Clinicopathological Predictors of Positive Resection Margins in Breast-Conserving Surgery.

BACKGROUND: Ductal carcinoma in situ (DCIS) is associated with risk of positive resection margins following breast-conserving surgery (BCS) and subsequent reoperation. Prior reports grossly underestimate the risk of margin positivity with IBC containing a DCIS component (IBC + DCIS) due to patient-level rather than margin-level analysis. OBJECTIVE: The aim of this study was to delineate the relative risk of IBC + DCIS compared with pure IBC (without a DCIS component) on margin positivity through detailed margin-level interrogation. METHODS: A single institution, retrospective, observational cohort study was conducted in which pathology databases were evaluated to identify patients who underwent BCS over 5 years (2014-2019). Margin-level interrogation included granular detail into the extent, pathological subtype and grade of disease at each resection margin. Predictors of a positive margin were computed using multivariate regression analysis. RESULTS: Clinicopathological details were examined from 5454 margins from 909 women. The relative risk of a positive margin with IBC + DCIS versus pure IBC was 8.76 (95% confidence interval [CI] 6.64-11.56) applying UK Association of Breast Surgery guidelines, and 8.44 (95% CI 6.57-10.84) applying the Society of Surgical Oncology/American Society for Radiation Oncology guidelines. Independent predictors of margin positivity included younger patient age (0.033, 95% CI 0.006-0.060), lower specimen weight (0.045, 95% CI 0.020-0.069), multifocality (0.256, 95% CI 0.137-0.376), lymphovascular invasion (0.138, 95% CI 0.068-0.208) and comedonecrosis (0.113, 95% CI 0.040-0.185). CONCLUSIONS: Compared with pure IBC, the relative risk of a positive margin with IBC + DCIS is approximately ninefold, significantly higher than prior estimates. This margin-level methodology is believed to represent the impact of DCIS more accurately on margin positivity in IBC.

Mastitis and Mammary Abscess Management Audit (MAMMA) in the UK and Ireland.

BACKGROUND: The aim of this multicentre prospective audit was to describe the current practice in the management of mastitis and breast abscesses in the UK and Ireland, with a specific focus on rates of surgical intervention. METHODS: This audit was conducted in two phases from August 2020 to August 2021; a phase 1 practice survey and a phase 2 prospective audit. Primary outcome measurements for phase 2 included patient management pathway characteristics and treatment type (medical/radiological/surgical). RESULTS: A total of 69 hospitals participated in phase 2 (1312 patients). The key findings were a high overall rate of incision and drainage (21.0 per cent) and a lower than anticipated proportion of ultrasound-guided aspiration of breast abscesses (61.0 per cent). Significant variations were observed regarding the rate of incision and drainage (range 0-100 per cent; P < 0.001) and the rate of needle aspiration (range 12.5-100 per cent; P < 0.001) between individual units. Overall, 22.5 per cent of patients were admitted for inpatient treatment, out of whom which 72.9 per cent were commenced on intravenous antibiotics. The odds of undergoing incision and drainage for a breast abscess or being admitted for inpatient treatment were significantly higher if patients presented at the weekend compared with a weekday (P ≤ 0.023). Breast specialists reviewed 40.9 per cent of all patients directly, despite the majority of patients (74.2 per cent) presenting within working hours on weekdays. CONCLUSIONS: Variation in practice exists in the management of mastitis and breast abscesses, with high rates of incision and drainage in certain regions of the UK. There is an urgent need for a national best-practice toolbox to minimize practice variation and standardize patient care.

Mastitis and breast abscess is a painful infection of the breast. It is an extremely common breast problem. One in three women can get this condition at some stage in their life. To treat a breast abscess, the pus inside should be drained out of the body. This can be done either by cutting into the breast using surgery or by inserting a fine needle using an ultrasonography scan (which uses ultrasound). Fine-needle drainage has the benefit that it does not require admission to hospital. Surgery can cause the breast to look misshapen. It is unknown which method is used more often in the UK and Ireland. The aim of this study was to describe how mastitis and breast abscesses are treated in the UK and Ireland. This study involved a survey of practice (phase 1) and collection of data, which are routinely recorded for these patients (phase 2). This study involved 69 hospitals and 1312 patient records. One in five women had an operation for a breast abscess. This was higher than expected. Six in 10 women had a pus drainage using a fine needle. The chance of having an operation depended on the hospital. Women that came to hospital at the weekend were almost twice as likely to have an operation. One in five women were admitted to hospital. The chances of that more than doubled if a woman came to hospital at the weekend. There are differences in treatment of mastitis and breast abscesses across the UK and Ireland. Changes need to be put in place to make access to treatment more equal.

Alanine metabolism in Bacillus subtilis.

Both isomeric forms of alanine play a crucial role in bacterial growth and viability; the L-isomer of this amino acid is one of the building blocks for protein synthesis, and the D-isomer is incorporated into the bacterial cell wall. Despite a long history of genetic manipulation of Bacillus subtilis using auxotrophic markers, the genes involved in alanine metabolism have not been characterized fully. In this work, we genetically characterized the major enzymes involved in B. subtilis alanine biosynthesis and identified an alanine permease, AlaP (YtnA), which we show has a major role in the assimilation of D-alanine from the environment. Our results provide explanations for the puzzling fact that growth of B. subtilis does not result in the significant accumulation of extracellular D-alanine. Interestingly, we find that in B. subtilis, unlike E. coli where multiple enzymes have a biochemical activity that can generate alanine, the primary synthetic enzyme for alanine is encoded by alaT, although a second gene, dat, can support slow growth of an L-alanine auxotroph. However, our results also show that Dat mediates the synthesis of D-alanine and its activity is influenced by the abundance of L-alanine. This work provides valuable insights into alanine metabolism that suggests that the relative abundance of D- and L-alanine might be linked with cytosolic pool of D and L-glutamate, thereby coupling protein and cell envelope synthesis with the metabolic status of the cell. The results also suggest that, although some of the purified enzymes involved in alanine biosynthesis have been shown to catalyze reversible reactions in vitro, most of them function unidirectionally in vivo.

Diagnostic Accuracy of Nipple Discharge Fluid Cytology: A Meta-Analysis and Systematic Review of the Literature.

BACKGROUND: Nipple discharge is the third most frequent complaint of women attending rapid diagnostic breast clinics. Nipple smear cytology remains the single most used diagnostic method for investigating fluid content. This study aimed to conduct a systematic review and meta-analysis of the diagnostic accuracy of nipple discharge fluid assessment. METHODS: The study incorporated searches for studies interrogating the diagnostic data of nipple discharge fluid cytology compared with the histopathology gold standard. Data from studies published from 1956 to 2019 were analyzed. The analysis included 8648 cytology samples of women with a presenting complaint of nipple discharge. Both hierarchical and bivariate models for diagnostic meta-analysis were used to attain overall pooled sensitivity and specificity. RESULTS: Of 837 studies retrieved, 45 fulfilled the criteria for inclusion. The diagnostic accuracy of the meta-analysis examining nipple discharge fluid had a sensitivity of 75 % (95 % confidence interval [CI], 0.74-0.77) and a specificity of 87 % (95 % CI, 0.86-0.87) for benign breast disease. For breast cancer, it had a sensitivity of 62 % (95 % CI, 0.53-0.71) and a specificity 71 % (95 % CI, 0.57-0.81). Furthermore, patients presenting with blood-stained discharge yielded an overall malignancy rate of 58 % (95 % CI, 0.54-0.60) with a positive predictive value (PPV) of 27 % (95 % CI, 0.17-0.36). CONCLUSIONS: Pooled data from studies encompassing nipple discharge fluid assessment suggest that nipple smear cytology is of limited diagnostic accuracy. The authors recommend that a tailored approach to diagnosis be required given the variable sensitivities of currently available tests.

Neuroenhancement of surgeons during robotic suturing.

BACKGROUND: The initial phases of robotic surgical skills acquisition are associated with poor technical performance, such as low knot-tensile strength (KTS). Transcranial direct-current stimulation (tDCS) can improve force and accuracy in motor tasks but research in surgery is limited to open and laparoscopic tasks in students. More recently, robotic surgery has gained traction and is now the most common approach for certain procedures (e.g. prostatectomy). Early-phase robotic suturing performance is dependent on prefrontal cortex (PFC) activation, and this study aimed to determine whether performance can be improved with prefrontal tDCS. METHODS: Fifteen surgical residents were randomized to either active then sham tDCS or sham then active tDCS, in two counterbalanced sessions in a double-blind crossover study. Within each session, participants performed a robotic suturing task repeated in three blocks: pre-, intra- and post-tDCS. During the intra-tDCS block, participants were randomized to either active tDCS (2 mA for 15 min) to the PFC or sham tDCS. Primary outcome measures of technical quality included KTS and error scores. RESULTS: Significantly faster completion times were observed longitudinally, regardless of active (p < 0.001) or sham stimulation (p < 0.001). KTS was greater following active compared to sham stimulation (median: active = 44.35 N vs. sham = 27.12 N, p < 0.001). A significant reduction in error scores from "pre-" to "post-" (p = 0.029) were only observed in the active group. CONCLUSION: tDCS could reduce error and enhance KTS during robotic suturing and warrants further exploration as an adjunct to robotic surgical training.

Experimental evaluation of respiratory droplet spread to rooms connected by a central ventilation system.

This article presents results from an experimental study to ascertain the transmissibility of the SARS-CoV-2 virus between rooms in a building that are connected by a central ventilation system. Respiratory droplet surrogates made of mucus and virus mimics were released in one room in a test building, and measurements of concentration levels were made in other rooms connected via the ventilation system. The paper presents experimental results for different ventilation system configurations, including ventilation rate, filtration level (up to MERV-13), and fractional outdoor air intake. The most important finding is that respiratory droplets can and do transit through central ventilation systems, suggesting a mechanism for viral transmission (and COVID-19 specifically) within the built environment in reasonable agreement with well-mixed models. We also find the deposition of small droplets (0.5-4 µm) on room walls to be negligibly small.

Diagnostic Accuracy of Nipple Aspirate Fluid Cytology in Asymptomatic Patients: A Meta-analysis and Systematic Review of the Literature.

PURPOSE: To calculate the diagnostic accuracy of nipple aspirate fluid (NAF) cytology. BACKGROUND: Evaluation of NAF cytology in asymptomatic patients conceptually offers a non-invasive method for either screening for breast cancer or else predicting or stratifying future cancer risk. METHODS: Studies were identified by performing electronic searches up to August 2019. A meta-analysis was conducted to attain an overall pooled sensitivity and specificity of NAF for breast cancer detection. RESULTS: A search through 938 studies yielded a total of 19 studies. Overall, 9308 patients were examined, with cytology results from 10,147 breasts [age (years), mean ± SD = 49.73 ± 4.09 years]. Diagnostic accuracy meta-analysis of NAF revealed a pooled specificity of 0.97 (95% CI 0.97-0.98), and sensitivity of 0.64 (95% CI 0.62-0.66). CONCLUSIONS: The diagnostic accuracy of nipple smear cytology is limited by poor sensitivity. If nipple fluid assessment is to be used for diagnosis, then emerging technologies for fluid biomarker analysis must supersede the current diagnostic accuracy of NAF cytology.

Langer's arch: A rare but important consideration for axillary surgery with implications for training.

Langer's arch (LA), although rare, is an important anatomical anomaly in the axilla that may be encountered during axillary lymph node dissection (ALND). Failure to recognize this anomaly may cause disorientation during ALND, resulting in inadequate clearance, with implications for local disease recurrence and inaccurate staging. Here, we present a case that highlights the confusion LA can cause even in experienced surgeons. With indications for ALND decreasing, resulting in lower operative numbers, surgeons are less likely to be exposed to this structure during their training. Improving knowledge and understanding of LA is important to ensure breast surgeons' preparedness for axillary surgery.

Can we see what is invisible? The role of MRI in the evaluation and management of patients with pathological nipple discharge.

INTRODUCTION: The aim of this study was to determine the ability of MRI to identify and assess the extent of disease in patients with pathological nipple discharge (PND) with an occult malignancy not evident on standard pre-operative evaluation with mammography and ultrasound. METHODS: Patients presenting to the breast unit of Imperial College Healthcare NHS Trust between December 2009 and December 2018 with PND and normal imaging were enrolled in the study. Pre-operative bilateral breast MRI was performed in all patients as part of our protocol and all patients were offered diagnostic microdochectomy. RESULTS: A total of 82 patients fulfilled our selection criteria and were enrolled in our study. The presence of an intraductal papilloma (IDP) was identified as the cause of PND in 38 patients (46.3%), 14 patients had duct ectasia (DE-17%) and 5 patients had both an IDP and DE. Other benign causes were identified in 11 patients (13.4%). Despite normal mammography and ultrasound a malignancy was identified in 14 patients (17%). Eleven patients had DCIS (13.4%), two had invasive lobular carcinoma and one patient had an invasive ductal carcinoma. The sensitivity of MRI in detecting an occult malignancy was 85.71% and the specificity was 98.53%. The positive predictive value was 92.31% and the negative predictive value was 97.1%. CONCLUSIONS: Although a negative MRI does not exclude the presence of an occult malignancy the high sensitivity and specificity of this diagnostic modality can guide the surgeon and alter the management of patients with PND.

Functional redundancy of division specific penicillin-binding proteins in Bacillus subtilis.

Bacterial cell division involves the dynamic assembly of a diverse set of proteins that coordinate the invagination of the cell membrane and synthesis of cell wall material to create the new cell poles of the separated daughter cells. Penicillin-binding protein PBP 2B is a key cell division protein in Bacillus subtilis proposed to have a specific catalytic role in septal wall synthesis. Unexpectedly, we find that a catalytically inactive mutant of PBP 2B supports cell division, but in this background the normally dispensable PBP 3 becomes essential. Phenotypic analysis of pbpC mutants (encoding PBP 3) shows that PBP 2B has a crucial structural role in assembly of the division complex, independent of catalysis, and that its biochemical activity in septum formation can be provided by PBP 3. Bioinformatic analysis revealed a close sequence relationship between PBP 3 and Staphylococcus aureus PBP 2A, which is responsible for methicillin resistance. These findings suggest that mechanisms for rescuing cell division when the biochemical activity of PBP 2B is perturbed evolved prior to the clinical use of ß-lactams.

Streamlining Decision Making in Contralateral Risk-Reducing Mastectomy: Impact of PREDICT and BOADICEA Computations.

INTRODUCTION: Patients with sporadic breast cancer (BC) have low contralateral breast cancer risk (CLBCR; approximately 0.7% per annum) and contralateral prophylactic mastectomy (CPM) offers no survival advantage. CPM with autologous reconstruction (AR) has major morbidity and resource implications. OBJECTIVE: The aim of this study was to review the impact of PREDICT survival estimates and lifetime CLBCR scores on decision making for CPM in patients with unilateral BC. METHODS: Of n = 272 consecutive patients undergoing mastectomy and AR, 252 were included. Five- and 10-year survival was computed with the PREDICT(V2) online prognostication tool, using age and clinicopathological factors. Based on family history (FH) and tumor biology, CLBCR was calculated using validated BODICEA web-based software. Survival scores were correlated against CLBCR estimates to identify patients receiving CPM with 'low' CLBCR (< 30% lifetime risk) and poor prognosis (5-year survival < 80%). Patients with 'high' CLBCR receiving unilateral mastectomy (UM) were similarly identified (UK National Institute of Health and Care Excellence [NICE] criteria for CPM, ≥ 30% lifetime BC risk). Justifications motivating CPM were investigated. RESULTS: Of 252 patients, 215 had UM and 37 had bilateral mastectomy and AR. Only 23 (62%) patients receiving CPM fulfilled the NICE criteria. Of 215 patients, 5 (2.3%) failed to undergo CPM despite high CLBCR and good prognosis. CPMs were performed, at the patient's request, for no clear justification (n = 8), contralateral non-invasive disease, and/or FH (n = 5), FH alone (n = 4) and ipsilateral cancer recurrence-related anxiety (n = 3). CONCLUSION: In the absence of prospective risk estimates of CLBCR and prognosis, certain patients receive CPM and reconstruction despite modest CLBCR, yet a proportion of patients with good prognoses and substantial risk are not undergoing CPM.

Review of the Scientific Understanding of Radioactive Waste at the U.S. DOE Hanford Site.

This Critical Review reviews the origin and chemical and rheological complexity of radioactive waste at the U.S. Department of Energy Hanford Site. The waste, stored in underground tanks, was generated via three distinct processes over decades of plutonium extraction operations. Although close records were kept of original waste disposition, tank-to-tank transfers and conditions that impede equilibrium complicate our understanding of the chemistry, phase composition, and rheology of the waste. Tank waste slurries comprise particles and aggregates from nano to micro scales, with varying densities, morphologies, heterogeneous compositions, and complicated responses to flow regimes and process conditions. Further, remnant or changing radiation fields may affect the stability and rheology of the waste. These conditions pose challenges for transport through conduits or pipes to treatment plants for vitrification. Additionally, recalcitrant boehmite degrades glass quality and the high aluminum content must be reduced prior to vitrification for the manufacture of waste glass of acceptable durability. However, caustic leaching indicates that boehmite dissolves much more slowly than predicted given surface normalized rates. Existing empirical models based on ex situ experiments and observations generally only describe material balances and have not effectively predicted process performance. Recent advances in the areas of in situ microscopy, aberration-corrected transmission electron microscopy, theoretical modeling across scales, and experimental methods for probing the physics and chemistry at mineral-fluid and mineral-mineral interfaces are being implemented to build robustly predictive physics-based models.

Free Flap Head and Neck Reconstruction with an Emphasis on Postoperative Care.

Microsurgical free tissue transfer represents the mainstay of care in both ablative locoregional management and the simultaneous reconstruction of a defect. Advances in microsurgical techniques have helped balance the restoration of both form and function-decreasing the significant morbidity once associated with large ablative, traumatic, or congenital defects-while providing immediate reconstruction enabling early aesthetic and functional rehabilitation. There are a multitude of perioperative measures and considerations that aim to maximize the success of free tissue transfer. These include nutritional support, tight glycemic control, acknowledgment of psychological and psychiatric factors, intraoperative surgical technique, and close postoperative monitoring of the patients' hemodynamic physiology. While the success rates of free tissue transfer in experienced hands are comparable to alternative options, the consequences of flap failure are catastrophic-with the potential for significant patient morbidity, prolonged hospital stay (and associated increased financial implications), and increasingly limited options for further reconstruction. Success is entirely dependent on a continuous arterial inflow and venous outflow until neovascularization occurs. Flap failure is multifactorial and represents a dynamic process from the potentially reversible failing flap to the necrotic irreversibly failed flap-necessitating debridement, prolonged wound care, and ultimately decisions concerned with future reconstruction. The overriding goal of free flap monitoring is therefore the detection of microvascular complications prior to permanent injury occurring-identifying and intervening within that critical period between the failing flap and the failed flap-maximizing the potential for salvage. With continued technique refinement, microvascular free flap reconstruction offers patients the chance for both reliable functional and aesthetic restoration in the face of significant ablative defects. The caveat to this optimism is the requirement for considered perioperative care and the optimization of those factors that may offer the difference between success and failure.

"Contemplating the Next Maneuver": Functional Neuroimaging Reveals Intraoperative Decision-making Strategies.

OBJECTIVE: To investigate differences in the quality, confidence, and consistency of intraoperative surgical decision making (DM) and using functional neuroimaging expose decision systems that operators use. SUMMARY BACKGROUND DATA: Novices are hypothesized to use conscious analysis (effortful DM) leading to activation across the dorsolateral prefrontal cortex, whereas experts are expected to use unconscious automation (habitual DM) in which decisions are recognition-primed and prefrontal cortex independent. METHODS: A total of 22 subjects (10 medical student novices, 7 residents, and 5 attendings) reviewed simulated laparoscopic cholecystectomy videos, determined the next safest operative maneuver upon video termination (10 s), and reported decision confidence. Video paradigms either declared ("primed") or withheld ("unprimed") the next operative maneuver. Simultaneously, changes in cortical oxygenated hemoglobin and deoxygenated hemoglobin inferring prefrontal activation were recorded using Optical Topography. Decision confidence, consistency (primed vs unprimed), and quality (script concordance) were assessed. RESULTS: Attendings and residents were significantly more certain (P < 0.001), and decision quality was superior (script concordance: attendings = 90%, residents = 78.3%, and novices = 53.3%). Decision consistency was significantly superior in experts (P < 0.001) and residents (P < 0.05) than novices (P = 0.183). During unprimed DM, novices showed significant activation of the dorsolateral prefrontal cortex, whereas this activation pattern was not observed among residents and attendings. During primed DM, significant activation was not observed in any group. CONCLUSIONS: Expert DM is characterized by improved quality, consistency, and confidence. The findings imply attendings use a habitual decision system, whereas novices use an effortful approach under uncertainty. In the presence of operative cues (primes), novices disengage the prefrontal cortex and seem to accept the observed operative decision as correct.

Diagnostic Accuracy of Intraoperative Techniques for Margin Assessment in Breast Cancer Surgery: A Meta-analysis.

OBJECTIVE: The aim of this study was to conduct a systematic review and meta-analysis to clarify the diagnostic accuracy of intraoperative breast margin assessment (IMA) techniques against which the performance of emerging IMA technologies may be compared. SUMMARY OF BACKGROUND DATA: IMA techniques have failed to penetrate routine practice due to limitations, including slow reporting times, technical demands, and logistics. Emerging IMA technologies are being developed to reduce positive margin and re-excision rates and will be compared with the diagnostic accuracy of existing techniques. METHOD: Studies were identified using electronic bibliographic searches up to January 2016. MESH terms and all-field search terms included "Breast Cancer" AND "Intraoperative" AND "Margin." Only clinical studies with raw diagnostic accuracy data as compared with final permanent section histopathology were included. A bivariate model for diagnostic meta-analysis was used to attain overall pooled sensitivity and specificity. RESULTS: Eight hundred thirty-eight unique studies revealed 35 studies for meta-analysis. Pooled sensitivity (Sens), specificity (Spec), and area under the receiver operating characteristic curve (AUROC) values were calculated per group (Sens, Spec, AUROC): frozen section = 86%, 96%, 0.96 (n = 9); cytology = 91%, 95%, 0.98 (n = 11); intraoperative ultrasound = 59%, 81%, 0.78 (n = 4); specimen radiography = 53%, 84%, 0.73 (n = 9); optical spectroscopy = 85%, 87%, 0.88 (n = 3). CONCLUSIONS: Pooled data suggest that frozen section and cytology have the greatest diagnostic accuracy. However, these methods are resource intensive and turnaround times for results have prevented widespread international adoption. Emerging technologies need to compete with the diagnostic accuracy of existing techniques while offering advantages in terms of speed, cost, and reliability.

The Preservation of Cued Recall in the Acute Mentally Fatigued State: A Randomised Crossover Study.

PURPOSE: The objective of this study is to investigate the impact of acute mental fatigue on the recall of clinical information in the non-sleep-deprived state. Acute mental fatigue in the non-sleep-deprived subject is rarely studied in the medical workforce. Patient handover has been highlighted as an area of high risk especially in fatigued subjects. This study evaluates the deterioration in recall of clinical information over 2 h with cognitively demanding work in non-sleep-deprived subjects. METHOD: A randomised crossover study involving twenty medical students assessed free (presentation) and cued (MCQ) recall of clinical case histories at 0 and 2 h under low and high cognitive load using the N-Back task. Acute mental fatigue was assessed through the Visual Analogue Scale, Stanford Scale and NASA-TLX Mental Workload Rating Scale. RESULTS: Free recall is significantly impaired by increased cognitive load (p < 0.05) with subjects demonstrating perceived mental fatigue during the high cognitive load assessment. There was no significant difference in the amount of information retrieved by cued recall under high and low cognitive load conditions (p = 1). DISCUSSION: This study demonstrates the loss of clinical information over a short time period involving a mentally fatiguing, high cognitive load task. Free recall for the handover of clinical information is unreliable. Memory cues maintain recall of clinical information. This study provides evidence towards the requirement for standardisation of a structured patient handover. The use of memory cues (involving recognition memory and cued recall methodology) would be beneficial in a handover checklist to aid recall of clinical information and supports evidence for their adoption into clinical practice.

A widespread family of bacterial cell wall assembly proteins.

Teichoic acids and acidic capsular polysaccharides are major anionic cell wall polymers (APs) in many bacteria, with various critical cell functions, including maintenance of cell shape and structural integrity, charge and cation homeostasis, and multiple aspects of pathogenesis. We have identified the widespread LytR-Cps2A-Psr (LCP) protein family, of previously unknown function, as novel enzymes required for AP synthesis. Structural and biochemical analysis of several LCP proteins suggest that they carry out the final step of transferring APs from their lipid-linked precursor to cell wall peptidoglycan (PG). In Bacillus subtilis, LCP proteins are found in association with the MreB cytoskeleton, suggesting that MreB proteins coordinate the insertion of the major polymers, PG and AP, into the cell wall.