The crosstalk between neuropilin-1 and tumor necrosis factor-α in endothelial cells.

Tumor necrosis factor-α (TNFα) is a master cytokine which induces expression of chemokines and adhesion molecules, such as intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1), in endothelial cells to initiate the vascular inflammatory response. In this study, we identified neuropilin-1 (NRP1), a co-receptor of several structurally diverse ligands, as a modulator of TNFα-induced inflammatory response of endothelial cells. NRP1 shRNA expression suppressed TNFα-stimulated leukocyte adhesion and expression of ICAM-1 and VCAM-1 in human umbilical vein endothelial cells (HUVECs). Likewise, it reduced TNFα-induced phosphorylation of MAPK p38 but did not significantly affect other TNF-induced signaling pathways, such as the classical NFκB and the AKT pathway. Immunofluorescent staining demonstrated co-localization of NRP1 with the two receptors of TNF, TNFR1 and TNFR2. Co-immunoprecipitation further confirmed that NRP1 was in the same protein complex or membrane compartment as TNFR1 and TNFR2, respectively. Modulation of NRP1 expression, however, neither affected TNFR levels in the cell membrane nor the receptor binding affinities of TNFα. Although a direct interface between NRP1 and TNFα/TNFR1 appeared possible from a protein docking model, a direct interaction was not supported by binding assays in cell-free microplates and cultured cells. Furthermore, TNFα was shown to downregulate NRP1 in a time-dependent manner through TNFR1-NFκB pathway in HUVECs. Taken together, our study reveals a novel reciprocal crosstalk between NRP1 and TNFα in vascular endothelial cells.

Pifithrin-µ Induces Stress Granule Formation, Regulates Cell Survival, and Rewires Cellular Signaling.

(1) Background: Stress granules (SGs) are cytoplasmic protein-RNA condensates that assemble in response to various insults. SG production is driven by signaling pathways that are relevant to human disease. Compounds that modulate SG characteristics are therefore of clinical interest. Pifithrin-µ is a candidate anti-tumor agent that inhibits members of the hsp70 chaperone family. While hsp70s are required for granulostasis, the impact of pifithrin-µ on SG formation is unknown. (2) Methods: Using HeLa cells as model system, cell-based assays evaluated the effects of pifithrin-µ on cell viability. Quantitative Western blotting assessed cell signaling events and SG proteins. Confocal microscopy combined with quantitative image analyses examined multiple SG parameters. (3) Results: Pifithrin-µ induced bona fide SGs in the absence of exogenous stress. These SGs were dynamic; their properties were determined by the duration of pifithrin-µ treatment. The phosphorylation of eIF2α was mandatory to generate SGs upon pifithrin-µ exposure. Moreover, the formation of pifithrin-µ SGs was accompanied by profound changes in cell signaling. Pifithrin-µ reduced the activation of 5'-AMP-activated protein kinase, whereas the pro-survival protein kinase Akt was activated. Long-term pifithrin-µ treatment caused a marked loss of cell viability. (4) Conclusions: Our study identified stress-related changes in cellular homeostasis that are elicited by pifithrin-µ. These insights are important knowledge for the appropriate therapeutic use of pifithrin-µ and related compounds.

SC912 inhibits AR-V7 activity in castration-resistant prostate cancer by targeting the androgen receptor N-terminal domain.

Androgen deprivation therapies (ADT) are the mainstay treatments for castration-resistant prostate cancer (CRPC). ADT suppresses the androgen receptor (AR) signaling by blocking androgen biosynthesis or inhibiting AR with antiandrogens that target AR's ligand-binding domain (LBD). However, the ADT's effect is short-lived, as the AR signaling inevitably arises again, which is frequently coupled with AR-V7 overexpression. AR-V7 is a truncated form of AR that lacks the LBD, thus being constitutively active in the absence of androgens and irresponsive to AR-LBD-targeting inhibitors. Though compelling evidence has tied AR-V7 to drug resistance in CRPC, pharmacological inhibition of AR-V7 is still an unmet need. Here, we discovered a small molecule, SC912, which binds to full-length AR as well as AR-V7 through AR N-terminal domain (AR-NTD). This pan-AR targeting relies on the amino acids 507-531 in the AR-NTD. SC912 also disrupted AR-V7 transcriptional activity, impaired AR-V7 nuclear localization and DNA binding. In the AR-V7 positive CRPC cells, SC912 suppressed proliferation, induced cell-cycle arrest, and apoptosis. In the AR-V7 expressing CRPC xenografts, SC912 attenuated tumor growth and antagonized intratumoral AR signaling. Together, these results suggested the therapeutic potential of SC912 for CRPC.

The influence of posterior acromial morphology on outcomes and return to pushups in young patients undergoing arthroscopic posterior capsulolabral repair.

Background: Prior evidence has identified specific posterior acromial morphology as significantly associated with unidirectional posterior shoulder instability. The purpose of this study is to determine the influence of posterior acromial morphology on the outcomes of arthroscopic posterior capsulolabral repair (APCLR) for unidirectional posterior shoulder instability. Additionally, we sought to determine the influence of posterior acromial morphology on the rate and time to return to pushups following APCLR. Methods: We performed a retrospective review of prospectively collected data. The study included consecutive patients undergoing APCLR. Data collected included demographics, radiographic measurements including posterior acromial height (PAH) and posterior acromial tilt on preoperative scapular-Y radiographs, and patient-reported outcome measures at the preoperative and postoperative visits. In addition, starting at 6 months postoperative, patients were asked if they could perform pushups defined as at least 10 repetitions. At the final follow-up, we collected the number of pushups patients were able to perform. Results: Thirty-two consecutive patients underwent APCLR with a mean follow-up of 26 months (range, 12-41). Significant improvement from preoperative to 2 years postoperative was demonstrated in Subjective Shoulder Value (50-85), VAS (6-2.5), American Shoulder and Elbow Surgeons (48 to 83), and Western Ontario Shoulder Instability (WOSI) (1437-777), P = .001. The recurrent instability rate was 3/32 (9%). Patients with PAH > 23 (N = 17) had a recurrent instability rate of 18% (3/17) versus PAH ≤ 23 (N = 15) 0% (0/15), worse WOSI scores (P = .41), and a lower number of pushups (P = .48). The percentage of patients reporting the ability to perform pushups was (6 months/1 year/2 years) (50%/78%/95%). The mean number of pushups reported at the final follow-up was 33 (range, 1-60). Discussion: Following APCLR, approximately 50% of patients resume pushups at 6 months postoperatively, and 80% return at 1 year. Patients reported performing a mean of 33 pushups following APCLR at the final follow-up. Patients with a PAH greater than 23 on preoperative scapular-Y radiographs had a higher rate of recurrent posterior instability, worse WOSI scores, and lower return to pushups; however, the results did not meet statistical significance. Therefore, future larger studies are needed to determine if posterior acromial morphology is independently associated with worse outcomes and increased recurrent instability rates following APCLR.

A High Rate of Return to Running Is Seen After Both Arthroscopic and Open Shoulder Surgery.

Purpose: To determine the percentage of patients who report the ability to run 1 mile at various time points after arthroscopic and open shoulder surgery. Methods: We performed a retrospective review of prospectively collected data for all active-duty military patients aged 18 to 45 years who underwent shoulder surgery at a single institution over a 2-year period. The rehabilitation protocol discouraged running before 3 months, but all patients were able to return to unrestricted running at 3 months postoperatively. Patients were excluded if they lacked 1-year follow-up data. Parameters collected included demographic information and validated patient-reported outcome measures at the preoperative and short-term postoperative visits, as well as patients' ability to run at least 1 mile postoperatively. Results: A total of 126 patients were identified who underwent shoulder surgery with return-to-running data. Compared with baseline, significant improvements in patient-reported outcomes were shown at 1 and 2 years postoperatively (P = .001). The percentage of patients reporting the ability to run 1 mile postoperatively was 59% at 3 months, 74% at 4.5 months, 79% at 6 months, 83% at 12 months, and 91% at 24 months. There was no significant difference in patients undergoing shoulder surgery for instability versus non-instability diagnoses or in patients undergoing open versus arthroscopic anterior stabilization. All 11 patients unable to return to running at final follow-up had chronic lower-extremity diagnoses limiting their running ability. Conclusions: Young military athletes undergoing arthroscopic and open shoulder surgery have a high rate of early return to running. Approximately 60% of patients report the ability to run 1 mile at 3 months postoperatively, and three-quarters of patients do so at 4.5 months. Age, sex, military occupation, underlying diagnosis or type of surgery did not influence the rate of return to running after shoulder surgery. Level of Evidence: Level IV, therapeutic case series.

Oral follicle-stimulating hormone receptor agonist affects granulosa cells differently than recombinant human FSH.

OBJECTIVE: To determine whether TOP5300, a novel oral follicle-stimulating hormone (FSH) receptor (FSHR) allosteric agonist, elicits a different cellular response than recombinant human FSH (rh-FSH) in human granulosa cells from patients undergoing in vitro fertilization. DESIGN: Basic science research with a preclinical allosteric FSHR agonist. SETTING: University hospital. PATIENT(S): Patients with infertility at a single academic fertility clinic were recruited under an Institutional Review Board-approved protocol. Primary granulosa cell cultures were established for 41 patients, of whom 8 had normal ovarian reserve (NOR), 17 were of advanced reproductive age (ARA), 12 had a diagnosis of polycystic ovary syndrome (PCOS), and 4 had a combination of diagnoses, such as ARA and PCOS. INTERVENTION(S): Primary granulosa-lutein (GL) cell cultures were treated with rh-FSH, TOP5300, or vehicle. MAIN OUTCOME MEASURE(S): Estradiol (E2) production using enzyme-linked immunosorbent assay, steroid pathway gene expression of StAR and aromatase using quantitative polymerase chain reaction, and FSHR membrane localization using immunofluorescence were measured in human GL cells. RESULT(S): TOP5300 consistently stimulated E2 production among patients with NOR, ARA, and PCOS. Recombinant FSH was the more potent ligand in GL cells from patients with NOR but was ineffective in cells from patients with ARA or PCOS. The lowest level of FSHR plasma membrane localization was seen in patients with ARA, although FSHR localization was more abundant in cells from patients with PCOS; the highest levels were present in cells from patients with NOR. The localization of FSHR was not affected by TOP5300 relative to rh-FSH in any patient group. TOP5300 stimulated greater expression of StAR and CYP19A1 across cells from all patients with NOR, ARA, and PCOS combined, although rh-FSH was unable to stimulate StAR and aromatase (CYP19A1) expression in cells from patients with PCOS. TOP5300-induced expression of StAR and CYP19A1 mRNA among patients with ARA and NOR was consistently lower than that observed in cells from patients with PCOS. CONCLUSION(S): TOP5300 appears to stimulate E2 production and steroidogenic gene expression from GL cells more than rh-FSH in PCOS, relative to patients with ARA and NOR. It does not appear that localization of FSHR at cell membranes is a limiting step for TOP5300 or rh-FSH stimulation of steroidogenic gene expression and E2 production.

Systematic Exposure in Revision Total Hip Arthroplasty: The Posterior Approach.

As indications for total hip arthroplasty (THA) continue to expand, and patients continue to live longer with more active lifestyles, the incidence of revision THA is expected to rise. General orthopaedic surgeons are now beginning to consider doing revision THA surgery because of the increased revision burden being experienced nationwide. While classical approaches to the hip can be used for simple revisions, extensile exposure techniques in conjunction with selective soft-tissue releases are often required for adequate visualization for more complex revision cases. This review provides a systematic approach to surgical exposure for revision THA using the posterior approach. The surgeon should follow a stepwise progression to obtain safe, adequate, and reproducible visualization of both the acetabulum and the proximal femur.

Prosthetic Hip Dislocations in Direct Anterior Versus Posterior Approach in Patients With Instrumented Lumbar Fusion.

BACKGROUND: Instrumented posterior lumbar spinal fusion (IPLSF) has been demonstrated to contribute to instability following total hip arthroplasty (THA). It is unclear whether a supine direct anterior (DA) approach reduces the risk of instability. METHODS: A retrospective review of 1,773 patients who underwent THA through either a DA approach or a posterior approach at our institution over a 7-year period was performed. Radiographic and chart reviews were then used to identify our primary group of interest comprised of 111 patients with previous IPLSF. Radiographic review, chart review, and phone survey was performed. Dislocation rates in each approach group were then compared within this cohort of patients with IPLSF. RESULTS: Within the group of patients with IPLSF, 33.3% (n = 37) received a DA approach while 66.6% (n = 74) received a posterior approach. None of the 9 total dislocations in the DA group had IPLSF, whereas 4 of the 16 total dislocations in the posterior approach group had IPLSF (P = .78). When examining the larger group of patients, including those without IPLSF, patients undergoing a DA approach had a lower BMI and were likely have a smaller head size implanted (P < .001 for both). Using Fischer's exact test, fusion was associated with dislocation in the posterior approach group (P < .01), whereas fusion was not associated with dislocation in the anterior approach group (P = 1.0). CONCLUSIONS: While there was no significant difference in dislocation rates between posterior and anterior approach groups, in patients with IPLSF, the anterior approach had a lower percentage of dislocation events compared to the posterior approach.

Rate and time to return to shooting following arthroscopic and open shoulder surgery.

Background: There is limited information on return to shooting following shoulder surgery. The purpose of this study is to determine the rate and timing for resuming shooting a rifle following shoulder surgery. Methods: We performed a retrospective review of prospectively collected data. The study included patients undergoing arthroscopic and open shoulder stabilization for unidirectional shoulder instability, and arthroscopic surgery for rotator cuff tears, SLAP lesions, biceps tendinopathy, and acromioclavicular pathology. Data collected included the laterality of surgery, shooting dominance, and patient-reported outcome measures at the preoperative and postoperative visits. Starting at the 4.5-month clinic visit, patients were asked if they could shoot a military rifle. Results: One hundred patients were identified with arthroscopic and open shoulder surgery with a mean age of 30 years (range, 18-45) and a mean follow-up of 24 months (range, 12-32). The cohort consisted of patients undergoing arthroscopic Bankart repair (n = 23), arthroscopic posterior labral repair (n = 18), open Latarjet (n = 16), mini-open subpectoral biceps tenodesis (OBT) (n = 25), OBT with open distal clavicle resection (DCR) (n = 10), open DCR (n = 4), and arthroscopic rotator cuff repair with concomitant OBT (n = 4). Significant improvement in SSV, VAS, ASES, and WOSI was shown at 1-year postoperative, SSV 85, VAS 2, ASES 85, WOSI 239, P = .001. The percentage of patients reporting the ability to shoot a military rifle postoperatively were 47%, 63%, 85%, and 94% at 4.5 months, 6 months, 1 year, and 2 years, respectively. At 4.5 months postoperatively, patients who underwent surgery ipsilateral to their shooting dominance (n = 59) had a rate of return to shooting (33%) versus shoulder surgery on the contralateral side of shooting dominance (n = 41) (60%), P = .04. However, there was no significant difference in the groups at 6 months and 1 year. Additionally, there was a significant difference in the rate of return to shooting at 6 months in patients undergoing arthroscopic posterior labral repair versus the remainder of the cohort (posterior instability (33%) vs. (69%), P = .016), and a significant difference between posterior shoulder stabilization and anterior shoulder stabilization (70%), P = .03. Conclusion: Patients undergoing arthroscopic and open shoulder surgery have a high rate of return to shooting. Approximately 60% of patients resume shooting at 6 months postoperatively and 85% return at 1 year. Patients undergoing shoulder surgery on the contralateral side of their shooting dominance return to shooting significantly faster than those with shoulder surgery ipsilateral to their shooting dominance. Additionally, those undergoing arthroscopic posterior shoulder stabilization return to shooting at a slower rate than anterior stabilization surgery.

NEDD9 links anaplastic thyroid cancer stemness to chromosomal instability through integrated centrosome asymmetry and DNA sensing regulation.

Stemness and chromosomal instability (CIN) are two common contributors to intratumor heterogeneity and therapy relapse in advanced cancer, but their interplays are poorly defined. Here, in anaplastic thyroid cancer (ATC), we show that ALDH+ stem-like cancer cells possess increased CIN-tolerance owing to transcriptional upregulation of the scaffolding protein NEDD9. Thyroid patient tissues and transcriptomic data reveals NEDD9/ALDH1A3 to be co-expressed and co-upregulated in ATC. Compared to bulk ALDH- cells, ALDH+ cells were highly efficient at propagating CIN due to their intrinsic tolerance of both centrosome amplification and micronuclei. ALDH+ cells mitigated the fitness-impairing effects of centrosome amplification by partially inactivating supernumerary centrosomes. Meanwhile, ALDH+ cells also mitigated cell death caused by micronuclei-mediated type 1 interferon secretion by suppressing the expression of the DNA-sensor protein STING. Both mechanisms of CIN-tolerance were lost upon RNAi-mediated NEDD9 silencing. Both in vitro and in vivo, NEDD9-depletion attenuated stemness, CIN, cell/tumor growth, while enhancing paclitaxel effectiveness. Collectively, these findings reveal that ATC progression can involve an ALDH1A3/NEDD9-regulated program linking their stemness to CIN-tolerance that could be leveraged for ATC treatment.

Formal Orthopaedic Surgery "Boot Camp" Curriculum to Optimize Performance on Acting Internships.

Orthopaedic surgery is one of the most competitive residency specialties in the National Residency Matching Program. To improve the odds of matching, senior medical students applying in the field participate in orthopaedic surgery away rotations with programs across the country. Students who do well on these rotations have a higher likelihood of matching because clinical performance is a principal criterion used by admissions committees to rank applicants. On the other hand, these rotations can be physically and emotionally taxing on medical students because poor performance can negatively affect their application and, thus, chances of matching at that institution. Unfortunately, the resources provided by medical schools to prepare students for these high-stakes rotations are usually sparse and unstructured. To address this gap in training at our institution, we developed a formal "boot camp" offered through the university to prepare interested senior medical students for their orthopaedic surgery acting internships. This course focuses on building a solid foundation of musculoskeletal knowledge and exposing students to surgical and procedural skills that are fundamental to the practice of orthopaedic surgery. Over the 2 years, this course has been offered at our institution, and it has proven successful in outcome measures, such as student satisfaction and preparedness, student orthopaedic knowledge, program director evaluations, and match rate. This article describes the novel 1-month curriculum, which includes lectures, laboratory, and clinical experience.

Pharmacological Characterization of Low Molecular Weight Biased Agonists at the Follicle Stimulating Hormone Receptor.

Follicle-stimulating hormone receptor (FSHR) plays a key role in reproduction through the activation of multiple signaling pathways. Low molecular weight (LMW) ligands composed of biased agonist properties are highly valuable tools to decipher complex signaling mechanisms as they allow selective activation of discrete signaling cascades. However, available LMW FSHR ligands have not been fully characterized yet. In this context, we explored the pharmacological diversity of three benzamide and two thiazolidinone derivatives compared to FSH. Concentration/activity curves were generated for Gαs, Gαq, Gαi, ß-arrestin 2 recruitment, and cAMP production, using BRET assays in living cells. ERK phosphorylation was analyzed by Western blotting, and CRE-dependent transcription was assessed using a luciferase reporter assay. All assays were done in either wild-type, Gαs or ß-arrestin 1/2 CRISPR knockout HEK293 cells. Bias factors were calculated for each pair of read-outs by using the operational model. Our results show that each ligand presented a discrete pharmacological efficacy compared to FSH, ranging from super-agonist for ß-arrestin 2 recruitment to pure Gαs bias. Interestingly, LMW ligands generated kinetic profiles distinct from FSH (i.e., faster, slower or transient, depending on the ligand) and correlated with CRE-dependent transcription. In addition, clear system biases were observed in cells depleted of either Gαs or ß-arrestin genes. Such LMW properties are useful pharmacological tools to better dissect the multiple signaling pathways activated by FSHR and assess their relative contributions at the cellular and physio-pathological levels.

Follicle-Stimulating Hormone Induces Lipid Droplets via Gαi/o and ß-Arrestin in an Endometrial Cancer Cell Line.

Follicle-stimulating hormone (FSH) and its G protein-coupled receptor, FSHR, represents a paradigm for receptor signaling systems that activate multiple and complex pathways. Classically, FSHR activates Gαs to increase intracellular levels of cAMP, but its ability to activate other G proteins, and ß-arrestin-mediated signaling is well documented in many different cell systems. The pleiotropic signal capacity of FSHR offers a mechanism for how FSH drives multiple and dynamic downstream functions in both gonadal and non-gonadal cell types, including distinct diseases, and how signal bias may be achieved at a pharmacological and cell system-specific manner. In this study, we identify an additional mechanism of FSH-mediated signaling and downstream function in the endometrial adenocarcinoma Ishikawa cell line. While FSH did not induce increases in cAMP levels, this hormone potently activated pertussis toxin sensitive Gαi/o signaling. A selective allosteric FSHR ligand, B3, also activated Gαi/o signaling in these cells, supporting a role for receptor-mediated activation despite the low levels of FSHR mRNA. The low expression levels may attribute to the lack of Gαs/cAMP signaling as increasing FSHR expression resulted in FSH-mediated activation of the Gαs pathway. Unlike prior reports for FSH-mediated Gαs/cAMP signaling, FSH-mediated Gαi/o signaling was not affected by inhibition of dynamin-dependent receptor internalization. While chronic FSH did not alter cell viability, FSH was able to increase lipid droplet size. The ß-arrestins are key adaptor proteins known to regulate FSHR signaling. Indeed, a rapid, FSH-dependent increase in interactions between ß-arrestin1 and Gαi1 was observed via NanoBiT complementation in Ishikawa cells. Furthermore, both inhibition of Gαi/o signaling and siRNA knockdown of ß-arrestin 1/2 significantly reduced FSH-induced lipid droplet accumulation, implying a role for a Gαi/o/ß-arrestin complex in FSH functions in this cell type. As FSH/FSHR has been implicated in distinct hormone-dependent cancers, including endometrial cancer, analysis of the cancer genome database from 575 human endometrial adenocarcinoma tumors revealed that a subpopulation of samples expressed FSHR. Overall, this study highlights a novel mechanism for FSHR signal pleiotropy that may be exploited for future personalized therapeutic approaches.

Development of a spring-based weight system for halo gravity traction for complex pediatric spinal deformity.

STUDY DESIGN: Description of technique. OBJECTIVES: Describes the redesign of free-weight-based halo gravity traction wheelchairs and walkers to a spring-based system. The treatment of severe pediatric spinal deformities is challenging and associated with significant morbidity. Halo gravity traction (HGT) can be utilized to assist in the correction of severe spinal deformities. A time period of traction may also be used to medically optimize patients prior to surgery. Implementing HGT therapy requires specialized equipment, specially trained hospital staff, as well as significant commitment from caregivers. This study describes the transition from a free weight-based HGT equipment to a spring-based weight system. The new system is expected to decrease the burden of care and improve safety for patients and their families. METHODS: A thorough interview process was carried out to gather data on existing HGT systems. All stakeholders, including orthopedic technicians, nurses, surgeons, patients and their families were asked questions on how to improve the equipment and process. With colleagues at a partner children's hospital, new HGT wheelchairs and walkers with a spring-based weight system were designed and built. RESULTS: Spring-based weight HGT wheelchairs and walkers are more economical to build, safer, and easier to operate than free-weight-based systems. A cost analysis found that spring-based systems cost $780 less compared to free weight-based systems. In addition, the new spring-based wheelchairs and walkers are about 50% lighter with improved weight distribution making them safer to operate and easier to transport. There is consensus among surgeons, nurses, and families that the redesigned units are easier to operate. CONCLUSIONS: Spring-based weight HGT systems are economically viable and clinically effective for pre-operative traction for children with severe spinal deformities. They are well tolerated by patients and easy to care for by nursing and surgical staff. LEVELS OF EVIDENCE: III.

Discovery and Preclinical Development of Orally Active Small Molecules that Exhibit Highly Selective Follicle Stimulating Hormone Receptor Agonism.

An orally active follicle stimulating hormone receptor allosteric agonist would provide a preferred treatment for over 16 million infertile women of reproductive age in low complexity methods (ovulation induction-intrauterine insemination) or in high complexity methods (controlled ovarian stimulation-in vitro fertilization). We present two oral follicle stimulating hormone receptor allosteric agonist compounds that have the desired pharmacology, drug metabolism, pharmacokinetics, and safety profile for clinical use. These molecules provide a single agent suitable for ovulation induction-intrauterine insemination or controlled ovarian stimulation-in vitro fertilization that is more convenient for patients and achieves similar preclinical efficacy as rec-hFSH. TOP5668, TOP5300 were evaluated in vitro in Chinese hamster ovary cells transfected with individual glycoprotein receptors measuring cAMP (FSHR, LH/CGR, thyroid stimulating hormone receptor). TOP5668 was found to have solely follicle stimulating hormone receptor allosteric agonist activity while TOP5300 was found to have mixed follicle stimulating hormone receptor allosteric agonist and LHR-AA activity. Both compounds stimulated concentration-dependent increases in estradiol production from cultured rat granulosa cells in the presence or absence of low dose rec-hFSH, while only TOP5300 stimulated testosterone production from rat primary Leydig cells. In pooled human granulosa cells obtained from patients undergoing controlled ovarian stimulation-in vitro fertilization, TOP5300 stimulated 7-fold greater maximal estradiol response than rec-hFSH and TOP5668 was 10-fold more potent than TOP5300. Both TOP5300 and TOP5668 stimulated follicular development in immature rat to the same efficacy as recombinant follicle stimulating hormone. In mice treated with TOP5300, in the presence of low dose of follicle stimulating hormone, there were no differences in oocyte number, fertilization rate, and hatched blastocyst rate in mice with TOP5300 and low dose follicle stimulating hormone vs. reference proteins pregnant mare serum gonadotropin or high dose rec-hFSH. ADME/PK and safety profiles were favorable. In addition, there was no appreciable activity on thyroid hormones by TOP5300 in 14-days toxicological study in rat or dog. The selected lead compound, TOP5300 stimulated a more robust increase in estradiol production from granulosa-lutein cells from women with polycystic ovarian syndrome patient compared to rec-hFSH. Conclusions: Two novel oral FSHR allosteric agonist, TOP5668 and TOP5300, were found to mimic the biological activity of rec hFSH in preclinical studies. Both compounds led to folliculogenesis and superovulation in rat and mice. Specifically, TOP5300 led to a similar number of ovulated oocytes that fertilized and developed into hatched blastocysts in mice when compared to rec-hFSH. The safety profile demonstrated lack of toxicity.

High Resiliency Linked to Short-Term Patient Reported Outcomes and Return to Duty Following Arthroscopic Knee Surgery.

INTRODUCTION: Resilience is a psychometric construct of a patient's ability to recover from adversity and has been used to predict outcomes but its use in orthopedics has been limited. The purpose of this study was to examine the association between resilience and outcomes. MATERIALS AND METHODS: We performed a retrospective analysis of prospectively collected data of patient who underwent sports knee surgery at a single institution performed by 6 orthopedic surgeons from January 2017 to December 2017. We included active-duty patients with complete preoperative outcomes and a minimum of 6 month follow-up. All patients completed the Brief Resilience Scale (BRS), Veteran's Rand-12 (VR-12), Patient-Reported Outcomes Measurement Information System 43 (PROMIS-43), International Knee Documentation Committee function score (IKDC), and Knee Injury and Osteoarthritis Outcome Score (KOOS). Patients were divided into low resilience (LR) and high resilience (HR) groups based on a score of less than 24 for low and greater than or equal to 24 according to BRS. Outcomes were then compared. RESULTS: We identified 50 active-duty patients who had complete preoperative and postoperative outcomes at a minimum of 6 months. Mean preoperative and postoperative BRS were significantly different (25.8 HR v 18.6 LR, p < 0.001). We found a difference in postop KOOS in pain, sports, and short form (pain 70.9 HR v 55.7 LR, p = 0.03; sports 50.3 HR v 32.2 LR, p = 0.03; short form (72.1 HR v 62.5 LR, p = 0.04). Similarly, there was a significant difference in postoperative IKDC score (58.0 HR v 44.0 LR, p = 0.03). Similarly we found significant differences in postoperative PROMIS-43 (anxiety 44.4 HR v 60.3 LR, p = 0.004; depression 41.6 HR v 58.1 LR, p = 0.004; fatigue 45.1 HR v 58.6 LR, p = 0.001; sleep 52.6 HR v 62.5 LR, p = 0.02; social participation 36.2 HR v 47.6 LR, p < 0.001). Postoperative VR-12 mental was also statistically different between the two groups (53.5 HR v 41.6 LR; p = 0.01). In addition, 2.3% of the HR group changed MOS as a result of their sports knee surgery compared to 22.2% of the LR group. CONCLUSIONS: Active-military patients with high preoperative resilience appear to have significantly better early postoperative outcomes following sports knee surgery in terms of PROMIS-43, KOOS, and IKDC. There was also a lower rate of changing MOS secondary to sports knee surgery in patients with high resilience.

A novel orally available seleno-purine molecule suppresses triple-negative breast cancer cell proliferation and progression to metastasis by inducing cytostatic autophagy.

Patients with triple-negative breast cancer (TNBC) often have a poor prognosis largely due to lack of effective targeted therapy. Using a library of seleno-purines coupled to a high-throughput biochemical enzymatic assays we identified a potent pharmacological enhancer of autophagy (referred herein as SLLN-15) that selectively activated cytostatic macroautophagy/autophagy in TNBC preclinical models. SLLN-15 induced a dose-dependent anti-proliferative activity in the TNBC cell lines MDA-MB-231 and BT-20 via induction of autophagy and autophagic flux. This induction was associated with a selective inhibition of AKT-MTOR signaling. Conversely, rapamycin, a known autophagy inducer and MTOR inhibitor, was unable to duplicate SLLN-15's effect on TNBC cells. Inhibition of autophagy by siRNA-mediated targeting of the autophagy regulators, BECN1, ATG5 and ATG7 or using 3-methyladenine (3-MA), significantly protected against SLLN-15-induced inhibition of cell viability, further supporting that SLLN-15-induced inhibition of cancer cell proliferation was autophagy-dependent. SLLN-15-induced autophagy in TNBC cells was also associated with decreased AURKA expression, decreased AKT phosphorylation and subsequent blockage of the AKT-MTOR pathway. In vivo, oral SLLN-15 revealed a potent anticancer and anti-metastatic activity in mice bearing TNBC. Altogether, this study describes a novel regulator of mammalian autophagy, with potential utility as an experimental therapeutic for TNBCs. Abbreviations: 3-MA: 3-methyladenine; ATG5: autophagy related 5; ATG7: autophagy related 7; AURKA: aurora kinase A; AURKB: aurora kinase B; BECN1: beclin 1; CQ: chloroquine; DMSO: dimethyl sulfoxide; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescent protein; ERBB2: erb-b2 receptor tyrosine kinase 2; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MTOR: mechanistic target of rapamycin kinase; PARP1: poly(ADP-ribose) polymerase 1; PI: propidium iodide; SQSTM1/p62: sequestosome 1; TNBC: triple-negative breast cancer.