Behavioral characterization of G-protein-coupled receptor 160 knockout mice.

ABSTRACT: Neuropathic pain is a devastating condition where current therapeutics offer little to no pain relief. Novel nonnarcotic therapeutic targets are needed to address this growing medical problem. Our work identified the G-protein-coupled receptor 160 (GPR160) as a potential target for therapeutic intervention. However, the lack of small-molecule ligands for GPR160 hampers our understanding of its role in health and disease. To address this void, we generated a global Gpr160 knockout (KO) mouse using CRISPR-Cas9 genome editing technology to validate the contributions of GPR160 in nociceptive behaviors in mice. Gpr160 KO mice are healthy and fertile, with no observable physical abnormalities. Gpr160 KO mice fail to develop behavioral hypersensitivities in a model of neuropathic pain caused by constriction of the sciatic nerve. On the other hand, responses of Gpr160 KO mice in the hot-plate and tail-flick assays are not affected. We recently deorphanized GPR160 and identified cocaine- and amphetamine-regulated transcript peptide (CARTp) as a potential ligand. Using Gpr160 KO mice, we now report that the development of behavioral hypersensitivities after intrathecal or intraplantar injections of CARTp are dependent on GPR160. Cocaine- and amphetamine-regulated transcript peptide plays a role in various affective behaviors, such as anxiety, depression, and cognition. There are no differences in learning, memory, and anxiety between Gpr160 KO mice and their age-matched and sex-matched control floxed mice. Results from these studies support the pronociceptive roles of CARTp/GPR160 and GPR160 as a potential therapeutic target for treatment of neuropathic pain.

Hydrogel Delivery Device for the In Vitro and In Vivo Sustained Release of Active rhGALNS Enzyme.

Morquio A disease is a genetic disorder resulting in N-acetylgalactosamine-6-sulfate sulfatase (GALNS) deficiency, and patients are currently treated with enzyme replacement therapy via weekly intravenous enzyme infusions. A means of sustained enzyme delivery could improve patient quality of life by reducing the administration time, frequency of hospital visits, and treatment cost. In this study, we investigated poly(ethylene-glycol) (PEG) hydrogels as a tunable, hydrolytically degradable drug delivery system for the encapsulation and sustained release of recombinant human GALNS (rhGALNS). We evaluated hydrogel formulations that optimized hydrogel gelation and degradation time while retaining rhGALNS activity and sustaining rhGALNS release. We observed the release of active rhGALNS for up to 28 days in vitro from the optimized formulation. rhGALNS activity was preserved in the hydrogel relative to buffer over the release window, and encapsulation was found to have no impact on the rhGALNS structure when measured by intrinsic fluorescence, circular dichroism, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In vivo, we monitored the retention of fluorescently labeled rhGALNS in C57BL/6 albino mice when administered via subcutaneous injection and observed rhGALNS present for up to 20 days when delivered in a hydrogel versus 7 days in the buffer control. These results indicate that PEG hydrogels are suitable for the encapsulation, preservation, and sustained release of recombinant enzymes and may present an alternative method of delivering enzyme replacement therapies that improve patient quality of life.

Risk of Complications in Combined Plastic Surgery Procedures Using the Tracking Operations and Outcomes for Plastic Surgeons Database.

BACKGROUND: Combining multiple surgical procedures into a single operative session is widespread in the field of plastic surgery; however, the implications of this practice are not fully understood. OBJECTIVES: This study compared 30-day complication rates associated with combined plastic surgery procedures with the rates for index procedures. METHODS: This retrospective cohort analysis utilized the Tracking Operations and Outcomes for Plastic Surgeons database from 2016 to 2020 to identify the 3 most frequent combinations of augmentation mammaplasty, reduction mammaplasty, trunk liposuction, mastopexy, and abdominoplasty. RESULTS: The 30-day overall complication rate was 5.0% (1400 of 26,771 patients), with a higher complication rate for combined procedures compared with index (7.6% vs 4.2%, adjusted odd ratio [aOR], 1.91 [95% CI, 1.61-2.27], P < .001). There were no significant differences in complication rates for abdominoplasty or mastopexy combinations compared with index. Complication rates for reduction mammaplasty combinations compared with index were not statistically different after controlling for demographics (aOR, 1.02 [95% CI, 0.61-1.64], P = .93). Higher rates of minor and major complications were observed for combinations of trunk liposuction (aOR, 4.84 [95% CI, 3.31-7.21), P < .001) and augmentation mammaplasty (aOR, 1.60 [95% CI 1.13-2.22], P = .007) compared with index. CONCLUSIONS: Combinations with trunk liposuction or augmentation mammaplasty present with increased risk of complications compared with index, controlling for demographics. Abdominoplasty and mastopexy may be combined with other plastic surgery procedures without increased risk to patients. The complication risk of reduction mammaplasty combinations is mediated by other variables, suggesting the need for shared surgical decision-making when recommending these combinations to patients.

Predictors of Complications in Autologous Breast Reconstruction Using DIEP Flaps: Implications for Management.

BACKGROUND: Deep inferior epigastric perforator (DIEP) flaps are the standard for autologous breast reconstruction. This study investigated risk factors for DIEP complications in a large, contemporary cohort to optimize surgical evaluation and planning. METHODS: This retrospective study included patients who underwent DIEP breast reconstruction between 2016 and 2020 at an academic institution. Demographics, treatment, and outcomes were evaluated in univariable and multivariable regression models for postoperative complications. RESULTS: In total, 802 DIEP flaps were performed in 524 patients (mean age, 51.2 ± 9.6 years; mean body mass index, 29.3 ± 4.5). Most patients (87%) had breast cancer; 15% were BRCA -positive. There were 282 (53%) delayed and 242 (46%) immediate reconstructions and 278 (53%) bilateral and 246 (47%) unilateral reconstructions. Overall complications occurred in 81 patients (15.5%), including venous congestion (3.4%), breast hematoma (3.6%), infection (3.6%), partial flap loss (3.2%), total flap loss (2.3%), and arterial thrombosis (1.3%). Longer operative time was significantly associated with bilateral immediate reconstructions and higher body mass index. Prolonged operative time (OR, 1.16; P = 0.001) and immediate reconstruction (OR, 1.92; P = 0.013) were significant predictors of overall complications. Partial flap loss was associated with bilateral immediate reconstructions, higher body mass index, current smoking status, and longer operative time. CONCLUSIONS: Prolonged operative time is a significant risk factor for overall complications and partial flap loss in DIEP breast reconstruction. For each additional hour of surgical time, the risk of developing overall complications increases by 16%. These findings suggest that reducing operative time through co-surgeon approaches, consistency in surgical teams, and counseling patients with more risk factors toward delayed reconstructions may mitigate complications. CLINICAL QUESTION/LEVEL OF EVIDENCE: Risk, III.

WDR5 represents a therapeutically exploitable target for cancer stem cells in glioblastoma.

Glioblastomas (GBMs) are heterogeneous, treatment-resistant tumors driven by populations of cancer stem cells (CSCs). However, few molecular mechanisms critical for CSC population maintenance have been exploited for therapeutic development. We developed a spatially resolved loss-of-function screen in GBM patient-derived organoids to identify essential epigenetic regulators in the SOX2-enriched, therapy-resistant niche and identified WDR5 as indispensable for this population. WDR5 is a component of the WRAD complex, which promotes SET1 family-mediated Lys4 methylation of histone H3 (H3K4me), associated with positive regulation of transcription. In GBM CSCs, WDR5 inhibitors blocked WRAD complex assembly and reduced H3K4 trimethylation and expression of genes involved in CSC-relevant oncogenic pathways. H3K4me3 peaks lost with WDR5 inhibitor treatment occurred disproportionally on POU transcription factor motifs, including the POU5F1(OCT4)::SOX2 motif. Use of a SOX2/OCT4 reporter demonstrated that WDR5 inhibitor treatment diminished cells with high reporter activity. Furthermore, WDR5 inhibitor treatment and WDR5 knockdown altered the stem cell state, disrupting CSC in vitro growth and self-renewal, as well as in vivo tumor growth. These findings highlight the role of WDR5 and the WRAD complex in maintaining the CSC state and provide a rationale for therapeutic development of WDR5 inhibitors for GBM and other advanced cancers.

Incidence of Venous Thromboembolism Based on Caprini Score in Deep Inferior Epigastric Perforator Flap Breast Reconstruction.

BACKGROUND: Deep inferior epigastric perforator (DIEP) flaps are commonly used for autologous breast reconstruction, but reported rates of venous thromboembolism (VTE) are up to 6.8%. This study aimed to determine the incidence of VTE based on preoperative Caprini score following DIEP breast reconstruction. METHODS: This retrospective study included patients who underwent DIEP flaps for breast reconstruction between January 1, 2016 and December 31, 2020 at a tertiary-level, academic institution. Demographics, operative characteristics, and VTE events were recorded. Receiver operating characteristic analysis was performed to determine the area under the curve (AUC) of the Caprini score for VTE. Univariate and multivariate analyses assessed risk factors associated with VTE. RESULTS: This study included 524 patients (mean age 51.2 ± 9.6 years). There were 123 (23.5%) patients with the Caprini score of 0 to 4, 366 (69.8%) with scores 5 to 6, 27 (5.2%) with scores 7 to 8, and 8 (1.5%) patients with scores >8. Postoperative VTE occurred in 11 (2.1%) patients, at a median time of 9 days (range 1-30) after surgery. VTE incidence by the Caprini score was 1.9% for scores 3 to 4, 0.8% for scores 5 to 6, 3.3% for scores 7 to 8, and 13% for scores >8. The Caprini score achieved an AUC of 0.70. A Caprini score >8 was significantly predictive of VTE on multivariable analysis relative to scores 5 to 6 (odds ratio = 43.41, 95% confidence interval = 7.46-252.76, p < 0.001). CONCLUSION: In patients undergoing DIEP breast reconstruction, VTE incidence was highest (13%) in Caprini scores greater than eight despite chemoprophylaxis. Future studies are needed to assess the role of extended chemoprophylaxis in patients with high Caprini scores.

Novel Non-Opioid Based Therapeutics for Chronic Neuropathic Pain.

Chronic neuropathic pain is currently a major health issue in U.S. complicated by the lack of non-opioid analgesic alternatives. Our investigations led to the discovery of major signaling pathways involved in the transition of acute to chronic neuropathic pain and the identification of several targets for therapeutic intervention. Our translational approach has facilitated the advancement of novel medicines for chronic neuropathic pain that are in advanced clinical development and clinical trials.

The small G-protein RalA promotes progression and metastasis of triple-negative breast cancer.

BACKGROUND: Breast cancer (BC) is the most common cancer in women and the leading cause of cancer-associated mortality in women. In particular, triple-negative BC (TNBC) has the highest rate of mortality due in large part to the lack of targeted treatment options for this subtype. Thus, there is an urgent need to identify new molecular targets for TNBC treatment. RALA and RALB are small GTPases implicated in growth and metastasis of a variety of cancers, although little is known of their roles in BC. METHODS: The necessity of RALA and RALB for TNBC tumor growth and metastasis were evaluated in vivo using orthotopic and tail-vein models. In vitro, 2D and 3D cell culture methods were used to evaluate the contributions of RALA and RALB during TNBC cell migration, invasion, and viability. The association between TNBC patient outcome and RALA and RALB expression was examined using publicly available gene expression data and patient tissue microarrays. Finally, small molecule inhibition of RALA and RALB was evaluated as a potential treatment strategy for TNBC in cell line and patient-derived xenograft (PDX) models. RESULTS: Knockout or depletion of RALA inhibited orthotopic primary tumor growth, spontaneous metastasis, and experimental metastasis of TNBC cells in vivo. Conversely, knockout of RALB increased TNBC growth and metastasis. In vitro, RALA and RALB had antagonistic effects on TNBC migration, invasion, and viability with RALA generally supporting and RALB opposing these processes. In BC patient populations, elevated RALA but not RALB expression is significantly associated with poor outcome across all BC subtypes and specifically within TNBC patient cohorts. Immunohistochemical staining for RALA in patient cohorts confirmed the prognostic significance of RALA within the general BC population and the TNBC population specifically. BQU57, a small molecule inhibitor of RALA and RALB, decreased TNBC cell line viability, sensitized cells to paclitaxel in vitro and decreased tumor growth and metastasis in TNBC cell line and PDX models in vivo. CONCLUSIONS: Together, these data demonstrate important but paradoxical roles for RALA and RALB in the pathogenesis of TNBC and advocate further investigation of RALA as a target for the precise treatment of metastatic TNBC.

Dioxin Exposure Alters Molecular and Morphological Responses to Thyroid Hormone in Xenopus laevis Cultured Cells and Prometamorphic Tadpoles.

Amphibian metamorphosis is driven by thyroid hormone (TH). We used prometamorphic tadpoles and a cell line of the African clawed frog (Xenopus laevis) to examine immediate effects of dioxin exposure on TH. Gene expression patterns suggest cross-talk between the thyroid hormone receptor (TR) and aryl hydrocarbon receptor (AHR) signaling pathways. In XLK-WG cells, expression of Cytochrome P450 1A6 (cyp1A6), an AHR target, was induced 1000-fold by 100 nM TCDD (2, 3, 7, 8 tetrachlorodibenzo-p-dioxin). Krüppel-Like Factor 9 (klf9), the first gene induced in a cascade of TH responses tied to metamorphosis, was upregulated over 5-fold by 50 nM triiodothyronine (T3) and 2-fold by dioxin. Co-exposure to T3 and TCDD boosted both responses, further inducing cyp1A6 by 75% and klf9 about 60%. Additional canonical targets of each receptor, including trßa and trßb (TR) and udpgt1a (AHR) responded similarly. Induction of TH targets by TCDD in XLK-WG cells predicts that exposure could speed metamorphosis. We tested this hypothesis in two remodeling events: tail resorption and hind limb growth. Resorption of ex vivo cultured tails was accelerated by 10 nM T3, while a modest increase in resorption by 100 nM TCDD lacked statistical significance. Hind limbs doubled in length over four days following 1 nM T3 treatment, but limb length was unaffected by 100 nM TCDD. TCDD co-exposure reduced the T3 effect by nearly 40%, despite TCDD induction of klf9 in whole tadpoles, alone or with T3. These results suggest that tissue-specific TCDD effects limit or reverse the increased metamorphosis rate predicted by klf9 induction.

Undergraduates Learn Evolution Through Teaching Kindergartners About Blind Mexican Cavefish.

The development and implementation of a scientific outreach activity comes with a number of challenges. A successful outreach event must match the sophistication of content to the audience, be engaging, expand the knowledge base for participants, and be inclusive for a diverse audience. Ideally, a successful event will also convey the importance of scientific outreach for future scientists and citizens. In this paper, we present a simple, hands-on guide to a scientific outreach event targeted to kindergarten learners. This activity also pursued a second goal: the inclusion of undergraduate students in the development and delivery of the event. We provided a detailed set of four activities, focusing on the blind Mexican cavefish, which were enthusiastically received by kindergarten audiences. The engagement of undergraduate students in the development of this activity encouraged public outreach involvement and fostered new scientific and communication skills. The format of the outreach event we describe is flexible. We provide a set of guidelines and suggestions for adapting this approach to other biological topics. The activity and approach we describe enables the implementation of effective scientific outreach, using active learning approaches, which benefits both elementary school learners and undergraduate students.

Dynamic oxygenation measurements using a phosphorescent coating within a mammary window chamber mouse model.

Phosphorescent lifetime imaging was employed to measure the spatial and temporal distribution of oxygen partial pressure in tissue under the coverslip of a mammary window chamber breast cancer mouse model. A thin platinum-porphyrin coating, whose phosphorescent lifetime varies monotonically with oxygen partial pressure, was applied to the coverslip surface. Dynamic temporal responses to induced modulations in oxygenation levels were measured using this approach.

Multi-modality imaging of a murine mammary window chamber for breast cancer research.

Window chamber models have been developed and utilized as a means to study the complex microenvironment in which cancers develop, proliferate, and metastasize in small animals. Here we utilize rapid prototyping printer technology to construct a new plastic orthotopic mammary window chamber that is compatible with magnetic resonance imaging, nuclear imaging, and optical imaging. Optical imaging allows for high-resolution cellular and molecular level analysis of tissues; magnetic resonance imaging provides quantitative measures of tumor size, perfusion, diffusion, fat/water content relaxation parameters; and a nuclear imaging technique, called the Beta Imager, supports functional and metabolic imaging. Our demonstration of the multiple imaging capabilities of this model suggests that it can be used as a powerful platform for studying basic cancer biology and developing new cancer therapies.

Multimodality pH imaging in a mouse dorsal skin fold window chamber model.

Upregulate levels of expression and activity of membrane H+ ion pumps in cancer cells drives the extracellular pH (pHe,) to values lower than normal. Furthermore, disregulated pH is indicative of the changes in glycolytic metabolism in tumor cells and has been shown to facilitate extracellular tissue remodeling during metastasis Therefore, measurement of pHe could be a useful cancer biomarker for diagnostic and therapy monitoring evaluation. Multimodality in-vivo imaging of pHe in tumorous tissue in a mouse dorsal skin fold window chamber (DSFWC) model is described. A custom-made plastic window chamber structure was developed that is compatible with both imaging optical and MR imaging modalities and provides a model system for continuous study of the same tissue microenvironment on multiple imaging platforms over a 3-week period. For optical imaging of pHe, SNARF-1 carboxylic acid is injected intravenously into a SCID mouse with an implanted tumor. A ratiometric measurement of the fluorescence signal captured on a confocal microscope reveals the pHe of the tissue visible within the window chamber. This imaging method was used in a preliminary study to evaluate sodium bicarbonate as a potential drug treatment to reverse tissue acidosis. For MR imaging of pHe the chemical exchange saturation transfer (CEST) was used as an alternative way of measuring pHe in a DSFWC model. ULTRAVIST®, a FDA approved x-ray/CT contrast agent has been shown to have a CEST effect that is pH dependent. A ratiometric analysis of water saturation at 5.6 and 4.2 ppm chemical shift provides a means to estimate the local pHe.

Training and education in religion/spirituality within APA-accredited clinical psychology programs: 8 years later.

This study was a follow up investigation of Brawer et al.'s (Prof Psychol Res Pr 33(2):203-206, 2002) survey of education and training of clinical psychologists in religion/spirituality. Directors of clinical training were surveyed to determine whether changes had occurred in the coverage of religion and spirituality through course work, research, supervision, and in the systematic coverage of the content area. Results indicated an increased coverage in the areas of supervision, dedicated courses, inclusion as part of another course, and research. There was no increase in systematic coverage, but significantly more programs provided at least some coverage. The current study also assesses other areas of incorporation as well as directors' opinions regarding the importance of religion/spirituality in the field of psychology.