , y(1) w, y(1) w, nSyb-GAC#39171) , repo-GASC#7415) , repo-QFC#66477) , Shaw-GAC#60325) , Ddc-GALSC#7009) , TH-GAL4SC#8848) , Trh-GALC#38389) , UAS_H2AC#38389) , QUAS_H2C#38389) , brp-GALbrp gRNA pCDF3: two gRNA sequences targeting each side of the insertion location in exon 2 of brp, were selected using the FlyCRISPR algorithm (http://flycrispr.molbio.wisc.edu/), consisting of 20 nucleotides each (PAM excluded), and predicted to have minimal off-targets. Each individual 20-nucleotide gRNA sequence were inserted into pCFD3 plasmid  using the KLD enzyme mix (New England Biolabs). brp-GAL4 insertion construct: the seven following PCR amplified fragments were assembled using HIFI technology: (1) 1198 bp homology arm covering 5’ UTR until 5’ target site; (2) the region between 5’ target site and the start codon were amplified from Drosophila nos-cas9 (attp2) genomic DNA (a modified Pam sequence was inserted using overlapping primers); (3) Linker-T2A-GAL4.2 sequence was amplified from pBID-DSCP-G-GAL4 (Drosophila nos-cas9 (attp2) genomic DNA. Full length assembly was topo cloned in zero-blunt end pCR4 vector (Invitrogen), all constructs have been verified by sequencing  and injections were carried out into a nos-cas9 (attp2) strain  strain . CorrectpDESTP10 LexO-H2A-GFP template (Gift from Steve Stowers) with a synthetic 5’UTR sequence (syn21) added upstream to H2A on the forward primer; (2) sfGFP was amplified from template pHD-sfGFP Scareless dsRed (Addgene 80811); and (3) mKok amplified from pCS2 +ChMermaid S188 (Addgene 53617) with the CAAX membrane tag sequence (Thosea asigna virus 2 A(T2A) self-cleaving peptide sequence (PCR amplifications were performed using Platinium Superfi polymerase (Invitrogen). The three PCR fragments were assembled together using Hifi technology (Invitrogen): (1) Histone2A (H2A) cDNA was amplified from sequence  added atsequence , was inssequence  using LRMiMIC lines generated by the group of Hugo Bellen  were acqWandering third instar larvae were dissected in 1 × PBS (Mediatech) and fixed with 4% formaldehyde (Sigma-Aldrich) for 20 mins. 1 × PBS were added to remove the fixative, and then the CNS was dissected  and rins3 were removed from analysis by segmentation filter to avoid unspecific signals. Subsequently, the number of nuclei and the x, y, z coordinates of the geometric center of each nucleus were output from Vision4D. Where manual counting was employed (brp-GAL4 labelled CNS), Vision4D was used to visualise and iteratively proceed through and manually annotate the dataset. Example whole CNS datasets where functional neurons or glia are labelled are available , whose coordinates encode the birth and death of homological features. For the filtration in the figure above, the persistence diagram that tracks one-dimensional features (i.e. unfilled loops) contains only a single point with coordinates . Here, the first coordinate, x, is the radius at which the loop is first formed, and the second coordinate, y, is the radius at which the loop has just been filled in. In the example it is clear that a<x<b<y< c.In the parlance of TDA, we refer to this appearance and disappearance of topological structures as the birth and death of homology classes in various degrees. We capture the whole life cycle with a mathematical object called the As multisets of points in the plane, persistence diagrams are not immediately usable as features for SVMs. One way to vectorise persistence diagrams and thus render them digestible by SVMs is to define kernels based on the diagrams, with the heat kernel  being anIn this analysis, we calculated the persistent homology of the alpha complex  of the pThe hyperparameters involved, i.e., the SVM regulariser and the heat kernel bandwidth, were determined by a parameter search in the following way. Six point clouds from males and six from females were randomly selected. All 100 subsampled versions of each of these 12 constituted a training set, for a total of 1200 training point clouds. The remaining 1900 subsampled point clouds constituted the testing set. The Pearson’s correlation between the gender predicted by the SVM on the testing set and the ground truth was computed for each choice of hyperparameters, and a choice in a stable region with high correlation was selected: a regularisation parameter c=10 in the notation of 9EH10 , visualised with goat anti mouse Alexa Fluor 488  together with conjugated goat anti HRP Alexa Fluor 647  and mounted in VectaShield (Vector Laboratories). Duel colour Z-stack images of stage 15/16 and late stage 17 embryos were obtained on a CSU-W1 Confocal Scanner Unit  using two prime BSI express cameras (Teledyne Photometrics). For motor neuron and adult brain preparations, larval fillets from the third instar larvae, or brains from adults were dissected and fixed with 4% formaldehyde (Sigma-Aldrich) for 20 mins. After fixation, samples were rinsed with 1 × PBS and were washed in PBT overnight at 4°C, and then mounted in Vectashield antifade mounting medium. Z-stack images were obtained with a Leica SP8 upright confocal microscope.Embryos were collected and staged at 25°C on apple agar plates supplemented with yeast paste. Standard methods were used for dechorionation, removal of the vitelline membrane and fixation . EmbryosFor Deadpan staining, the CNS from the third instar larvae were dissected out and fixed with 4% PFA for 20 mins. After fixation, samples were rinsed with 1 × PBS, and permealised with PBT (1 × PBS + 4% Triton-X 100). Antibody stainings were done in PBT +5% normal goat serum. The dilution for chicken anti-GFP (Abcam) was 1:500, for rat anti-Deadpan (Abcam) was 1:50. Goat anti-chicken Alexa Fluor 488, and goat anti-rat Alexa Fluor 594 secondary antibodies were obtained from ThermoFisher and used at the 1:500 dilutions. The CNS were mounted in VectaShield antifade mounting medium, and imaged using a Leica SP8 upright confocal microscope.https://doi.org/10.5281/zenodo.6574838 and plotted as a histogram of distance distribution.Column statistics analyses were performed using GraphPad Prism 9 (GraphPad Software). For  Drosophila brain. The authors uncover unexpected sexual dimorphism at this early stage. This paper will be of interest to neuroscientists – from those who use larval Drosophila as their study model to others who are generally interested in connectomics and transcriptomics.This manuscript describes a pipeline involving whole brain imaging, automated neuronal segmentation, and topographical analysis, to assess the number of specific cell types in the larval  public reviews designed to be posted alongside the preprint for the benefit of readers; ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: i) Decision letter after peer review:Drosophila Whole Brain Cellular Quantitation reveals Sexual Dimorphism" for consideration by eLife. Your article has been reviewed by 2 peer reviewers, including Sonia Sen as Reviewing Editor and Reviewer #1, and the evaluation has been overseen by K VijayRaghavan as the Senior Editor.Thank you for submitting your article "Intact The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:While noting the value and general rigour of this work, we had a few concerns that the authors could address. These are listed below.1) The new Gal4 lines: These lines form the basis of this study and we anticipate that they will be of value to the broader community. For both these reasons, we request that the authors verify them better, for example, with neuroblast-specific markers and/or co-labels with markers of mature neurons.2) Comparison with previous studies: We were concerned about the comparison with previous studies – please see detailed reviews below. Could the authors please revisit the text surrounding this, and perhaps provide a systematic comparison of the previous studies that have 'counted' cell numbers?3) Interpretation of some of the findings: Some of the findings are interesting, but we were not sure what they meant (please see the detailed comments below). Could the authors discuss this? If they feel it would be too speculative, they could perhaps use the Ideas and Speculation section within discussion to do this.4) Description of methods: Could the authors please describe some of their methods in more detail? Please see the detailed reviews below, but, for example, the procedures leading to the topographical comparisons, or the comparison of 3D coordinates across brains.Reviewer #1 (Recommendations for the authors):– The authors have generated invaluable Gal4 lines through their study. We would have liked to have seen a better characterization of them particularly because we think they will be of value to the broader community. For example with neuroblast-specific markers or co-labels with markers of mature neurons.– It would be nice to have a more systematic description of previous studies that have estimated cell numbers in larval and adult brains. The authors have done this, but the larval and adult studies are listed together, making it hard to assess the different approaches and their advantages and limitations. Could the authors please look into discussing it systematically in this way?– The authors quote previous studies suggesting glia constitute 10% of the cell in the brain – this is for the adult, isn't it? Significant neurogenesis occurs between early L3 and the adult and so is it possible that the 30% observed in this study transitions to 10%?– I did not quite understand how one can compare 3D coordinates of nuclei across brains if there isn't a common reference? Could the authors please elaborate on this?– What do the authors interpret from the nuclear distance distribution patterns of neurons and glia?– Similarly, the authors say that the sexual dimorphism extends to deeper features, is subtle, but widespread. Could the authors speculate what these subtle, widespread features might be?Reviewer #2 (Recommendations for the authors):The conclusions of this paper are supported mainly by data, but some aspects of data analysis and interpretation need to be clarified and extended to solidify their findings.1) The paper is generally written well, but some data interpretations are based on inaccurate and shaky estimates, which are better avoided. In my understanding, none of the precedents formally publish the number of neurons and glia in the whole nervous system of third instar larvae. As mentioned in the text, there are indeed some estimates that range from 10,000 to 15,000 , but they are not published as data and are not of the same age as their animal. Therefore, it is better for the authors to avoid direct comparisons with this wide range of statistics to draw numerical arguments . Instead, those studies collectively suggest the present results are in good agreement with them, solidifying their work rather than contradicting.2) Usage of the term "brain" has to be strict to avoid any future confusion (cf. title and abstract). While I agree that they found sexual dimorphism in the larval nervous system, I am unsure whether it is about the brain. The central nervous system of larvae consists of the brain and ventral nervous system, and the methodology of the current study analyzes both. Numerically speaking, the latter is the majority (4~5 times more), suggesting that the difference is somewhat related to other peripheral parts of the nervous system than the brain. I suggest the authors revise the relevant points and put their work in the proper context.3) Text writing in the methodological part could work better to substantiate their main findings further. In particular, the procedures leading to the topographical comparisons that contribute to their main conclusion (P8. 4th paragraph – P9. 2nd paragraph) uses many unfamiliar mathematical terms without proper explanations that make it difficult for non-specialists to follow.4) In the distribution of inter-nuclei distances , I see fractions of the cells fall into the range of distance 0, suggestive of double counting. Some questions about nuclei counting accuracy.5) I would like to know the specific citation behind their notion that "genes likely to be expressed only in active larval neurons with synaptic connections but not by neuroblasts or by immature neurons". Brp is known to be expressed in the developing embryo .6) (P9. L192) "As the Sh gene is x-linked, we carried out our quantification analysis in male brains only to avoid potential gene dosage effects."How does the dosage effect affect their cell counting? In other words, is this data appropriate being here without contributing the main conclusion of sexual dimorphism?7) A list of questions for methodological clarifications.– What genotypes were used for the topographical comparisons ?– (P8. L160) What are the "multi-scale topological structures"?– (P8. L162-164) I do not follow this part. The authors need to elaborate on the explanation.– What is the voxel resolution of their images?– How are inter-nuclei distances calculated ?– (P16. L356) How is the volume cutoff (15um3) determined?– What is coded by the color in Figure 2a-2d and others?– What is the unit of the abscissa in Figure 2e?– (P8. L137) What is the denominator to get the value (~15 to 30%)?8)  GeneName>GAL4. Is this the standard way to describe the genotype? Reviewer #1 (Recommendations for the authors):– The authors have generated invaluable Gal4 lines through their study. We would have liked to have seen a better characterization of them particularly because we think they will be of value to the broader community. For example with neuroblast-specific markers or co-labels with markers of mature neurons.brp-Gal4, Syt1-Gal4 and nSyb-Gal4 were mutually exclusive from Deadpan labelling . Finally, we also examined the expression of all three lines in the adult CNS and found that all three also appear to express in neurons in the adult brain .We thank the reviewer for their kind and insightful comments. To address their concern, we have carried out several additional experiments to further characterise these lines. We first examined the expression of these lines during embryo development . We observed no expression at stage 15-16 or stages prior to this . However, at stage 17 all three lines had neuronal expression . This is in good agreement with the onset of synaptic activity in embyros (Baines and Bate 1998). We also examined the expression of Deadpan, a transcription factor that labels neuroblast nuclei, in the larval CNS together with histoneGFP expression labelling driven by all three neuronal lines. Consistent with our lines not labelling neuroblasts, we found that the expression of – It would be nice to have a more systematic description of previous studies that have estimated cell numbers in larval and adult brains. The authors have done this, but the larval and adult studies are listed together, making it hard to assess the different approaches and their advantages and limitations. Could the authors please look into discussing it systematically in this way?As correctly described by reviewer #2, most of these prior estimates seem to be rough approximations and are not based on data or at least not explicitly linked to data in the source manuscripts. We have however rewritten this section as the reviewer requested. Larval and adult estimates are now separated and additional references added to support the range of numbers proposed (lines 16-23 revised manuscript).– The authors quote previous studies suggesting glia constitute 10% of the cell in the brain – this is for the adult, isn't it? Significant neurogenesis occurs between early L3 and the adult and so is it possible that the 30% observed in this study transitions to 10%?This is an excellent point. We have added this potential explanation of divergence in our measurements to this discussion (line 305-309 revised manuscript).– I did not quite understand how one can compare 3D coordinates of nuclei across brains if there isn't a common reference? Could the authors please elaborate on this?Reviewer #2 also asked for clarification about this novel biological data analysis method. Topological summaries indeed lack common reference points but nonetheless can be compared by standard methods. They can be thought of as multi-scale descriptions of the shape of the data set. In addition to adding several basic references to topological data analysis to aid biologists, we added this ‘motivating example’ or thought experiment to help illustrate the underlying principle to the mathematics methods section (revised manuscript lines 401-409)“For a motivating example of the principles underlying topological summaries, one could think of pearls forming a necklace. Topological summaries express the global structure of the necklace formed by the relationships between the positions of the individual pearls, but are invariant under translation and rotation of the necklace overall. Two such necklaces have topological summaries that are comparable even if the pearls in one have no relationship to the pearls in the other. It is the global structures – such as its circular shape on a large scale, or bulges on a smaller scale – formed by the relationships of the individual pearls of each one that matter”– What do the authors interpret from the nuclear distance distribution patterns of neurons and glia?We interpret these results as yet another confirmation that cells labelled are different between the neuronal Gal4 lines and the glial lines. We have edited the manuscript to make this point explicit (line 135-137 revised manuscript)– Similarly, the authors say that the sexual dimorphism extends to deeper features, is subtle, but widespread. Could the authors speculate what these subtle, widespread features might be?We are very reluctant to speculate as we have no data on which to propose a hypothesis. Topology analysis is telling us the differences between the female and male CNS is subtle, widespread and clearly detectible but also difficult to identify with simpler analysis approaches. An interesting future direction might be to examine CNS gynandromorphs to determine if these differences could be more discreetly localised.Reviewer #2 (Recommendations for the authors):The conclusions of this paper are supported mainly by data, but some aspects of data analysis and interpretation need to be clarified and extended to solidify their findings.We thank the reviewer for their supportive comments and have sought to address their concerns.1) The paper is generally written well, but some data interpretations are based on inaccurate and shaky estimates, which are better avoided. In my understanding, none of the precedents formally publish the number of neurons and glia in the whole nervous system of third instar larvae. As mentioned in the text, there are indeed some estimates that range from 10,000 to 15,000 , but they are not published as data and are not of the same age as their animal. Therefore, it is better for the authors to avoid direct comparisons with this wide range of statistics to draw numerical arguments . Instead, those studies collectively suggest the present results are in good agreement with them, solidifying their work rather than contradicting.This point was also raised Reviewer #1. The reviewer is correct that previous estimates were not published as data and have a broad range. Nonetheless they have been widely cited with the most recent estimates in higher range ~15K .We have rewritten this section (lines 13-23 revised manuscript) to make the point that the cited numbers are approximations more explicit and revised our conclusions as the reviewer suggested to indicate our data supports neuron numbers at the lowest end of previous estimates. As the reviewer suggested, we have removed all direct numerical comparisons to previous approximations.2) Usage of the term "brain" has to be strict to avoid any future confusion (cf. title and abstract). While I agree that they found sexual dimorphism in the larval nervous system, I am unsure whether it is about the brain. The central nervous system of larvae consists of the brain and ventral nervous system, and the methodology of the current study analyzes both. Numerically speaking, the latter is the majority (4~5 times more), suggesting that the difference is somewhat related to other peripheral parts of the nervous system than the brain. I suggest the authors revise the relevant points and put their work in the proper context.Drosophila CNS and our experiments include measurements of the brain and VNC combined (line 13-16 revised manuscript).The reviewer is absolutely correct. We have edited the title, abstract, text and figure legends to use the term ‘central nervous system (CNS)’ when appropriate, which is the case for the majority of our experiments. We make explicit that the 3) Text writing in the methodological part could work better to substantiate their main findings further. In particular, the procedures leading to the topographical comparisons that contribute to their main conclusion (P8. 4th paragraph – P9. 2nd paragraph) uses many unfamiliar mathematical terms without proper explanations that make it difficult for non-specialists to follow.Indeed, there are many terms here that may be unfamiliar to those of us outside mathematics fields. To address the reviewers concern, we have made a number of edits. In the main text, we have added additional accessible references to serve as general primers for non-specialists and added an additional explanation of the general approach (lines 177-183 revised manuscript). We have also expanded the methods section to include further general topology references and a ‘motivating example’ or thought experiment to help illustrate the underlying principle given that this type of topological data analysis is new for neuroscience microscopy data (lines 401-409 revised manuscript).4) In the distribution of inter-nuclei distances , I see fractions of the cells fall into the range of distance 0, suggestive of double counting. Some questions about nuclei counting accuracy.The plot in Figure 2e is derived from a regression analysis . As the reviewer correctly points out the input data are distances, so we should have should have set the range of the analysis outputs to positive values. We have rerun the analysis making this correction and edited the text and figure.5) I would like to know the specific citation behind their notion that "genes likely to be expressed only in active larval neurons with synaptic connections but not by neuroblasts or by immature neurons". Brp is known to be expressed in the developing embryo .This point was also raised Reviewer #1. To address this concern, we have carried out several additional experiments to further characterise these lines. We first examined the expression of these lines during embryo development . We observed no expression at stage 15-16 or stages prior to this . However, at stage 17 all three lines had expression . This is in good agreement with the onset of synaptic activity in embyros (Baines and Bate 1998).We have edited the statement cited to read ‘genes likely to be expressed only in active neurons with synaptic connections but not by neuroblasts or by immature neurons’ (line 90 revised manuscript) to define our rationale and subsequently confirm these lines indeed initiate expression when synaptic activity begins  and lines 110-121 in the revised manuscript.6) (P9. L192) "As the Sh gene is x-linked, we carried out our quantification analysis in male brains only to avoid potential gene dosage effects."How does the dosage effect affect their cell counting? In other words, is this data appropriate being here without contributing the main conclusion of sexual dimorphism?+ channels. Our concern was that well documented X-linked gene dosage effects could influence expression of the Sh-Gal4 line (which in on the X chromosome) which is why we choose to carry out these experiments in only one sex. We think these experiments are useful to illustrate another utility of the technique independently of other findings related to sexual dimorphism in the manuscript. We have edited the abstract to make this additional goal clear.Our goal with this series of experiments was to illustrate the utility and application of the technique to investigate the frequency of expression of a gene family throughout the CNS and we believe we have made some surprising observations into the relative abundance of K7) A list of questions for methodological clarifications.– What genotypes were used for the topographical comparisons ?All point clouds from all CNS lines were used. This has been clarified to the methods (revised manuscript line 413)– (P8. L160) What are the "multi-scale topological structures"?We have added additional references that explain these terms, see also answer to question 3 above.– (P8. L162-164) I do not follow this part. The authors need to elaborate on the explanation.We have added additional explanation in the main text and methods, see also answer to question 3 above.– What is the voxel resolution of their images?Our voxel resolution is 0.317um. We had added this to the methods (revised manuscript line 383)– How are inter-nuclei distances calculated ?Arivis4D outputs the three dimensional coordinates of the geometric center of every labelled nuclei. These co-ordinates were then processed using a Matlab code we generated to calculate internuclei the distance in 3D volume. This code is publicly available to download at https://doi.org/10.5281/zenodo.6574838 and this has been added to the revised methods.– (P16. L356) How is the volume cutoff (15um3) determined?πr!). This was empirically validated by volume measurements of subsets of neurons. This has been clarified in the methods.The volume was calculated by as the component of the radius of a the nucleus -1.5um producing a spherical volume – 15um3 .– What is the unit of the abscissa in Figure 2e?um. We have corrected this error.– (P8. L137) What is the denominator to get the value (~15 to 30%)?We have removed this statement and calculation as described above based on the reviewers valid criticism.8)  GeneName>GAL4. Is this the standard way to describe the genotype?-,. , _ or > have been used to describe a promoter upstream of a Gal4 in various manuscripts. However, as suggested, we have replaced > with – as this seems the most widely employed shorthand.Unfortunately, there is no ‘standard’ way to describe these shorthand genotypes (full genotypes are included in the methods and key resources file). A ReferencesDrosophila Embryo. J Neurosci 18:4673–4683. doi:10.1523/JNEUROSCI.18-12-04673.1998Baines RA, Bate M. 1998. Electrophysiological Development of Central Neurons in the


Menaquinone is essential in electron transport and ATP generation in all Gram-positive, and anaerobically respiring Gram-negative bacteria. By inhibiting menaquinone production in target organisms, bactericidal action can be achieved irrespective of the organisms' growth phase. This pathway is absent in human cells, as menaquinone is obtained only through the diet. This paper provides a succinct review of major advancements, where present, at all enzymatic steps of the biosynthetic pathway of menaquinone. Structure–activity relationships are evaluated, as well as results translating these relationships to growth inhibition studies. Menaquinone is essential in electron transport and ATP generation in all Gram-positive, and anaerobically respiring Gram-negative bacteria. Inhibition of menaquinone production at different steps of the biosynthesis pathway has shown promising novel antibacterial action. In these steps, MK (1) is reduced by two electrons to produce menaquinol (MKH2 (2)) and shuttles these electrons to an acceptor in the next step of the chain.7 This substrate oxidation process provides the energy needed to maintain the proton gradient and potential energy used by the F0F1ATPase complex to convert ADP into ATP. This is done through a proton ion channel that allows the protons to flow down the gradient, which causes a rotational conformational change in the complex resulting in the movements that produce and release ATP.8 Without this shuttling of protons the gradient would collapse and ATP synthesis would cease. Unlike bacteria, humans utilise ubiquinone within their electron transport chain in order to shuttle electrons. Therefore MK (1) biosynthesis is absent, with 1 being obtained through dietary sources.9Menaquinone   plays an1) is unavailable.10–12Although MK's (1) role in humans is not completely understood, it is hypothesised that 1 is utilised to produce clotting factors if phylloquinone  converts 3 into isochorismate (4), which in turn is converted to 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate  by MenD.16 The aromatic intermediate OSB (7) is produced in a two-step process involving MenH and MenC via the intermediate compound SHCHC (6). The carboxyl group in 7 is then activated by MenE through an ATP dependent process to produce O-succinylbenzyl-CoA , which allows MenB to catalyse the cyclization of this product forming the naphthalenoid skeleton DHNA-CoA (9). This skeleton is then hydrolysed through an unknown Men enzyme creating DHNA (10) which is decarboxylated and prenylated by MenA to produce DMK (11).17 The final step in the pathway involves MenG adding a methyl group to position 3 of the naphthalenoid ring of 11 creating MK (1).The synthesis of 1 by bacteria is a seven-step process  which st16 Interestingly, not all of the enzymes have had significant investigation into potential inhibitors.18–26 To date the most promising results have come from inhibitors targeting the enzymes further downstream of the synthesis pathway. Major inhibitors that have been discovered to date will be reviewed in this article.All enzymes of this pathway are enticing targets for inhibition as they are extremely conserved in all Gram-positive and facultative anaerobic Gram-negative bacteria.1,14 Investigations into inhibitions of this enzyme have been conducted by Fang et al.13 by using acylphosphonate derivatives as false substrates of α-ketoglutarate. Of the eleven compounds that they synthesised, two compounds were effective inhibitors.MenD, the second enzyme involved in the biosynthesis of 1 is a thiamin diphosphate-dependent decarboxylase that catalyses the creation of SEPHCHC (5) by a ping-pong mechanism involving α-ketoglutarate.Ki) at 0.7 μM and 16 μM concentrations, respectively. It was observed that although a great improvement in the inhibitory action is achieved by addition of an alkyl group to the phosphonate, when larger alkyl groups were substituted at this position less inhibitory action was produced. Similarly, it was shown that esterification of the carboxylate group by alkyl addition rendered the inhibitor ineffective. Whilst investigating additions to this ester group, a phenyl ester showed improved inhibitory action when compared to its alkyl ester counterparts. This was hypothesised by Fang et al.13 to be due to potential π-stacking with amino acid residues in the active site corresponding to the aromatic groups position, however with a Ki value of 160 μM, this is much less effective than simply keeping the carboxylate group present.These two compounds , methyl 27 who managed to synthesise thiamine diphosphate analogues with activity against MenD. Four analogues were successfully synthesised showing moderate enzyme inhibition, however when it came to growth inhibition the results were less promising. Only one analogue showed growth inhibition at concentrations <200 μM, with the others inhibiting growth at concentrations 3–10 times more than this. It can be concluded that these analogues require further investigation to translate their moderate enzyme inhibition to growth inhibition. Specific structures of these thiamine diphosphate analogues are available in Xu's publication.27Further work into inhibition of MenD has also been carried out by Xu,via an OSB-AMP intermediate.15,23,25,28–30 After initial location and characterization of the MenE gene by Sharma,29 varying approaches have been undertaken to attempt to inhibit this enzyme. The reaction mechanisms of MenE have been proposed by Tian et al.15 where they describe the reaction as a Bi Uni Uni Bi ping-pong mechanism in the presence of ATP, via an acyl-AMP intermediate.15 The adenylation of OSB by ATP creates the OSB-AMP intermediate, before ligation of CoA via thioesterification produces OSB-CoA.The fifth enzyme in the bacterial synthesis of 1, MenE catalyses the addition of S-CoA to OSB et al.23 initially created inhibitors by using this approach. Three inhibitors were synthesised which showed ability to inhibit MenE. In these created compounds, linkages mimicking the phosphate AMP linkage were replaced by bioisosteres sulphamate 14, sulphamide 15 and vinyl sulphonamide groups 16  in bacteria, catalysing the creation of DHNA-CoA (9) from O-succinylbenzoate-CoA via a Claisen condensation. The structure of MenB was first reported by Truglio et al.32 in 2003 after a crystal structure of this enzyme was obtained from enzyme extracted from M. tuberculosis. The crystals were able to be soaked in acetoacetyl-CoA and an X-ray diffraction pattern produced. This not only allowed structure determination of the quaternary structure, but also allowed the active site to be catagorised and a catalytic mechanism proposed. Obtaining crystals of natural substrate bound into the active sight was not able to be achieved as the substrate is too unstable to exist for long periods of time, reacting with itself to form spironolactone moieties. From the obtained enzymatic structures, it was determined that MenB is a member of a superfamily of enzymes called the crotonase superfamily.32,331,4-Dihydroxy-2-naphthoyl-CoA synthase is the 6et al.34 where a coupled assay was used to screen over 105 000 drug-like molecules with a large structural diversity. From this bank of molecules, 455 molecules showed at least 30% enzyme inhibition relative to control, of which four compounds with a benzoxazine scaffold were chosen for further investigation. Using this scaffold structure as a theme, 13 compounds (18a–m) were synthesised to attempt to increase inhibitory action  is important in coordination of substrates towards this active site to allow the reaction to occur.36 This tyrosine motif is suspected to interact with the isoprenyl side chain to draw the chain into the active site to allow the catalytic reaction to occur, and as such lipophilicity at this site for any potential inhibitor is important. Coordination of DHNA (10) into the active site is primarily achieved through the presence of the carbonyl group. This carbonyl also plays a part in the proposed reaction mechanism, providing an environment which promotes electron movement into the ring structure of DHNA (10).MenA is involved in the penultimate step in the synthesis of MK (1). It has been proposed that this enzyme has structural and functional similarities in its catalytic mechanism to the UbiA enzyme, a member of a class and family very similar to the MenA enzyme, which was deduced from amino acid sequence analysis.et al.17 and Debnath et al.37 who have based much of their efforts on the creation of structures with similar SARs to aurachin RE, a natural product that shows bactericidal activity through a proposed dual mechanism of MenA and respiratory chain inhibition.38 Debnath et al.37 have been able to synthesise over 400 molecules of interest with >95% purity which have been screened in bacterial growth inhibitory assays, with varying results. The compounds synthesised have been based around a benzophenone core structure, with a highly varied lipophilic side chain substitution. It has been discovered by Debnath et al.37 that the position of a nitrogen atom in this tail is important to its bactericidal activity, with the most anti-mycobacterial activity achieved with a nitrogen 13 carbon-spaces away from the carbonyl on the benzophenone structure, yielding MIC of 0.85 mg L−1. Many variations of these compounds are displayed in their results tables, all catagorised into five broad classes (Classes A–E) of agents  and oxygen recovery assays against non-replicating M. tuberculosis (determining direct respiratory chain inhibition).39 Whilst the chirality of the variations on Class A inhibitors effected MIC values, chirality was not shown to have an effect on the activity of group B, C, D and E molecules.Of these classes, Class A showed to be the most effective, with the above-mentioned MIC values recorded from elements of this species of compound. The classes of compounds categorised differ in their variation in the backbone of the side chain and substitution of functional groups. It is also stated that the presence of a halogen on the first benzene ring improves bacterial killing potential, however the mechanisms for this have not been able to be explained clearly. These compounds were found to act et al.37 who found that initial effects on mammalian cell lines showed IC50 levels of 1–6.5 mg L−1. Through the use of an O-methyl oxime substitution this was reduced to an IC50 of 25 mg L−1. It is hypothesised that this substitution reduced the benzophenone electrophilicity allowing it to interfere less with the mammalian cell redox systems, although its specific mechanism is still largely unknown.39 Although this substitution reduced toxicity to mammalian cells, further decrease in toxicity is desired.It is worth noting the synthesis of all of these compounds involves costly catalysts and many steps. Although yields of each step have been acceptable (approx. 80% per step) and the products quite pure after workup, room for improvement can be made in reducing overall cost of manufacture of derivatives. Cytotoxicity was also investigated by Debnath et al.37 has shown insight into the specific inhibition of MenA. It is also worth noting that extremely limited SAR studies have been characterised, as well as no literature showing the crystal structure of MenA is available. As such, active site interactions with substrates for this enzyme are not known with any clarity.To date only work published from the laboratories of Debnath et al.40 Here borinic ester derivatives were developed and found to have inhibitory action, and screened against known methyltransferase inhibitors sinefungin and S-adenosylhomocysteine (SAH). Whilst many derivatives were tested against B. subtilis' MenG methyltransferase, only one borinic ester 20 was found to have activity.MenG is the final step in the bacterial production of MK (1). It is responsible for the addition of the methyl group onto the aromatic ring of DMK (11). Although the structural similarities present with ubiquinone, MenG has no functional equivalence to ubiquinone methyltransferases. Work involving the discovery of therapeutic agents which inhibit MenG has been investigated by Benkovic meta-chloro derivative of the species of compounds. This showed activity comparable to sinefungin, the natural inhibitor already known. The results of their assays showed that at 100 μM concentration of this compound, only 40% of enzyme activity remained. When this chloro group was moved to a different position on the aromatic ring the activity was greatly limited, or in the case of removal, activity lost completely. It is worth noting that even with the chloro group present this structure was not as effective as SAH in inhibiting MenG, as inhibition with SAH resulted in <10% of enzyme activity remaining, but both SAH and sinefungin are not appropriate for therapeutic use due to their levels of toxicity to eukaryotic cells. Although the other borinic esters did not return as promising results with specific regards to MenG inhibition, a complete list of structures investigated can be found in their supplementary material, of which most had respectable MIC values against a range of bacterial species.40As shown in et al.41 Lysocin E is a cyclic peptide, that shows potent bactericidal activity against a variety of Gram-positive bacteria. This bactericidal activity results from bacterial membrane disruption, as menaquinone is present in small amounts in the bacterial cell membrane. Cytotoxicity studies were also conducted using mice models, and lysocin E was found to have low toxicity, with an acute toxic dose of >400 mg kg−1. Although this shows potential for therapeutic use, spontaneous mutants showing resistance to lysocin E were observed in a laboratory setting.26,41In a unique approach, a direct MK (1) binding agent, lysocin E, was discovered by Hamamoto Great strides have been achieved in the progress towards inhibition of MK (1) biosynthesis in bacteria as a result of combined efforts of laboratories around the world. Advances in MenA inhibition provide the greatest developments into both potency and selectivity of many classes of novel antibacterial agents at this current stage. Although the efforts into MenA inhibition have proved fruitful, progress towards MenB and MenE inhibitors should also be followed closely as they too hold promising futures for research. As stated previously, this follows the observed trend of enzymes further down the synthesis pathway providing the most successful inhibitors. As such, more investigation into the earlier synthetic pathway enzymes would fill a current gap in the research.The authors declare there are no conflicts of interest.