α-catenin middle- and actin-binding domain unfolding mutants differentially impact epithelial strength and sheet migration.

α-catenin (α-cat) displays force-dependent unfolding and binding to actin filaments through direct and indirect means, but features of adherens junction structure and function most vulnerable to loss of these allosteric mechanisms have not been directly compared. By reconstituting an α-cat F-actin-binding domain unfolding mutant known to exhibit enhanced binding to actin (α-cat-H0-FABD+) into α-cat knockout Madin Darby Canine Kidney (MDCK) cells, we show that partial loss of the α-cat catch bond mechanism (via an altered H0 α-helix) leads to stronger epithelial sheet integrity with greater colocalization between the α-cat-H0-FABD+ mutant and actin. α-cat-H0-FABD+ -expressing cells are less efficient at closing scratch-wounds, suggesting reduced capacity for more dynamic cell-cell coordination. Evidence that α-cat-H0-FABD+ is equally accessible to the conformationally sensitive α18 antibody epitope as WT α-cat and shows similar vinculin recruitment suggests this mutant engages lower tension cortical actin networks, as its M-domain is not persistently open. Conversely, α-cat-M-domain salt-bridge mutants with persistent recruitment of vinculin and phosphorylated myosin light chain show only intermediate monolayer adhesive strengths, but display less directionally coordinated and thereby slower migration speeds during wound-repair. These data show α-cat M- and FABD-unfolding mutants differentially impact cell-cell cohesion and migration properties, and suggest signals favoring α-cat-cortical actin interaction without persistent M-domain opening may improve epithelial monolayer strength through enhanced coupling to lower tension actin networks.

Outcomes Following Recent and Distant Neoadjuvant Radiation in Rectal Cancer: An Institutional Retrospective Review and Analysis of NSQIP.

BACKGROUND: Neoadjuvant chemoradiotherapy (nCRT) is the standard of care in locally advanced rectal cancer (LARC). However, radiation therapy is thought to increase operative difficulty due to induction of fibrosis. Total neoadjuvant therapy (TNT) protocols increase the time between completion of radiation and surgical resection which may lead to increased operative difficulty and complications. METHODS: A single institution retrospective review of patients ≥18 years with LARC undergoing nCRT from 2015 to 2022. Patients were dichotomized in 2 cohorts: <90 days from radiation to surgery (recent radiation), and ≥90 days from radiation to surgery (distant radiation). Institutional data was compared to National Surgical Quality Improvement Program (NSQIP) rectal cancer data from 2016 to 2020. Outcomes included intraoperative complications, 30-day morbidity, and oncologic outcomes. RESULTS: One hundred forty-six institutional patients included, 120 had recent radiation, 26 had distant radiation. Thirty-day morbidity and intraoperative complications did not differ. There was greater radial margin positivity (7% vs. 24%), fewer lymph nodes harvested (17 ± 5 vs. 15 ± 6), and a lower rate of complete mesorectal dissection (88% vs. 65%,) in distant radiation patients 3059 patients were included in NSQIP analysis, 2029 completed radiation <90 days before surgery and 1030 without radiation 90 days before surgery. Patients without radiation 90 days preoperatively had more radial margin positivity (9.2% vs. 4.6%), organ space infection (8.6% vs. 6.4%), and pneumonia (2.2% vs. 0.9%). CONCLUSION: The present study suggests that increased time between radiation and surgery results in more challenging dissection with less complete mesorectal dissection and increased radial margin positivity without increasing technical complications.

α-catenin mechanosensitivity as a route to cytokinesis failure through sequestration of LZTS2.

Epithelial cells can become polyploid upon tissue injury, but mechanosensitive cues that trigger this state are poorly understood. Using α-catenin (α-cat) knock-out Madin Darby Canine Kidney (MDCK) cells reconstituted with wild-type and mutant forms of α-cat as a model system, we find that an established α-cat actin-binding domain unfolding mutant designed to reduce force-sensitive binding to F-actin (α-cat-H0-FABD+) can promote cytokinesis failure, particularly along epithelial wound-fronts. Enhanced α-cat coupling to cortical actin is neither sufficient nor mitotic cell-autonomous for cytokinesis failure, but critically requires the mechanosensitive Middle-domain (M1-M2-M3) and neighboring cells. Disease relevant α-cat M-domain missense mutations known to cause a form of retinal pattern dystrophy (α-cat E307K or L436P) are associated with elevated binucleation rates via cytokinesis failure. Similar binucleation rates are seen in cells expressing an α-cat salt-bridge destabilizing mutant (R551A) designed to promote M2-M3 domain unfurling at lower force thresholds. Since binucleation is strongly enhanced by removal of the M1 as opposed to M2-M3 domains, cytokinetic fidelity is most sensitive to α-cat M2-M3 domain opening. To identify α-cat conformation-dependent proximity partners that contribute to cytokinesis, we used a biotin-ligase approach to distinguished proximity partners that show enhanced recruitment upon α-cat M-domain unfurling (R551A). We identified Leucine Zipper Tumor Suppressor 2 (LZTS2), an abscission factor previously implicated in cytokinesis. We confirm that LZTS2 enriches at the midbody, but discover it also localizes to tight and tricellular junctions. LZTS2 knock-down promotes binucleation in both MDCK and Retinal Pigmented Epithelial (RPE) cells. α-cat mutants with persistent M2-M3 domain opening showed elevated junctional enrichment of LZTS2 from the cytosol compared α-cat wild-type cells. These data implicate LZTS2 as a mechanosensitive effector of α-cat that is critical for cytokinetic fidelity. This model rationalizes how persistent mechano-activation of α-cat may drive tension-induced polyploidization of epithelia post-injury and suggests an underlying mechanism for how pathogenic α-cat mutations drive macular dystrophy.

The impact of underrepresented minority or marginalized identity status on training outcomes of MD-PhD students.

Dual-degree MD-PhD programs have historically lacked diversity of race, ethnicity, gender, sexual orientation, and other facets of identity. Like MD- and PhD-granting programs, MD-PhD program training environments are also marked by structural barriers that negatively impact measurable academic outcomes of underrepresented and/or marginalized students in academic medicine (racial and ethnic minority groups considered underrepresented by the National Institute of Health, sexual and gender minorities, individuals with disabilities, and individuals of low socioeconomic status). In this article, we review the existing literature on MD-PhD program disparities affecting students from these groups and provide recommendations grounded on the reviewed evidence. Our literature review identified four generalizable barriers that can impact the training outcomes of students from these marginalized and/or underrepresented groups: 1) discrimination and bias, 2) impostor syndrome and stereotype threat, 3) lack of identity-similar mentors, and 4) suboptimal institutional policies and procedures. We propose goal-oriented interventions that may begin to ameliorate the disparities present in MD-PhD program training environments that affect students from marginalized and/or underrepresented groups in academic medicine.

Impact of National Accreditation Program for Rectal Cancer guidelines on surgical margin status.

BACKGROUND: The American College of Surgeons established the National Accreditation Program for Rectal Cancer (NAPRC) to standardize rectal cancer care. We sought to assess the impact of NAPRC guidelines at a tertiary care center on surgical margin status. MATERIALS AND METHODS: The Institutional NSQIP database was queried for patients with rectal adenocarcinoma undergoing surgery for curative intent two years prior to and following implementation of NAPRC guidelines. Primary outcome was surgical margin status before (pre-NAPRC) versus after (post-NAPRC) implementation of NAPRC guidelines. RESULTS: Surgical pathology in five (5%) pre-NAPRC and seven (8%) post-NAPRC patients had positive radial margins (p = 0.59); distal margins were positive in three (3%) post-NAPRC and six (7%) post-NAPRC patients (p = 0.37). Local recurrence was observed in seven (6%) pre-NAPRC patients, there were no recurrences to date in post-NAPRC patients (p = 0.15). Metastasis was observed in 18 (17%) pre-NAPRC patients and four (4%) post-NAPRC patients (p = 0.55). CONCLUSION: NAPRC implementation was not associated with a change in surgical margin status for rectal cancer at our institution. However, the NAPRC guidelines formalize evidence-based rectal cancer care and we anticipate that improvements will be greatest in low-volume hospitals which may not utilize multidisciplinary collaboration.

Omission of or Poor Response to Preoperative Chemoradiotherapy Impacts Radial Margin Positivity Rates in Locally Advanced Rectal Cancer.

BACKGROUND: In the setting of multidisciplinary standardized care of locally advanced rectal cancer, preoperative chemoradiotherapy and total mesorectal excision have become the mainstay treatment. OBJECTIVE: This study aimed to evaluate whether the lack of preoperative chemoradiotherapy or poor response to it is associated with higher radial margin disease involvement in patients with locally advanced rectal cancer. DESIGN: This is a retrospective cohort study using a publicly available database. SETTING: Data were collected from the proctectomy-targeted National Surgical Quality Improvement Project file from 2016 to 2017. PATIENTS: A total of 1161 patients were analyzed. They were categorized into 3 groups: patients who did not receive any preoperative chemoradiotherapy (28.6%), patients who received and responded to preoperative chemoradiotherapy (41.2%), and patients who received but did not respond to preoperative chemoradiotherapy (30.2%). MAIN OUTCOME MEASURES: Response to treatment was determined by using the American Joint Committee on Cancer pretreatment and final pathological staging. Circumferential radial margin was extracted from the targeted proctectomy file. RESULTS: Disease-involved positive circumferential radial margin was found in 86 (7.4%) cases. Positive radial margin was noted in 11 of 479 patients (2.3%) who underwent preoperative chemoradiotherapy and responded to treatment, 30 of 350 patients (8.6%) who did not respond or had a poor response to preoperative chemoradiotherapy, and 45 of 332 patients (13.6%) who did not receive preoperative chemoradiotherapy (p < 0.001). Regression analysis demonstrated that patients who do not receive preoperative chemoradiotherapy or have poor response to it have 6.6 and 4 times higher chances of having a positive radial margin. LIMITATIONS: There is a risk of selection bias, unidentified confounders, and missing data despite the use of a nationwide cohort. CONCLUSIONS: Omission of indicated preoperative chemoradiotherapy or poor response to it is associated with increased risk of radial margin positivity. More efforts are needed for standardized rectal cancer care with the appropriate use of preoperative chemoradiotherapy. See Video Abstract at http://links.lww.com/DCR/B467. LA OMISIN O LA ESCASA RESPUESTA A QUIMIORADIOTERAPIA PREOPERATORIA, AFECTA LAS TASAS DE POSITIVIDAD DEL MARGEN RADIAL, EN EL CNCER RECTAL LOCALMENTE AVANZADO: ANTECEDENTES:En el contexto de la atención multidisciplinaria estandarizada del cáncer rectal localmente avanzado, la quimioradioterapia preoperatoria y la escisión mesorrectal total, se han convertido en el tratamiento principal.OBJETIVO:Evaluar si la omisión de quimioradioterapia preoperatoria o la escasa respuesta, se asocia con mayor enfermedad del margen radial, en pacientes con cáncer rectal localmente avanzado.DISEÑO:Estudio de cohorte retrospectivo utilizando una base de datos disponible públicamente.AJUSTE:Se recopilaron datos del archivo del Proyecto Nacional de Mejora de la Calidad Quirúrgica dirigido a la proctectomía de 2016-2017.PACIENTES:Se analizaron un total de 1161 pacientes. Clasificados en tres grupos: pacientes que no recibieron quimioradioterapia preoperatoria (28,6%), pacientes que recibieron y respondieron a quimioradioterapia preoperatoria (41,2%) y pacientes que recibieron pero no respondieron a la quimioradioterapia preoperatoria (30,2%).PRINCIPALES MEDIDAS DE RESULTADO:La respuesta al tratamiento se determinó utilizando el pre tratamiento y la estatificación patológica final, del American Joint Committee on Cancer. El margen radial circunferencial se extrajo del archivo de proctectomía dirigida.RESULTADOS:Se encontró enfermedad que abarcaba el margen radial circunferencial +, en el 86 (7,4%) casos. Se observó el margen radial +, en 11 de 479 pacientes (2,3%) que se sometieron a quimioradioterapia preoperatoria y respondieron al tratamiento, 30 de 350 pacientes (8,6%) que no respondieron o tuvieron una mala respuesta con quimioradioterapia preoperatoria y en 45 de 332 pacientes (13,6%) que no recibieron quimioradioterapia preoperatoria (p <0,001). El análisis de regresión demostró que los pacientes que no reciben quimioradioterapia preoperatoria o que tienen escasa respuesta, presentan respectivamente, 6,6 y 4 veces más probabilidades de tener un margen radial +.LIMITACIONES:Existe el riesgo de sesgo de selección, factores de confusión no identificados y datos faltantes a pesar del uso de una cohorte nacional.CONCLUSIONES:La omisión de la quimioradioterapia preoperatoria indicada o la escasa respuesta, se asocian a un mayor riesgo de positividad del margen radial. Se necesitan mayores esfuerzos en la atención estandarizada del cáncer rectal, con el uso adecuado de quimioradioterapia preoperatoria. Consulte Video Resumen en http://links.lww.com/DCR/B467.

Size Analysis of C9orf72 Dipeptide Repeat Proteins Expressed in Drosophila melanogaster Using Semidenaturing Detergent Agarose Gel Electrophoresis.

This chapter supplements Chapter 6 on sample preparation and analysis using an analytical ultracentrifuge with fluorescence detection. In this related chapter, we describe how semidenaturing detergent agarose gel electrophoresis can be used to complement the analytical ultracentrifugation work, often as a prelude to careful biophysical analysis to help screen conditions to improve the success of sedimentation velocity experiments. We describe preparation of crude lysates made using Drosophila melanogaster and provide a protocol giving detailed instructions for successful fractionation of protein aggregates using SDD-AGE. While limited in resolving power, this method can identify fractionation in three pools based on sample migration in the gel: that of a monomer or limiting small oligomer species; intermediate aggregation pools, which are typically heterogeneous, represented as high retention smears; and large-scale aggregation, found caught up in the wells.

Aggregation Profiling of C9orf72 Dipeptide Repeat Proteins Transgenically Expressed in Drosophila melanogaster Using an Analytical Ultracentrifuge Equipped with Fluorescence Detection.

The recent development of a fluorescence detection system for the analytical ultracentrifuge has allowed for the characterization of protein size and aggregation in complex mixtures. Protocols are described here to analyze protein aggregation seen in various human neurodegenerative diseases as they are presented in transgenic animal model systems. Proper preparation of crude extracts in appropriate sample buffers is critical for success in analyzing protein aggregation using sedimentation velocity methods. Furthermore, recent advances in sedimentation velocity analysis have led to data collection using single multispeed experiments, which may be analyzed using a wide distribution analysis approach. In this chapter, we describe the use of these new sedimentation velocity methods for faster determination of a wider range of sizes. In Chapter 7 of this book, we describe how agarose gel electrophoresis can be used to complement the analytical ultracentrifugation work, often as a prelude to careful biophysical analysis to help screen conditions in order to improve the success of sedimentation velocity experiments.

p5RHH nanoparticle-mediated delivery of AXL siRNA inhibits metastasis of ovarian and uterine cancer cells in mouse xenografts.

Ovarian and uterine serous cancers are extremely lethal diseases that often present at an advanced stage. The late-stage diagnosis of these patients results in the metastasis of their cancers throughout the peritoneal cavity leading to death. Improving survival for these patients will require identifying therapeutic targets, strategies to target them, and means to deliver therapies to the tumors. One therapeutic target is the protein AXL, which has been shown to be involved in metastasis in both ovarian and uterine cancer. An effective way to target AXL is to silence its expression with small interfering RNA (siRNA). We investigate the ability of the novel siRNA delivery platform, p5RHH, to deliver anti-AXL siRNA (siAXL) to tumor cells both in vitro and in vivo as well as examine the phenotypic effects of this siRNA interference. First, we present in vitro assays showing p5RHH-siAXL treatment reduces invasion and migration ability of ovarian and uterine cancer cells. Second, we show p5RHH nanoparticles target to tumor cells in vivo. Finally, we demonstrate p5RHH-siAXL treatment reduces metastasis in a uterine cancer mouse xenograft model, without causing an obvious toxicity. Collectively, these findings suggest that this novel therapy shows promise in the treatment of ovarian and uterine cancer patients.

Therapeutic Inhibition of the Receptor Tyrosine Kinase AXL Improves Sensitivity to Platinum and Taxane in Ovarian Cancer.

Ovarian cancer, one of the deadliest malignancies in female cancer patients, is characterized by recurrence and poor response to cytotoxic chemotherapies. Fewer than 30% of patients with resistant disease will respond to additional chemotherapy treatments. This study aims to determine whether and how inhibition of the receptor tyrosine kinase AXL can restore sensitivity to first-line platinum and taxane therapy in ovarian cancer. AXL staining was quantified in a patient tissue microarray and correlated with chemoresponse of patients. We used small hairpin RNAs to knock down AXL expression and the small-molecule inhibitor BGB324 to inhibit AXL and assessed sensitivity of cell lines and primary patient-derived cells to chemotherapy. We quantified platinum accumulation by inductivity-coupled plasma phase mass spectrometry. Finally, we treated chemoresistant patient-derived xenografts with chemotherapy, BGB324, or chemotherapy plus BGB324 and monitored tumor burden. AXL expression was higher in chemoresistant patient tumors and cell lines than in chemosensitive tumors and cell lines. AXL staining significantly predicted chemoresponse. Knockdown and inhibition of AXL dose-dependently improved response to paclitaxel and carboplatin in both cell lines and primary cells. AXL inhibition increased platinum accumulation by 2-fold (*, P < 0.05). In vivo studies indicated that AXL inhibition enhanced the ability of chemotherapy to prevent tumor growth (****, P < 0.0001). AXL contributes to platinum and taxane resistance in ovarian cancer, and inhibition of AXL improves chemoresponse and accumulation of chemotherapy drugs. This study supports continued investigation into AXL as a clinical target.

SQ1274, a novel microtubule inhibitor, inhibits ovarian and uterine cancer cell growth.

OBJECTIVE: Paclitaxel, a microtubule inhibitor, is subject to tumor resistance while treating high-grade serous ovarian and uterine cancer. This study aims to directly compare the effects of SQ1274, a novel microtubule inhibitor that binds to the colchicine-binding site on tubulin, and paclitaxel in high-grade serous ovarian and uterine cancer cell lines both in vitro and in vivo. METHODS: We assessed the sensitivity of ovarian (OVCAR8) and uterine (ARK1) cancer cell lines to SQ1274 and paclitaxel using XTT assays. We used western blot and quantitative real-time PCR to analyze changes in AXL RNA and protein expression by SQ1274 and paclitaxel. Differences in cell-cycle arrest and apoptosis were investigated using flow cytometry. Finally, we treated ovarian and uterine xenograft models with vehicle, paclitaxel, or SQ1274. RESULTS: First, we demonstrate that SQ1274 has a much lower IC50 than paclitaxel in both ARK1 (1.26 nM vs. 15.34 nM, respectively) and OVCAR8 (1.34 nM vs. 10.29 nM, respectively) cancer cell lines. Second, we show SQ1274 decreases both RNA and protein expression of AXL. Third, we show that SQ1274 causes increased cell-cycle arrest and apoptosis compared to paclitaxel. Finally, we report that SQ1274 more effectively inhibits tumor growth in vivo compared to paclitaxel. CONCLUSIONS: SQ1274 presents as a viable alternative to paclitaxel for treating ovarian and uterine cancer. This study supports the development of SQ1274 as a chemotherapeutic to treat ovarian and uterine cancer.

Uncovering protein-protein interactions through a team-based undergraduate biochemistry course.

How can we provide fertile ground for students to simultaneously explore a breadth of foundational knowledge, develop cross-disciplinary problem-solving skills, gain resiliency, and learn to work as a member of a team? One way is to integrate original research in the context of an undergraduate biochemistry course. In this Community Page, we discuss the development and execution of an interdisciplinary and cross-departmental undergraduate biochemistry laboratory course. We present a template for how a similar course can be replicated at other institutions and provide pedagogical and research results from a sample module in which we challenged our students to study the binding interface between 2 important biosynthetic proteins. Finally, we address the community and invite others to join us in making a larger impact on undergraduate education and the field of biochemistry by coordinating efforts to integrate research and teaching across campuses.

Inhibition of the Receptor Tyrosine Kinase AXL Restores Paclitaxel Chemosensitivity in Uterine Serous Cancer.

Uterine serous cancer (USC) is aggressive, and the majority of recurrent cases are chemoresistant. Because the receptor tyrosine kinase AXL promotes invasion and metastasis of USC and is implicated in chemoresistance in other cancers, we assessed the role of AXL in paclitaxel resistance in USC, determined the mechanism of action, and sought to restore chemosensitivity by inhibiting AXL in vitro and in vivo We used short hairpin RNAs and BGB324 to knock down and inhibit AXL. We assessed sensitivity of USC cell lines to paclitaxel and measured paclitaxel intracellular accumulation in vitro in the presence or absence of AXL. We also examined the role of the epithelial-mesenchymal transition (EMT) in AXL-mediated paclitaxel resistance. Finally, we treated USC xenografts with paclitaxel, BGB324, or paclitaxel plus BGB324 and monitored tumor burden. AXL expression was higher in chemoresistant USC patient tumors and cell lines than in chemosensitive tumors and cell lines. Knockdown or inhibition of AXL increased sensitivity of USC cell lines to paclitaxel in vitro and increased cellular accumulation of paclitaxel. AXL promoted chemoresistance even in cells that underwent the EMT in vitro Finally, in vivo studies of combination treatment with BGB324 and paclitaxel showed a greater than 51% decrease in tumor volume after 2 weeks of treatment when compared with no treatment or single-agent treatments (P < 0.001). Our results show that AXL expression mediates chemoresistance independent of EMT and prevents accumulation of paclitaxel. This study supports the continued investigation of AXL as a clinical target, particularly in chemoresistant USC. Mol Cancer Ther; 16(12); 2881-91. ©2017 AACR.

Multiple Bacteriophage Selection Strategies for Improved Affinity of a Peptide Targeting ERBB2.

Due to the heterogeneity of ERBB2-expression between tumors and over the course of treatment, a non-invasive molecular imaging agent is needed to accurately detect overall ERBB2 status. Peptides are a highly advantageous platform for molecular imaging, since they have excellent tumor penetration and rapid pharmacokinetics. One limitation of peptides however, is their traditionally low target affinity, and consequently, tumor uptake. The peptide KCCYSL was previously selected from a bacteriophage (phage) display library to bind ERBB2 and did so with moderate affinity of 295 nM. In order to enhance tumor uptake and clinical utility of the peptide, a novel phage microlibrary was created by flanking the parent sequence with random amino acids, followed by reselection using parallel strategies for high affinity and specific ERBB2 binding in an attempt to affinity maturate the peptide. One limitation of traditional phage display selections is difficulty in releasing the highest affinity phages from the target by incubation of acidic buffer. In an attempt to recover high affinity second-generation peptides from the ERBB2 microlibrary, two elution strategies, sonication and target elution, were undertaken. Sonication resulted in an approximately 50-fold enhancement in recovered phage per round of selection in comparison to target elution. Despite the differences in elution efficiency, the affinities of phage-displayed peptides selected from either strategy were relatively similar. Although both selections yielded peptides with significantly improved affinity in comparison to KCCYSL, the improvements were modest, most likely because the parental peptide binding cannot be improved by additional amino acids.

Gabapentin significantly decreases posthemorrhoidectomy pain: a prospective study.

PURPOSE: Surgery for hemorrhoidectomy remains a painful procedure despite advances in pain management. Gabapentin is widely used for control of acute and chronic pain. Our aim was to evaluate the effect of gabapentin on posthemorrhoidectomy pain and opioid use. METHODS: A prospective, open-label study. Patients requiring hemorrhoid surgery were recruited to be in control (standard of care) or treatment group (standard of care plus daily gabapentin). RESULTS: Twenty-one treatment and 18 control patients were recruited. One patient from study group and two patients from control group were excluded due to failure to follow up. Pain levels for gabapentin group were significantly lower on postoperative days 1, 7, and 14 compared to the standard treatment group (3.68 vs. 6.82 p < 0.01, 2.68 vs. 5 p = 0.02 and 0.75 vs. 3.64 p < 0.001 respectively). There was a trend toward less opioids taken in gabapentin group for postoperative days 1, 7, and 14 (4.69 vs. 6.36; 2.13 vs. 2.73, and 0.125 vs. 0.9) but it did not reach statistical significance. The average hemorrhoidal grade and number of hemorrhoidal complexes removed was slightly higher in gabapentin group. Five control group patients experienced postoperative complications versus two gabapentin group patients. No gabapentin related complications were seen in the treatment group. The average cost of gabapentin course was $5.34 per patient. CONCLUSIONS: Daily use of gabapentin in perioperative period significantly decreased reported levels of postoperative pain. This effective, inexpensive addition improves pain after hemorrhoid surgery. Randomized placebo-controlled studies would better define the usefulness of this medication for posthemorrhoidectomy pain.