Hypoxia Is Associated with Increased Immune Infiltrates and Both Anti-Tumour and Immune Suppressive Signalling in Muscle-Invasive Bladder Cancer.

Hypoxia and a suppressive tumour microenvironment (TME) are both independent negative prognostic factors for muscle-invasive bladder cancer (MIBC) that contribute to treatment resistance. Hypoxia has been shown to induce an immune suppressive TME by recruiting myeloid cells that inhibit anti-tumour T cell responses. Recent transcriptomic analyses show hypoxia increases suppressive and anti-tumour immune signalling and infiltrates in bladder cancer. This study sought to investigate the relationship between hypoxia-inducible factor (HIF)-1 and -2, hypoxia, and immune signalling and infiltrates in MIBC. ChIP-seq was performed to identify HIF1α, HIF2α, and HIF1ß binding in the genome of the MIBC cell line T24 cultured in 1% and 0.1% oxygen for 24 h. Microarray data from four MIBC cell lines (T24, J82, UMUC3, and HT1376) cultured under 1%, 0.2%, and 0.1% oxygen for 24 h were used. Differences in the immune contexture between high- and low-hypoxia tumours were investigated using in silico analyses of two bladder cancer cohorts (BCON and TCGA) filtered to only include MIBC cases. GO and GSEA were used with the R packages "limma" and "fgsea". Immune deconvolution was performed using ImSig and TIMER algorithms. RStudio was used for all analyses. Under hypoxia, HIF1α and HIF2α bound to ~11.5-13.5% and ~4.5-7.5% of immune-related genes, respectively (1-0.1% O2). HIF1α and HIF2α both bound to genes associated with T cell activation and differentiation signalling pathways. HIF1α and HIF2α had distinct roles in immune-related signalling. HIF1 was associated with interferon production specifically, whilst HIF2 was associated with generic cytokine signalling as well as humoral and toll-like receptor immune responses. Neutrophil and myeloid cell signalling was enriched under hypoxia, alongside hallmark pathways associated with Tregs and macrophages. High-hypoxia MIBC tumours had increased expression of both suppressive and anti-tumour immune gene signatures and were associated with increased immune infiltrates. Overall, hypoxia is associated with increased inflammation for both suppressive and anti-tumour-related immune signalling and immune infiltrates, as seen in vitro and in situ using MIBC patient tumours.

Do meniscal repairs with meniscus cyst do better than meniscectomy?-a systematic review of meniscal cyst treatment.

Background: This systematic review aims to determine the best modality for the management of meniscal cysts and its associated meniscus tear; whether the meniscal cyst treated via arthroscopy or open methods and whether meniscal debridement or repair achieves better results. Methods: This systematic review was performed using PRISMA guidelines. A literature search of PubMed, EMBASE and Cochrane was carried out in July 2020 using the search terms 'meniscal cyst' and 'treatment'. All clinic studies that included filters for papers in the last 20 years, English language, and meniscal cysts found in humans were included. Studies that contained case reports, were in any language other than English, and with subjects that were not humans were excluded. The methodology quality assessment was performed through the modified Coleman methodology score (CMS). Results: A total of 166 results were obtained from PubMed, Cochrane library and EMBASE. Of them, 12 duplicates were identified across the databases and removed from consideration. Six papers were found relevant from EMBASE in which 1 was eventually included in this paper. In total, 12 papers were used in this study. The weighted mean age of the patients was 35.1 years, with total of 523 meniscal cysts, of which 488 of these cysts are associated with meniscal tears (93.31%). The studies included performed cystectomies and/or decompression of meniscal cysts while some left the meniscal cyst alone and dealt with the meniscal lesion instead. All clinical scores showed significant improvement following surgical procedures. Conclusions: Both arthroscopic and open methods can be used for meniscal cysts treatment. Open cystectomy rather than decompression seemed to confer lower risk of cyst recurrences and complications. It is inconclusive to whether meniscal repair or meniscus debridement influenced recurrence and outcome scores. A recommendation for meniscus repair cannot be made due to insufficient high-quality level I or II trials.

Tumour infiltrating B cells discriminate checkpoint blockade-induced responses.

BACKGROUND: Immune cell-driven anti-cancer activity is paramount for effective responses to checkpoint inhibitors (ICB). However, the contribution of the different immune cell subsets in the circulation and within the tumour is poorly understood. MATERIALS AND METHODS: To elucidate the role of the different cell subsets in anti-tumour responses elicited by ICB, we performed single-cell analysis of the transcriptome and surface proteome of paired pre- and early on-treatment metastatic melanoma tumour biopsies and matched peripheral blood mononuclear cell samples. We next compared the survival of metastatic melanoma patients treated with ICB according to the abundance of pre-treatment tumour-infiltrating B cell clonotypes. RESULTS: We identified cell clusters associated with disease control or progression, defined differential expression of biological pathways likely involved in the immune awakening against the tumour and examined how cell-cell communication patterns between the tumour cell subsets change during treatment. Furthermore, we discovered that B cells (immunoglobulin expression and abundance of B cell clonotypes) discriminate the clinical response after ICB and propose that B cells likely contribute to anti-tumour immunity by antigen presentation through major histocompatibility complex molecules. Finally, we demonstrated that the abundance of tumour-infiltrating B cell clonotypes at baseline identifies two distinct risk groups, a finding that we confirmed in an independent cohort. CONCLUSIONS: Our exploratory translational study provides new insights on the mechanistic role of B cells in anti-melanoma immunity during treatment with ICB. Additionally, we support pre-treatment B cell tumour infiltration as a promising prognostic biomarker to be further validated as a tool for clinical risk stratification.

Return to sport and patient satisfaction after arthroscopic Bankart repair: a single-institution experience.

Introduction: Arthroscopic Bankart repair is a widely accepted procedure to treat recurrent shoulder dislocation. This study aims to describe our experience with arthroscopic Bankart repair and its functional outcome. Methods: 107 patients who underwent arthroscopic Bankart repair from 2008 to 2013 were followed up for a minimum of three years and reviewed by an independent observer. 80 consented to being interviewed using the Oxford Shoulder Instability Score (OSIS) and Simple Shoulder Test. Results: 82 shoulders (two bilateral) were studied. Mean age at first dislocation was 19.4 ± 3.4 (12.0-31.0) years. Mean follow-up was 4.4 ± 1.3 (3.0-9.0) years and 2.5 ± 3.0 (0.1-15.4) years elapsed from first dislocation to surgery. 41 (50.0%) patients played overhead or contact sports and 44 (53.7%) played competitive sports before injury; 8 (9.8%) patients reported recurrence of dislocation, which was significantly associated with playing competitive sports before injury (p <0.039), 5 (6.1%) underwent revision surgery and 22 (26.8%) reported residual instability after surgery. 49 (59.8%) patients returned to playing sports, 75 (91.5%) were satisfied with their surgery and 79 (96.3%) were willing to undergo the surgery again. 74 (90.2%) patients had two-year good/excellent OSIS, which was significantly associated with playing competitive sports before injury (p = 0.039), self-reported stability after surgery (p = 0.017), satisfaction with surgery (p = 0.018) and willingness to undergo surgery again (p = 0.024). Conclusion: Arthroscopic Bankart repair yields good functional outcomes and is associated with high patient satisfaction, although not all patients return to sports.

MPFL reconstruction corrects patella alta: a cohort study.

PURPOSE: Patella alta is an established risk factor for recurrent lateral patella dislocations. Medial patellofemoral ligament (MPFL) reconstruction has been shown to reduce patella height. Our hypothesis is that isolated MPFL reconstruction corrects patella alta, thereby reducing the risk of recurrent patella dislocation. METHODS: A prospective cohort study of 33 knees which underwent MPFL reconstruction for recurrent patella dislocation. The pre-operative and post-operative patella height and Kujala knee outcome scores, as well as tibial tuberosity-trochlear groove (TT-TG) distance, and the grade of trochlear dysplasia were recorded. RESULTS: The mean age was 21.5 years (16-34 years). There was significant reduction in patella height in all patients (p < 0.001), a 67% normalisation of patella alta (CD ≥ 1.2) (p < 0.004), and improvement in the Kujala scores 57.1 (pre-operatively) to 94.8 (post-operatively, p < 0.0001). The recurrent dislocation rate was 3%. CONCLUSION: MPFL reconstruction alone results in correction in patella alta and improvement in clinical outcomes.

A KRAS-responsive long non-coding RNA controls microRNA processing.

Wild-type KRAS (KRASWT) amplification has been shown to be a secondary means of KRAS activation in cancer and associated with poor survival. Nevertheless, the precise role of KRASWT overexpression in lung cancer progression is largely unexplored. Here, we identify and characterize a KRAS-responsive lncRNA, KIMAT1 (ENSG00000228709) and show that it correlates with KRAS levels both in cell lines and in lung cancer specimens. Mechanistically, KIMAT1 is a MYC target and drives lung tumorigenesis by promoting the processing of oncogenic microRNAs (miRNAs) through DHX9 and NPM1 stabilization while halting the biogenesis of miRNAs with tumor suppressor function via MYC-dependent silencing of p21, a component of the Microprocessor Complex. KIMAT1 knockdown suppresses not only KRAS expression but also KRAS downstream signaling, thereby arresting lung cancer growth in vitro and in vivo. Taken together, this study uncovers a role for KIMAT1 in maintaining a positive feedback loop that sustains KRAS signaling during lung cancer progression and provides a proof of principle that interfering with KIMAT1 could be a strategy to hamper KRAS-induced tumorigenesis.

CKAP2L Promotes Non-Small Cell Lung Cancer Progression through Regulation of Transcription Elongation.

Chromosomal instability (CIN) is a driver of clonal diversification and intratumor heterogeneity, providing genetic diversity that contributes to tumor progression. It is estimated that approximately 80% of solid cancers, including non-small cell lung cancer (NSCLC), exhibit features of CIN, which affects tumor growth and response to therapy. However, the molecular mechanisms connecting CIN to tumor progression are still poorly understood. Through an RNAi screen performed on genes involved in CIN and overexpressed in human lung adenocarcinoma samples, we identified the cytoskeleton-associated protein 2-like (CKAP2L) as a potential oncogene that promotes lung cancer proliferation and growth in vitro and in vivo. Mechanistically, CKAP2L directly interacted with RNA Pol II and regulated transcription elongation of key genes involved in spindle assembly checkpoint, chromosome segregation, cell cycle, and E2F signaling. Furthermore, depletion of CKAP2L increased the sensitivity of NSCLC cells to alvocidib, a pan-CDK inhibitor, leading to a significant reduction of cell proliferation and an increase in cell death. Altogether, these findings shed light on the molecular mechanisms through which CKAP2L, a protein involved in CIN, promotes cancer progression and suggest that its inhibition represents a novel therapeutic strategy in NSCLC. SIGNIFICANCE: These findings demonstrate the oncogenic function of CKAP2L through regulation of transcription elongation and suggest that targeting CKAP2L could enhance therapeutic response in patients with NSCLC.

Timing of magnetic resonance imaging affects the accuracy and interobserver agreement of anterolateral ligament tears detection in anterior cruciate ligament deficient knees.

PURPOSE: We aimed to identify the anterolateral ligament (ALL) tears in anterior cruciate ligament (ACL)-deficient knees using standard 1.5-Tesla magnetic resonance imaging (MRI). METHODS: We included all patients who underwent primary ACL reconstruction at our center between 2012 and 2015. Exclusion criteria included patients with multiple ligament injuries, lateral collateral ligament, posterolateral corner, and infections, and patients who underwent MRI more than 2 months after their injury. All patients (n = 148) had ACL tears that were subsequently arthroscopically reconstructed. The magnetic resonance (MR) images of the injured knees performed within 2 months of injury were reviewed by a musculoskeletal radiologist and an orthopedic surgeon. The patients were divided into two groups. The first group of patients had MRI performed within 1 month of injury. The second group of patients had MRI performed 1-2 months after the index injury. Both assessors were blinded and the MR mages were read separately to assess the presence of ALL, presence of a tear and the location of the tear. Based on their readings, interobserver agreement (kappa statistic (K)), sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy were compared. RESULTS: The ALL was identified in 100% of the patients. However, there was a discrepancy of up to 15% in the identification of tear of the ALL. In the first group in which MRI scans were performed within 1 month of injury, the ALL tear was identified by the radiologist in 92% of patients and by the surgeon in 90% of patients (Κ = 0.86). In the second group in which MRI scans were performed within 1-2 months of the injury, the ALL tear was identified by the radiologist in 78% of patients and by the surgeon in 93% of patients (K = 0.62). CONCLUSION: The ALL can be accurately identified on MRI, but the presence and location of ALL tear and its location cannot be reliably identified on MRI. The accuracy in identification and characterization of a tear was affected by the interval between the time of injury and the time when the MRI was performed. LEVEL OF EVIDENCE: Diagnostic, level IIIb, retrospective.

Novel manifestations of immune dysregulation and granule defects in gray platelet syndrome.

Gray platelet syndrome (GPS) is a rare recessive disorder caused by biallelic variants in NBEAL2 and characterized by bleeding symptoms, the absence of platelet α-granules, splenomegaly, and bone marrow (BM) fibrosis. Due to the rarity of GPS, it has been difficult to fully understand the pathogenic processes that lead to these clinical sequelae. To discern the spectrum of pathologic features, we performed a detailed clinical genotypic and phenotypic study of 47 patients with GPS and identified 32 new etiologic variants in NBEAL2. The GPS patient cohort exhibited known phenotypes, including macrothrombocytopenia, BM fibrosis, megakaryocyte emperipolesis of neutrophils, splenomegaly, and elevated serum vitamin B12 levels. Novel clinical phenotypes were also observed, including reduced leukocyte counts and increased presence of autoimmune disease and positive autoantibodies. There were widespread differences in the transcriptome and proteome of GPS platelets, neutrophils, monocytes, and CD4 lymphocytes. Proteins less abundant in these cells were enriched for constituents of granules, supporting a role for Nbeal2 in the function of these organelles across a wide range of blood cells. Proteomic analysis of GPS plasma showed increased levels of proteins associated with inflammation and immune response. One-quarter of plasma proteins increased in GPS are known to be synthesized outside of hematopoietic cells, predominantly in the liver. In summary, our data show that, in addition to the well-described platelet defects in GPS, there are immune defects. The abnormal immune cells may be the drivers of systemic abnormalities such as autoimmune disease.

All-Inside Lateral Meniscal Repair via Anterolateral Portal Increases Risk of Vascular Injury: A Cadaveric Study.

PURPOSE: To compare the distance from the device tip to the neurovascular structures during an all-inside medial and lateral meniscal repair using anteromedial and anterolateral portals in a fresh-frozen cadaveric study. METHODS: Ten fresh-frozen cadaveric knees were studied. The popliteal artery, popliteal vein, and tibial nerve were identified after dissection via a posterior approach. An all-inside meniscal repair device was set to a 20-mm depth limit and inserted into a fixed point in the posterior horn at the meniscocapsular junction. This was performed for medial and lateral menisci via anteromedial and anterolateral arthroscopic portals. The distances between the device tip and the neurovascular structures were measured. We performed t tests to determine statistical significance. RESULTS: The distance between the device and popliteal artery was significantly closer when aimed at the posterior horn of the lateral meniscus via the anterolateral portal (4.7 ± 2.3 mm) versus the anteromedial portal (13.0 ± 8.0 mm, P = .010). The distance to the popliteal vein was closer via the anterolateral portal (6.7 ± 2.9 mm) versus the anteromedial portal (13.9 ± 5.8 mm, P = .004). For medial meniscal repair, the distance to the popliteal artery was significantly closer via the anteromedial portal (12.8 ± 11.3 mm) versus the anterolateral portal (23.8 ± 7.7 mm, P = .022). The distance to the popliteal vein was closer via the anteromedial portal (16.5 ± 11.3 mm) versus the anterolateral portal (28.3 ± 8.2 mm, P = .017). No significant difference was found in the distance to the tibial nerve when aimed at either meniscus via either portal. CONCLUSION: For all-inside meniscal repair, the popliteal vein is at risk and the popliteal artery is at high risk of injury when the posterior horn of the lateral meniscus is repaired via an anterolateral working portal. CLINICAL RELEVANCE: The popliteal artery and vein are at risk of injury when the posterior horn of the lateral meniscus undergoes all-inside repair via the anterolateral portal. Surgeons need to be aware of the risks when performing this repair.

Reliability of magnetic resonance imaging in evaluating meniscal and cartilage injuries in anterior cruciate ligament-deficient knees.

PURPOSE: The accuracy of magnetic resonance (MR) imaging in assessing meniscal and cartilage injuries in anterior cruciate ligament (ACL)-deficient knees as compared to arthroscopy was evaluated in the present study. METHODS: The results of all preoperative MR imaging performed within 3 months prior to the ACL reconstruction were compared against intraoperative arthroscopic findings. A total of 206 patients were identified. The location and type of meniscal injuries as well as the location and grade of the cartilage injuries were studied. The negative predictive value, positive predictive value, sensitivity, specificity and accuracy of MR imaging for these 206 cases were calculated and analysed. RESULTS: In patients with an ACL injury, the highest incidence of concomitant injury was that of medial meniscus tears, 124 (60.2 %), followed by lateral meniscus tears, 105 (51.0 %), and cartilage injuries, 66 (32.0 %). Twenty-three (11.2 %) patients sustained injuries to all of the previously named structures. MR imaging was most accurate in detecting medial meniscus tears (85.9 %). MR imaging for medial meniscus tears also had the highest sensitivity (88.0 %) and positive predictive value (88.7 %), while MR imaging for cartilage injuries had the largest specificity (84.1 %) and negative predictive value (87.1 %). It was least accurate in evaluating lateral meniscus tears (74.3 %). The diagnostic accuracy of medial meniscus imaging is significantly influenced by age and the presence of lateral meniscus tears, while the duration between MR imaging and surgery has greater impact on the likelihood of lateral meniscus and cartilage injuries actually being present during surgery. The majority of meniscus tears missed by MR imaging affected the posterior horn and were complex in nature. Cartilage injuries affecting the medial femoral condyle or medial patella facet were also often missed by MR imaging. CONCLUSION: MR imaging remains a reliable tool for assessing meniscus tears and cartilage defects preoperatively. It is most accurate when evaluating medial meniscus tears. However, MR imaging should be used with discretion especially if there is a high index of suspicion of lateral meniscus tears. LEVEL OF EVIDENCE: IV.

Consideration of growth factors and bio-scaffolds for treatment of combined grade II MCL and ACL injury.

The literature suggests that a Grade II medial collateral ligament (MCL) injury in combination with anterior cruciate ligament (ACL) injury will heal naturally and not compromise patient outcome following ACL reconstruction. Evidence based on bone-patella tendon-bone autograft use is stronger than evidence supporting anatomically placed soft tissue graft use. Current ACL reconstruction practices make greater use of soft tissue grafts, differing fixation methods, and anatomically lower placement on the inner wall of the lateral femoral condyle. Anatomical graft placement aligns the femoral bone tunnel more directly with valgus knee loading forces. Differences in the soft tissue graft-bone tunnel integration and ligamentization timetable following ACL reconstruction also increase concerns regarding residual Grade II MCL laxity and functional deficiency during accelerated functional rehabilitation. MCL dysfunction may increase susceptibility to early ACL graft slippage, elongation, outright failure, and medial femoral condyle lift-off with valgus knee loading. This concept paper discusses the potential role of growth factors and bio-scaffolds for improving Grade II MCL injury healing and mechanical integrity when the injury occurs in combination with an ACL injury that is reconstructed with a soft tissue graft and an anatomical surgical approach.

Repeat probing for treatment of persistent nasolacrimal duct obstruction.

Repeat probing of the nasolacrimal duct is one treatment option for children with persistent symptoms of nasolacrimal duct obstruction (NLDO) following an initial probing. The authors conducted a prospective, multicenter study in which 20 subjects age 6 to <48 months underwent a repeat probing for symptomatic NLDO and had follow-up visits 1 month and 6 months after surgery. Treatment success was defined as the absence of all three clinical signs of nasolacrimal duct obstruction-epiphora, increased tear lake, and mucous discharge. Repeat probing was successful in 56%(95% CI, 33%-76%) of cases.

Return to sports after anterior cruciate ligament reconstruction - a review of patients with minimum 5-year follow-up.

INTRODUCTION: It is difficult to counsel the anterior cruciate ligament (ACL) deficient patient considering surgical reconstruction on the likelihood of eventual return to sports as information on this is lacking, especially in the Asian context. We wanted to determine how many of our patients who had ACL surgery returned to their previous levels of sports, 5 years after their surgery. For those that had not returned to their previous levels of sports, we wanted to identify their reasons for not doing so. MATERIALS AND METHODS: Based on our inclusion criteria of a minimum 5-year follow-up after primary ACL reconstruction, 146 patients were identified for assessment. Sixty-four patients were successfully recalled. The mean age of our patients was 24.8 years (range, 18 to 40). The patients completed the Lysholm Knee, Tegner activity and the Subjective International Knee Documentation Committee (IKDC) questionnaires. Clinical examination of the operated knee was performed according to the Objective IKDC evaluation form and with a KT-1000 arthrometer. RESULTS: The mean Lysholm score was 85.2 and the mean subjective IKDC score was 79.5. 81.2% of our patients had normal or nearly normal knees (IKDC A or B) with the remaining 18.8% at IKDC grade C. The mean side-side difference for anterior translation using the KT-1000 arthrometer was 1.2 mm. The median pre-injury Tegner activity level was 7 and the median 5-year post-surgery Tegner activity level was 6. Nineteen patients did not return to their pre-injury sports levels because of social reasons and were excluded. From the remaining 45 patients, 28 patients (62.2%) returned to their previous level of sports and 17 patients (28.8%) did not return to their previous level of sports. Of whom, 9 (20%) said that they did not return due to fear of re-injury and the remaining 8 (17.8%) said they had not returned because of knee instability and pain. At 5 years, the subgroup of patients who had returned to sport had the best scores: Lysholm (88.5), subjective IKDC (84.6) and IKDC Grade A&B (89.3%). When we compared this with the subgroups that did not return to sport because of fear of re-injury and because of an unstable knee, we found that the difference in knee outcome scores between these 3 groups were statistically significant. CONCLUSION: Sixty-two per cent of our patients returned to their previous level of sport at 5 years after ACL reconstruction. Fear of reinjury is an important psychological factor for these patients not returning to sports. Our results would allow the attending surgeon to counsel the ACL deficient patient who is considering surgical reconstruction the likelihood of eventual return to sports.

Steroid hypopyon following intravitreal triamcinolone acetonide injection in a pseudophakic patient.

PURPOSE: A 64-year-old woman presenting with the complaint of 'white stuff' in her left eye one day after intravitreal triamcinolone injection for chronic pseudophakic cystoid macular edema after cataract extraction and anterior chamber intraocular lens implant is presented. METHODS: Differential diagnosis included post-injection endophthalmitis versus 'steroid hypopyon.' RESULTS: The patient was observed closely and the hypopyon cleared in four days. Three weeks after the injection, her visual acuity had improved from 20/200 to 20/70 with complete resolution of the macular edema. CONCLUSION: Patients having no capsular barrier presenting with hypopyon one day after IVTA may be observed closely for a few days for resolution of hypopyon prior to being treated as endophthalmitis.

Scleral dellen after bilateral adjustable suture medial rectus muscle resection.

Ocular complications after strabismus surgery in a large study population were recently reported. The most frequent of these complications included pseudoptosis, corneal dellen, conjunctival folds, and punctate epithelial keratopathy. We report the occurrence of a rare complication, scleral dellen, occurring in a healthy 30-year-old man after bilateral adjustable suture medial rectus muscle resection for residual exotropia.

Congenital blindness and osteoporosis-pseudoglioma syndrome.

Isteoporosis-pseudoglioma syndrome (OPPG) is a rare heritable entity that features severe osteoporosis and many variable ophthalmic findings leading to congenital or juvenile blindness. These include microphthalmos, cataracts, bilateral pseudogliomatous retinal detachments, and phthisis bulbi. OPPG is usually not suspected until fractures occur, frequently after seemingly minor trauma. We report the ophthalmic findings of an infant girl with OPPG.

Severe pupil distortion following transchamber repair of a cyclodialysis cleft.

This report describes a potential complication following the repair of a traumatic cyclodialysis cleft in a pediatric patient using a novel technique. A healthy 11-year-old boy suffered a blunt traumatic tennis ball injury to his left eye. He underwent repair of a retinal dialysis with detachment shortly after the injury. Postoperatively, he developed persistent hypotony, shallow anterior chamber, and hypotony maculopathy. An occult cyclodialysis cleft was suspected. Examination under anesthesia revealed a small cyclodialysis deft. The cleft was closed by transchamber placement of prolene sutures across the cleft under direct visualization through a Tano lens. Postoperative course was complicated by severe pupillary distortion despite subsequent suture removal.