Phage anti-CRISPR control by an RNA- and DNA-binding helix-turn-helix protein.

In all organisms, regulation of gene expression must be adjusted to meet cellular requirements and frequently involves helix-turn-helix (HTH) domain proteins1. For instance, in the arms race between bacteria and bacteriophages, rapid expression of phage anti-CRISPR (acr) genes upon infection enables evasion from CRISPR-Cas defence; transcription is then repressed by an HTH-domain-containing anti-CRISPR-associated (Aca) protein, probably to reduce fitness costs from excessive expression2-5. However, how a single HTH regulator adjusts anti-CRISPR production to cope with increasing phage genome copies and accumulating acr mRNA is unknown. Here we show that the HTH domain of the regulator Aca2, in addition to repressing Acr synthesis transcriptionally through DNA binding, inhibits translation of mRNAs by binding conserved RNA stem-loops and blocking ribosome access. The cryo-electron microscopy structure of the approximately 40 kDa Aca2-RNA complex demonstrates how the versatile HTH domain specifically discriminates RNA from DNA binding sites. These combined regulatory modes are widespread in the Aca2 family and facilitate CRISPR-Cas inhibition in the face of rapid phage DNA replication without toxic acr overexpression. Given the ubiquity of HTH-domain-containing proteins, it is anticipated that many more of them elicit regulatory control by dual DNA and RNA binding.

Type III CRISPR-Cas provides resistance against nucleus-forming jumbo phages via abortive infection.

Bacteria have diverse defenses against phages. In response, jumbo phages evade multiple DNA-targeting defenses by protecting their DNA inside a nucleus-like structure. We previously demonstrated that RNA-targeting type III CRISPR-Cas systems provide jumbo phage immunity by recognizing viral mRNA exported from the nucleus for translation. Here, we demonstrate that recognition of phage mRNA by the type III system activates a cyclic triadenylate-dependent accessory nuclease, NucC. Although unable to access phage DNA in the nucleus, NucC degrades the bacterial chromosome, triggers cell death, and disrupts phage replication and maturation. Hence, type-III-mediated jumbo phage immunity occurs via abortive infection, with suppression of the viral epidemic protecting the population. We further show that type III systems targeting jumbo phages have diverse accessory nucleases, including RNases that provide immunity. Our study demonstrates how type III CRISPR-Cas systems overcome the inaccessibility of jumbo phage DNA to provide robust immunity.

Antibacterial synergy between a phage endolysin and citric acid against the Gram-negative kiwifruit pathogen Pseudomonas syringae pv. actinidiae.

Horticultural diseases caused by bacterial pathogens provide an obstacle to crop production globally. Management of the infection of kiwifruit by the Gram-negative phytopathogen Pseudomonas syringae pv. actinidiae (Psa) currently includes copper and antibiotics. However, the emergence of bacterial resistance and a changing regulatory landscape are providing the impetus to develop environmentally sustainable antimicrobials. One potential strategy is the use of bacteriophage endolysins, which degrade peptidoglycan during normal phage replication, causing cell lysis and the release of new viral progeny. Exogenous use of endolysins as antimicrobials is impaired by the outer membrane of Gram-negative bacteria that provides an impermeable barrier and prevents endolysins from accessing their target peptidoglycan. Here, we describe the synergy between citric acid and a phage endolysin, which results in a reduction of viable Psa below detection. We show that citric acid drives the destabilization of the outer membrane via acidification and sequestration of divalent cations from the lipopolysaccharide, which is followed by the degradation of the peptidoglycan by the endolysin. Scanning electron microscopy revealed clear morphological differences, indicating cell lysis following the endolysin-citric acid treatment. These results show the potential for citric acid-endolysin combinations as a possible antimicrobial approach in agricultural applications. IMPORTANCE: The phytopathogen Pseudomonas syringae pv. actinidiae (Psa) causes major impacts to kiwifruit horticulture, and the current control strategies are heavily reliant on copper and antibiotics. The environmental impact and increasing resistance to these agrichemicals are driving interest in alternative antimicrobials including bacteriophage-derived therapies. In this study, we characterize the endolysin from the Otagovirus Psa374 which infects Psa. When combined with citric acid, this endolysin displays an impressive antibacterial synergy to reduce viable Psa below the limit of detection. The use of citric acid as a synergistic agent with endolysins has not been extensively studied and has never been evaluated against a plant pathogen. We determined that the synergy involved a combination of the chelation activity of citric acid, acidic pH, and the specific activity of the ΦPsa374 endolysin. Our study highlights an exciting opportunity for alternative antimicrobials in agriculture.

Quorum Sensing Controls Adaptive Immunity through the Regulation of Multiple CRISPR-Cas Systems.

Bacteria commonly exist in high cell density populations, making them prone to viral predation and horizontal gene transfer (HGT) through transformation and conjugation. To combat these invaders, bacteria possess an arsenal of defenses, such as CRISPR-Cas adaptive immunity. Many bacterial populations coordinate their behavior as cell density increases, using quorum sensing (QS) signaling. In this study, we demonstrate that QS regulation results in increased expression of the type I-E, I-F, and III-A CRISPR-Cas systems in Serratia cells in high-density populations. Strains unable to communicate via QS were less effective at defending against invaders targeted by any of the three CRISPR-Cas systems. Additionally, the acquisition of immunity by the type I-E and I-F systems was impaired in the absence of QS signaling. We propose that bacteria can use chemical communication to modulate the balance between community-level defense requirements in high cell density populations and host fitness costs of basal CRISPR-Cas activity.

Endotoxin, a novel biomarker for the rapid risk assessment of faecal contamination of coastal and transitional waters.

Current methods for testing water for faecal contamination rely on the culture of faecal indicator bacteria (FIB; Escherichia coli and Enterococci) that take 24-48 h, which leads to delays in taking proactive measures and poses a risk to public health. More rapid methods are therefore required. Here, we have tested a rapid, portable assay (Bacterisk) that detects the bacterial biomarker endotoxin in 30 min to quantify the bacterial biomass present, to evaluate 159 coastal water samples and to compare the results with the traditional culture of FIB. There was a significant correlation between the Bacterisk data given in endotoxin risk (ER) units and FIB culture that could accurately distinguish between poor and sufficient or good quality bathing water using the EU bathing directive values. Receiver operating characteristic analysis was used to determine the optimal ER threshold for coastal water samples, and the area under the curve was 0.9176 with a p-value of <0.0001. The optimal threshold was 7,300 ER units with a sensitivity of 95.45% and a specificity of 83.48%. In conclusion, we have shown that the Bacterisk assay provides a rapid and easy-to-use in situ method to assess bathing water quality.

A mobile restriction-modification system provides phage defence and resolves an epigenetic conflict with an antagonistic endonuclease.

Epigenetic DNA methylation plays an important role in bacteria by influencing gene expression and allowing discrimination between self-DNA and intruders such as phages and plasmids. Restriction-modification (RM) systems use a methyltransferase (MTase) to modify a specific sequence motif, thus protecting host DNA from cleavage by a cognate restriction endonuclease (REase) while leaving invading DNA vulnerable. Other REases occur solitarily and cleave methylated DNA. REases and RM systems are frequently mobile, influencing horizontal gene transfer by altering the compatibility of the host for foreign DNA uptake. However, whether mobile defence systems affect pre-existing host defences remains obscure. Here, we reveal an epigenetic conflict between an RM system (PcaRCI) and a methylation-dependent REase (PcaRCII) in the plant pathogen Pectobacterium carotovorum RC5297. The PcaRCI RM system provides potent protection against unmethylated plasmids and phages, but its methylation motif is targeted by the methylation-dependent PcaRCII. This potentially lethal co-existence is enabled through epigenetic silencing of the PcaRCII-encoding gene via promoter methylation by the PcaRCI MTase. Comparative genome analyses suggest that the PcaRCII-encoding gene was already present and was silenced upon establishment of the PcaRCI system. These findings provide a striking example for selfishness of RM systems and intracellular competition between different defences.

PADLOC: a web server for the identification of antiviral defence systems in microbial genomes.

Most bacteria and archaea possess multiple antiviral defence systems that protect against infection by phages, archaeal viruses and mobile genetic elements. Our understanding of the diversity of defence systems has increased greatly in the last few years, and many more systems likely await discovery. To identify defence-related genes, we recently developed the Prokaryotic Antiviral Defence LOCator (PADLOC) bioinformatics tool. To increase the accessibility of PADLOC, we describe here the PADLOC web server (freely available at https://padloc.otago.ac.nz), allowing users to analyse whole genomes, metagenomic contigs, plasmids, phages and archaeal viruses. The web server includes a more than 5-fold increase in defence system types detected (since the first release) and expanded functionality enabling detection of CRISPR arrays and retron ncRNAs. Here, we provide user information such as input options, description of the multiple outputs, limitations and considerations for interpretation of the results, and guidance for subsequent analyses. The PADLOC web server also houses a precomputed database of the defence systems in > 230,000 RefSeq genomes. These data reveal two taxa, Campylobacterota and Spriochaetota, with unusual defence system diversity and abundance. Overall, the PADLOC web server provides a convenient and accessible resource for the detection of antiviral defence systems.

Signatures of increasing environmental stress in bumblebee wings over the past century: Insights from museum specimens.

Determining when animal populations have experienced stress in the past is fundamental to understanding how risk factors drive contemporary and future species' responses to environmental change. For insects, quantifying stress and associating it with environmental factors has been challenging due to a paucity of time-series data and because detectable population-level responses can show varying lag effects. One solution is to leverage historic entomological specimens to detect morphological proxies of stress experienced at the time stressors emerged, allowing us to more accurately determine population responses. Here we studied specimens of four bumblebee species, an invaluable group of insect pollinators, from five museums collected across Britain over the 20th century. We calculated the degree of fluctuating asymmetry (FA; random deviations from bilateral symmetry) between the right and left forewings as a potential proxy of developmental stress. We: (a) investigated whether baseline FA levels vary between species, and how this compares between the first and second half of the century; (b) determined the extent of FA change over the century in the four bumblebee species, and whether this followed a linear or nonlinear trend; (c) tested which annual climatic conditions correlated with increased FA in bumblebees. Species differed in their baseline FA, with FA being higher in the two species that have recently expanded their ranges in Britain. Overall, FA significantly increased over the century but followed a nonlinear trend, with the increase starting c. 1925. We found relatively warm and wet years were associated with higher FA. Collectively our findings show that FA in bumblebees increased over the 20th century and under weather conditions that will likely increase in frequency with climate change. By plotting FA trends and quantifying the contribution of annual climate conditions on past populations, we provide an important step towards improving our understanding of how environmental factors could impact future populations of wild beneficial insects.

The Rsm (Csr) post-transcriptional regulatory pathway coordinately controls multiple CRISPR-Cas immune systems.

CRISPR-Cas systems provide bacteria with adaptive immunity against phages and plasmids; however, pathways regulating their activity are not well defined. We recently developed a high-throughput genome-wide method (SorTn-seq) and used this to uncover CRISPR-Cas regulators. Here, we demonstrate that the widespread Rsm/Csr pathway regulates the expression of multiple CRISPR-Cas systems in Serratia (type I-E, I-F and III-A). The main pathway component, RsmA (CsrA), is an RNA-binding post-transcriptional regulator of carbon utilisation, virulence and motility. RsmA binds cas mRNAs and suppresses type I and III CRISPR-Cas interference in addition to adaptation by type I systems. Coregulation of CRISPR-Cas and flagella by the Rsm pathway allows modulation of adaptive immunity when changes in receptor availability would alter susceptibility to flagella-tropic phages. Furthermore, we show that Rsm controls CRISPR-Cas in other genera, suggesting conservation of this regulatory strategy. Finally, we identify genes encoding RsmA homologues in phages, which have the potential to manipulate the physiology of host bacteria and might provide an anti-CRISPR activity.

Identification and classification of antiviral defence systems in bacteria and archaea with PADLOC reveals new system types.

To provide protection against viral infection and limit the uptake of mobile genetic elements, bacteria and archaea have evolved many diverse defence systems. The discovery and application of CRISPR-Cas adaptive immune systems has spurred recent interest in the identification and classification of new types of defence systems. Many new defence systems have recently been reported but there is a lack of accessible tools available to identify homologs of these systems in different genomes. Here, we report the Prokaryotic Antiviral Defence LOCator (PADLOC), a flexible and scalable open-source tool for defence system identification. With PADLOC, defence system genes are identified using HMM-based homologue searches, followed by validation of system completeness using gene presence/absence and synteny criteria specified by customisable system classifications. We show that PADLOC identifies defence systems with high accuracy and sensitivity. Our modular approach to organising the HMMs and system classifications allows additional defence systems to be easily integrated into the PADLOC database. To demonstrate application of PADLOC to biological questions, we used PADLOC to identify six new subtypes of known defence systems and a putative novel defence system comprised of a helicase, methylase and ATPase. PADLOC is available as a standalone package (https://github.com/padlocbio/padloc) and as a webserver (https://padloc.otago.ac.nz).

Laparoscopic removal of a synthetic retropubic mid-urethral sling (tension-free vaginal tape): step-by-step technique.

INTRODUCTION AND HYPOTHESIS: The synthetic mid-urethral sling (MUS) has been the pre-eminent surgical treatment option for stress urinary incontinence (SUI) in women in recent times. However, increasing numbers of patients are now requesting mesh removal, secondary to persisting symptoms attributed to their sling. We present a video demonstrating a combined vaginal and laparoscopic approach to this procedure; along with supporting information outlining essential pre-operative assessment, counselling, and governance considerations. METHODS: A 60-year-old woman presented with a 4-year history of pelvic pain. She ascribed this to her retropubic MUS (a tension-free vaginal tape). Following extensive work-up, the mesh was removed using the technique described. RESULTS: On review, 3 months post-operatively, the patient reported improvement in the vaginal discomfort she had experienced prior to the procedure-albeit with concomitant deterioration in her SUI. CONCLUSIONS: An open or laparoscopic approach can be employed to dissect out the retropubic arms of an MUS. The latter provides a superior view of the retropubic space and confers potential advantages regarding recovery and cosmesis. The surgical technique detailed is safe and effective, especially when augmented by thorough preparation and patient counselling.

Laparoscopic sacrohysteropexy versus vaginal hysterectomy and apical suspension: 7-year follow-up of a randomized controlled trial.

INTRODUCTION AND HYPOTHESIS: Laparoscopic mesh sacrohysteropexy offers a uterine-sparing alternative to vaginal hysterectomy with apical suspension, although randomised comparative data are lacking. This study was aimed at comparing the long-term efficacy of laparoscopic mesh sacrohysteropexy and vaginal hysterectomy with apical suspension for the treatment of uterine prolapse. METHODS: A randomised controlled trial comparing laparoscopic mesh sacrohysteropexy and vaginal hysterectomy with apical suspension for the treatment of uterine prolapse was performed, with a minimum follow-up of 7 years. The primary outcome was reoperation for apical prolapse. Secondary outcomes included patient-reported mesh complications, Pelvic Organ Prolapse Quantification, Patient Global Impression of Improvement in prolapse symptoms and the International Consultation on Incontinence Questionnaire Vaginal Symptoms, Female Lower Urinary Tract Symptoms (ICIQ-FLUTS) and PISQ-12 questionnaires. RESULTS: A total of 101 women were randomised and 62 women attended for follow-up at a mean of 100 months postoperatively (range 84-119 months). None reported a mesh-associated complication. The risk of reoperation for apical prolapse was 17.2% following vaginal hysterectomy (VH) and 6.1% following laparoscopic mesh sacrohysteropexy (LSH; relative risk 0.34, 95% CI 0.07-1.68, p = 0.17). Laparoscopic sacrohysteropexy was associated with a statistically significantly higher apical suspension (POP-Q point C -5 vs -4.25, p = 0.02) and longer total vaginal length (9 cm vs 6 cm, p < 0.001). There was no difference in the change in ICIQ-VS scores between the two groups (ICIQ-VS change -22 vs -25, p = 0.59). CONCLUSION: Laparoscopic sacrohysteropexy and vaginal hysterectomy with apical suspension have comparable reoperation rates and subjective outcomes. Potential advantages of laparoscopic sacrohysteropexy include a lower risk of apical reoperation, greater apical support and increased total vaginal length.

Crystal structure of the anti-CRISPR repressor Aca2.

Bacteria use adaptive CRISPR-Cas immune mechanisms to protect from invasion by bacteriophages and other mobile genetic elements. In response, bacteriophages and mobile genetic elements have co-evolved anti-CRISPR proteins to inhibit the bacterial defense. We and others have previously shown that anti-CRISPR associated (Aca) proteins can regulate this anti-CRISPR counter-attack. Here, we report the first structure of an Aca protein, the Aca2 DNA-binding transcriptional autorepressor from Pectobacterium carotovorum bacteriophage ZF40, determined to 1.34 Å. Aca2 presents a conserved N-terminal helix-turn-helix DNA-binding domain and a previously uncharacterized C-terminal dimerization domain. Dimerization positions the Aca2 recognition helices for insertion into the major grooves of target DNA, supporting its role in regulating anti-CRISPRs. Furthermore, database comparisons identified uncharacterized Aca2 structural homologs in pathogenic bacteria, suggesting that Aca2 represents the first characterized member of a more widespread family of transcriptional regulators.

Evolution of virulence in a novel family of transmissible mega-plasmids.

Some Serratia entomophila isolates have been successfully exploited in biopesticides due to their ability to cause amber disease in larvae of the Aotearoa (New Zealand) endemic pasture pest, Costelytra giveni. Anti-feeding prophage and ABC toxin complex virulence determinants are encoded by a 153-kb single-copy conjugative plasmid (pADAP; amber disease-associated plasmid). Despite growing understanding of the S. entomophila pADAP model plasmid, little is known about the wider plasmid family. Here, we sequence and analyse mega-plasmids from 50 Serratia isolates that induce variable disease phenotypes in the C. giveni insect host. Mega-plasmids are highly conserved within S. entomophila, but show considerable divergence in Serratia proteamaculans with other variants in S. liquefaciens and S. marcescens, likely reflecting niche adaption. In this study to reconstruct ancestral relationships for a complex mega-plasmid system, strong co-evolution between Serratia species and their plasmids were found. We identify 12 distinct mega-plasmid genotypes, all sharing a conserved gene backbone, but encoding highly variable accessory regions including virulence factors, secondary metabolite biosynthesis, Nitrogen fixation genes and toxin-antitoxin systems. We show that the variable pathogenicity of Serratia isolates is largely caused by presence/absence of virulence clusters on the mega-plasmids, but notably, is augmented by external chromosomally encoded factors.

The autoregulator Aca2 mediates anti-CRISPR repression.

CRISPR-Cas systems are widespread bacterial adaptive defence mechanisms that provide protection against bacteriophages. In response, phages have evolved anti-CRISPR proteins that inactivate CRISPR-Cas systems of their hosts, enabling successful infection. Anti-CRISPR genes are frequently found in operons with genes encoding putative transcriptional regulators. The role, if any, of these anti-CRISPR-associated (aca) genes in anti-CRISPR regulation is unclear. Here, we show that Aca2, encoded by the Pectobacterium carotovorum temperate phage ZF40, is an autoregulator that represses the anti-CRISPR-aca2 operon. Aca2 is a helix-turn-helix domain protein that forms a homodimer and interacts with two inverted repeats in the anti-CRISPR promoter. The inverted repeats are similar in sequence but differ in their Aca2 affinity, and we propose that they have evolved to fine-tune, and downregulate, anti-CRISPR production at different stages of the phage life cycle. Specific, high-affinity binding of Aca2 to the first inverted repeat blocks the promoter and induces DNA bending. The second inverted repeat only contributes to repression at high Aca2 concentrations in vivo, and no DNA binding was detectable in vitro. Our investigation reveals the mechanism by which an Aca protein regulates expression of its associated anti-CRISPR.

Validation of ROS1 by immunohistochemistry against fluorescent in situ hybridisation on cytology and small biopsy samples in a large teaching hospital.

OBJECTIVE: Rearranged ROS1, present in 1%-2% of non-small cell lung cancer (NSCLC) patients, usually young, never or light smokers, is assessed by fluorescence in situ hybridization (FISH) to determine eligibility for tyrosine kinase inhibitors (TKI). Immunohistochemistry (IHC) for the protein product of ROS1 rearrangement, a cost-effective alternative, is validated on cytology and small biopsy samples. METHODS: From 1 March to 31 December 2019, cytology cell blocks and small biopsy samples from a selected cohort of NSCLC patients were concurrently tested for ROS1 gene rearrangement by Vysis 6q22 Break Apart FISH probe and IHC using Cell Signalling D4D6 antibody. Mismatch cases were tested by an RNA fusion next generation sequencing (NGS) panel. RESULTS: In a prospective population of 95 cases, 91 were negative and two were positive by both FISH and IHC. Both dual positive cases were female never smokers and benefited from TKI treatment. Another two cases were positive by FISH but negative by IHC and repeat by NGS showed one to be negative but one failed. Turnaround time for IHC was 0 to 8 days from request to authorisation, whilst that of FISH was 9 to 42 days at a cost of £51 and £159 respectively. CONCLUSION: IHC to assess for the protein product of ROS1 gene rearrangement on cytology cell blocks and small biopsy samples in a routine setting is a promising screening method to assess eligibility for TKI treatment with positive and indeterminate cases confirmed by FISH or NGS as it has good negative predictive value, faster turnaround time and is cost effective, with proven technical and clinical validation.

Rethinking animal models of sepsis - working towards improved clinical translation whilst integrating the 3Rs.

Sepsis is a major worldwide healthcare issue with unmet clinical need. Despite extensive animal research in this area, successful clinical translation has been largely unsuccessful. We propose one reason for this is that, sometimes, the experimental question is misdirected or unrealistic expectations are being made of the animal model. As sepsis models can lead to a rapid and substantial suffering - it is essential that we continually review experimental approaches and undertake a full harm:benefit impact assessment for each study. In some instances, this may require refinement of existing sepsis models. In other cases, it may be replacement to a different experimental system altogether, answering a mechanistic question whilst aligning with the principles of reduction, refinement and replacement (3Rs). We discuss making better use of patient data to identify potentially useful therapeutic targets which can subsequently be validated in preclinical systems. This may be achieved through greater use of construct validity models, from which mechanistic conclusions are drawn. We argue that such models could provide equally useful scientific data as face validity models, but with an improved 3Rs impact. Indeed, construct validity models may not require sepsis to be modelled, per se. We propose that approaches that could support and refine clinical translation of research findings, whilst reducing the overall welfare burden on research animals.

Laparoscopic complete sacrocolpopexy mesh removal for right-sided gluteal pain and recurrent mesh erosion.

AIM OF VIDEO: The aim was to demonstrate laparoscopic complete excision of sacrocolpopexy mesh from a 65-year-old woman who had presented with delayed onset of persistent right-sided gluteal pain. METHOD: The patient was referred to our unit, having undergone a laparoscopic sacrocolpopexy for vault prolapse 7 years earlier, with a type 1 polypropylene mesh. Four years after the primary surgery, she first noticed symptoms of spontaneous vaginal pain together with deep dyspareunia, and right-sided gluteal pain. Clinical examination revealed mesh erosion at the vaginal vault. This was managed at her local hospital, with excision of the small exposed portion of the mesh and over sewing, from a vaginal approach. She continued to be symptomatic following this procedure. When her symptoms still failed to improve 3 years later, a tertiary referral was made to our unit. At laparoscopy, minimal adhesions between the bowel and the mesh were noted and divided. After carefully dissecting the right ureter and reflecting the bladder, the entire sacrocolpopexy mesh was removed with its ProTack fasteners. The entire specimen was retrieved in one piece through the open vault and the vagina was sutured with 2.0

A novel technique for complete laparoscopic excision of a transobturator sling with lower urinary tract mesh erosion.

INTRODUCTION AND HYPOTHESIS: To demonstrate a novel technique for complete laparoscopic removal of a transobturator sling for mesh erosion involving a large area of the urethra and bladder neck, without the need for concomitant vaginal dissection. METHOD: A 56-year-old woman had a transobturator sling inserted for stress urinary incontinence (SUI) in 2009. In 2017, 8 years following surgery, she experienced groin pain, exacerbated by exercise, and developed recurrent urinary tract infections with dysuria and urethral pain. A cystoscopy demonstrated mesh erosion from the midurethra to bladder neck with a 2-cm calculus formed around the mesh. After careful counselling and discussion at a multi-disciplinary meeting, a decision was made to proceed with laparoscopy with a view to remove the mesh completely. The mesh was removed from the points of erosion into the urethra through a total laparoscopic procedure. The patient made a good recovery with no ongoing pain or voiding difficulties. CONCLUSION: Combined approaches for complete excision of transobturator slings, including bilateral inguinal dissection, are relatively morbid with prolonged recovery time and in most centres will require involvement of plastic surgeons. The laparoscopic approach not only allows for the mesh to be removed in total (including the intramural portion of the mesh), but also provides magnified views compared with open surgery and thus allows for better identification of planes and dissection. It also has the added benefit of avoiding vaginal incisions and therefore reducing the risk of fistula formation between the urethra/bladder and vagina.