Cyanotriazoles are selective topoisomerase II poisons that rapidly cure trypanosome infections.

Millions who live in Latin America and sub-Saharan Africa are at risk of trypanosomatid infections, which cause Chagas disease and human African trypanosomiasis (HAT). Improved HAT treatments are available, but Chagas disease therapies rely on two nitroheterocycles, which suffer from lengthy drug regimens and safety concerns that cause frequent treatment discontinuation. We performed phenotypic screening against trypanosomes and identified a class of cyanotriazoles (CTs) with potent trypanocidal activity both in vitro and in mouse models of Chagas disease and HAT. Cryo-electron microscopy approaches confirmed that CT compounds acted through selective, irreversible inhibition of trypanosomal topoisomerase II by stabilizing double-stranded DNA:enzyme cleavage complexes. These findings suggest a potential approach toward successful therapeutics for the treatment of Chagas disease.

High-throughput CRISPR-mediated 3D enrichment platform for functional interrogation of chemotherapeutic resistance.

Cancer is a disease of somatic mutations. These cellular mutations compete to dominate their microenvironment and dictate the disease outcome. While a therapeutic approach to target-specific oncogenic driver mutations helps to manage the disease, subsequent molecular evolution of tumor cells threatens to overtake therapeutic progress. There is a need for rapid, high-throughput, unbiased in vitro discovery screening platforms that capture the native complexities of the tumor and rapidly identify mutations that confer chemotherapeutic drug resistance. Taking the example of the CDK4/6 inhibitor (CDK4/6i) class of drugs, we show that the pooled in vitro CRISPR screening platform enables rapid discovery of drug resistance mutations in a three-dimensional (3D) setting. Gene-edited cancer cell clones assembled into an organotypic multicellular tumor spheroid (MCTS), exposed to CDK4/6i caused selection and enrichment of the most drug-resistant phenotypes, detectable by next-gen sequencing after a span of 28 days. The platform was sufficiently sensitive to enrich for even a single drug-resistant cell within a large, drug-responsive complex 3D tumor spheroid. The genome-wide 3D CRISPR-mediated knockout screen (>18,000 genes) identified several genes whose disruptions conferred resistance to CDK4/6i. Furthermore, multiple novel candidate genes were identified as top hits only in the microphysiological 3D enrichment assay platform and not the conventional 2D assays. Taken together, these findings suggest that including phenotypic 3D resistance profiling in decision trees could improve discovery and reconfirmation of drug resistance mechanisms and afford a platform for exploring noncell autonomous interactions, selection pressures, and clonal competition.

Racial and ethnic disparities in access to minimally invasive gynecologic surgery for benign pathology.

PURPOSE OF REVIEW: To review current US literature and describe the extent, source, and impact of disparities that exist among Black, Indigenous, and people of color (BIPOC) in surgical route and outcomes for hysterectomy, myomectomy, and endometriosis surgery. RECENT FINDINGS: Despite the nationwide trend toward minimally invasive surgery (MIS), BIPOC women are disproportionally less likely to undergo MIS hysterectomy and myomectomy and have higher rates of perioperative complications. African American women, in particular, receive significantly disparate care. Contemporary literature on the prevalence of endometriosis in BIPOC women is lacking. Further, there is little data on the racial and ethnic differences in endometriosis surgery access and outcomes. SUMMARY: Racial and ethnic disparities in access to minimally invasive gynecologic surgery for benign pathology exist and these differences are not fully accounted for by patient, socioeconomic, or healthcare infrastructure factors. Initiatives that incentivize hiring surgeons trained to perform complex gynecologic surgery, standardized pathways for route of surgery, quality improvement focused on increased hospital MIS volume, and hospital-based public reporting of MIS volume data may be of benefit for minimizing disparities. Further, initiatives to reduce disparities need to address racism, implicit bias, and healthcare structural issues that perpetuate disparities.

A Clinically Applicable Prediction Model for the Risk of Transfusion in Women Undergoing Myomectomy.

STUDY OBJECTIVE: We sought to identify the variables independently associated with intra/postoperative blood transfusion at the time of myomectomy. We further hoped to develop an accurate prediction model using preoperative variables to categorize an individual's risk of blood transfusion during myomectomy. DESIGN: Case-control study. SETTING: Not applicable to this study, which used the American College of Surgeons National Surgical Quality Improvement Program (ACS-NSQIP) database. PATIENTS: Women who underwent an open/abdominal or laparoscopic (robotic or conventional) myomectomy between 2014 and 2017 at participating ACS-NSQIP sites. INTERVENTION: The primary dependent variable was occurrence of intra/postoperative bleeding requiring blood transfusion. Patient demographics, clinical characteristics, preoperative comorbidities, intraoperative variables, and additional 30-day postoperative outcomes were compared at the bivariable level. For the prediction-model development, only variables that can be reasonably known before surgery were included. Variables associated with intra/postoperative bleeding were entered into 2 separate multivariable logistic regression models. Validation of our prediction model was performed internally using 250 bootstrapped iterations of 50% subsamples drawn from the overall population of myomectomy cases from the ACS-NSQIP database. MEASUREMENTS AND MAIN RESULTS: We identified 6387 myomectomies performed during the defined study period. The most common race in our population was black/African American (45.7%), and most of the patients (57.5%) received an open/abdominal route of myomectomy. A total of 623 patients who underwent myomectomy (9.8%) experienced intraoperative/postoperative bleeding with a need for blood transfusion. At the bivariable level, we identified several variables independently associated with the need for blood transfusion at the time of myomectomy. In using only those variables that can be reasonably known before surgery to develop our prediction model, additional multivariable logistic regression elucidated black race, need for preoperative blood transfusion, planned abdominal/open route of surgery, and preoperative hematocrit value as independently associated with blood transfusion. CONCLUSION: We identified a number of perioperative variables associated with intraoperative or postoperative bleeding requiring blood transfusion at the time of myomectomy. We subsequently created a model that accurately predicts individual bleeding risk from myomectomy, using variables that are reasonably apparent preoperatively. Making this prediction model clinically available to gynecologic surgeons will serve to improve the care of women undergoing myomectomy.

Patterns of voiding following laparoscopic hysterectomy.

OBJECTIVE: Clarify the normal patterns of voiding after minimally invasive hysterectomy. We also aim to identify perioperative factors associated with delayed time to void immediately following hysterectomy. DESIGN: Retrospective cohort study SELECTION: Women undergoing laparoscopic hysterectomy between September 2012 to October 2018 at a single academic university hospital. RESULTS: 450 minimally invasive hysterectomies were included in the final analysis, 274 (60.9%) robotically-assisted, and 176 (39.1%) conventional laparoscopy. The overall median postoperative time-to-void following a retrograde bladder filling of 150 mL normal saline was 179 min. Based on the 50th percentile of the distribution of the time-to-void, two groups were created. Demographic characteristics between the groups were similar, except those who were above the 50th percentile were more likely to be older, have a reported history of previous myomectomy, and had a longer postoperative PACU stay compared to those below or equal to the 50th percentile. The mean time-to-void following conventional laparoscopic hysterectomy was less than that of robotic surgery (187.3 vs 200.5 min) however the difference was not statistically significant (p=.22). The use of hydromorphone intraoperatively and the combination of oxycodone-acetaminophen postoperatively were more likely to be associated with the group of patients above the 50th percentile but there was no significant difference in perioperative utilization of median morphine milliequivalents (MME) between the two groups. CONCLUSIONS: Following laparoscopic hysterectomy (either conventional or with robotic-assistance) with a retrograde bladder fill of 150 mL normal saline most patients will void within 4 h after surgery. This is consistent with historic data on normal voiding patterns facilitating safe same day discharge without prolonged time in the PACU.

Rates of anastomotic leak and fistula following surgical management of bowel endometriosis: a comparison of shaving, discoid excision, and segmental resection.

Endometriosis is a complex chronic inflammatory condition that can create a multitude of bothersome painful symptoms for women. Bowel endometriosis is often misdiagnosed or overlooked leading to years of suffering for many women. The surgical management of bowel endometriosis varies based on extent of disease as well as surgeon experience. Surgical treatment for bowel endometriosis is complex and a variety of intraoperative and postoperative complications must be considered. Two significant postoperative complications for bowel endometriosis include anastomotic leak and fistula formation. There is continued debate regarding the appropriate surgical treatment for bowel endometriosis. Aggressive surgery with segmental bowel resection is being utilized more cautiously, with an increase in less aggressive shaving or disc excision techniques. Historic beliefs regarding the limitations of shaving and disc excision are being challenged, and with a reduction in morbidity these less aggressive techniques are winning favor among gynecologic surgeons. Shaving, discoid excision, and segmental bowel resection are all feasible surgical management options for bowel endometriosis. Segmental resection is associated with the highest rates of both anastomotic leak and fistula formation, while shaving is associated with the lowest.

Reliability and Validity of 2 Surgical Prioritization Systems for Reinstating Nonemergent Benign Gynecologic Surgery during the COVID-19 Pandemic.

STUDY OBJECTIVE: Scientifically evaluate the validity and reproducibility of 2 novel surgical triaging systems, as well as offer modifications to the Medically-Necessary, Time-Sensitive (MeNTS) criteria for improved application in gynecologic surgeries. DESIGN: Retrospective cohort study. SETTING: Academic university hospital. PATIENTS: Ninety-seven patients with delayed benign gynecologic procedures owing to the coronavirus disease 2019 pandemic. INTERVENTION(S): Surgical prioritization was assessed using 2 novel scoring systems, the Gynecologic Medically-Necessary Time-Sensitive (Gyn-MeNTS) and modified Elective Surgery Acuity Scale (mESAS) systems for all 93 patients included. MEASUREMENTS AND MAIN RESULTS: The interrater reliability and validity of 2 novel surgical prioritization systems (Gyn-MeNTS and mESAS) were assessed. The Gyn-MeNTS scores were calculated by 3 raters and analyzed as continuous variables, with a lower score indicating more urgency/priority. The mESAS score was calculated by 2 raters and analyzed as a 3-level ordinal variable with a higher score indicating more urgency/priority. All 5 raters were blinded to reduce bias. The Gyn-MeNTS interrater reliability was tested using Spearman r and paired t tests were used to detect systematic differences between raters. Weighted κ indicated mESAS reliability. Concurrent validity with mESAS and surgeon self-prioritization (SSP) was examined with Spearman r and logistic regression. Spearman r's for all Gyn-MeNTS rater pairs were above 0.80 (0.84 for 1 vs 2; 0.82 for 1 vs 3; and 0.82 for 2 vs 3, all p <.001) indicating strong agreement. The weighted κ for the 2 mESAS raters was 0.57 (95% confidence interval, 0.40-0.73) indicating moderate agreement. When used together, both scores were significantly independently associated with SSP, with strong discrimination (area under the curve, 0.89). CONCLUSION: Interrater reliability is acceptable for both scoring systems, and concurrent validity of each is moderate for predicting SSP, but discrimination improves to a high level when they are used together.

Indolcarboxamide is a preclinical candidate for treating multidrug-resistant tuberculosis.

New chemotherapeutic compounds against multidrug-resistant Mycobacterium tuberculosis (Mtb) are urgently needed to combat drug resistance in tuberculosis (TB). We have identified and characterized the indolcarboxamides as a new class of antitubercular bactericidal agent. Genetic and lipid profiling studies identified the likely molecular target of indolcarboxamides as MmpL3, a transporter of trehalose monomycolate that is essential for mycobacterial cell wall biosynthesis. Two lead candidates, NITD-304 and NITD-349, showed potent activity against both drug-sensitive and multidrug-resistant clinical isolates of Mtb. Promising pharmacokinetic profiles of both compounds after oral dosing in several species enabled further evaluation for efficacy and safety. NITD-304 and NITD-349 were efficacious in treating both acute and chronic Mtb infections in mouse efficacy models. Furthermore, dosing of NITD-304 and NITD-349 for 2 weeks in exploratory rat toxicology studies revealed a promising safety margin. Finally, neither compound inhibited the activity of major cytochrome P-450 enzymes or the hERG (human ether-a-go-go related gene) channel. These results suggest that NITD-304 and NITD-349 should undergo further development as a potential treatment for multidrug-resistant TB.

Discovery of Q203, a potent clinical candidate for the treatment of tuberculosis.

New therapeutic strategies are needed to combat the tuberculosis pandemic and the spread of multidrug-resistant (MDR) and extensively drug-resistant (XDR) forms of the disease, which remain a serious public health challenge worldwide. The most urgent clinical need is to discover potent agents capable of reducing the duration of MDR and XDR tuberculosis therapy with a success rate comparable to that of current therapies for drug-susceptible tuberculosis. The last decade has seen the discovery of new agent classes for the management of tuberculosis, several of which are currently in clinical trials. However, given the high attrition rate of drug candidates during clinical development and the emergence of drug resistance, the discovery of additional clinical candidates is clearly needed. Here, we report on a promising class of imidazopyridine amide (IPA) compounds that block Mycobacterium tuberculosis growth by targeting the respiratory cytochrome bc1 complex. The optimized IPA compound Q203 inhibited the growth of MDR and XDR M. tuberculosis clinical isolates in culture broth medium in the low nanomolar range and was efficacious in a mouse model of tuberculosis at a dose less than 1 mg per kg body weight, which highlights the potency of this compound. In addition, Q203 displays pharmacokinetic and safety profiles compatible with once-daily dosing. Together, our data indicate that Q203 is a promising new clinical candidate for the treatment of tuberculosis.

The intramembrane protease Sppl2a is required for B cell and DC development and survival via cleavage of the invariant chain.

B cell development requires tight regulation to allow for the generation of a diverse repertoire while preventing the development of autoreactive cells. We report, using N-ethyl-N-nitrosourea (ENU)-induced mutagenesis, the identification of a mutant mouse (chompB) with a block in early B cell development. The blockade occurs after the transitional 1 (T1) stage and leads to a decrease in mature B cell subsets and deficits in T cell-dependent antibody responses. Additionally, chompB mice have decreases in myeloid dendritic cells (DCs). The mutation was mapped to the intramembrane protease signal peptide peptidase-like 2a (Sppl2a), a gene not previously implicated in immune cell development. Proteomic analysis identified the invariant chain (CD74) as a key substrate of Sppl2a and suggests that regulated intramembrane proteolysis of CD74 by Sppl2a contributes to B cell and DC survival. Moreover, these data suggest that modulation of Sppl2a may be a useful therapeutic strategy for treatment of B cell dependent autoimmune disorders.

CD4 count is associated with postoperative infection in patients with orthopaedic trauma who are HIV positive.

BACKGROUND: Since the advent of effective antiretroviral therapy, the number of people with AIDS has increased and a certain percentage of these patients will require emergent orthopaedic surgery. Little is known regarding orthopaedic infections and the association of CD4 counts with postoperative infection in patients with HIV infection who experience orthopaedic trauma. QUESTIONS/PURPOSES: We questioned whether the postoperative infection rate is higher after orthopaedic trauma surgery for patients who are HIV positive than for patients who are HIV negative undergoing similar surgery and aimed to identify preoperative variables that may be important in predicting postoperative infection in patients who are HIV positive. METHODS: We determined the postoperative infection rate in 64 patients who were HIV positive and who underwent orthopaedic surgery requiring instrumentation or an implant from January 2001 to May 2007. We compared this rate with historical control data from 2003 to 2007 for all orthopaedic procedures at Grady Memorial Hospital. We examined numerous preoperative variables for association with postoperative infection, including CD4 count, length of inpatient stay, polytrauma, and malnutrition. RESULTS: Of the 64 patients, 15 had postoperative infections develop with an infection rate of 23%, compared with the 3.9% rate for the historical control subjects. Analysis of the 64 patients who were HIV positive revealed CD4 counts less than 300 were associated with development of postoperative infection. Hospital stay, polytrauma, and low serum albumin also were found to be associated with postoperative infection. CONCLUSIONS: It is evident that patients who are HIV positive with low CD4 counts undergoing emergent orthopaedic intervention are a patient population at risk for infection. Further study is necessary to evaluate preoperative and perioperative interventions that may decrease infections in this population. LEVEL OF EVIDENCE: Level III, prognostic study. See Guidelines for Authors for a complete description of levels of evidence.