Case report: Persistent Müllerian duct syndrome and enlarged prostatic utricle in a male dog.

A 1-year-old male intact Miniature Schnauzer mix was presented for chronic intermittent hematuria. Abdominal ultrasonography revealed a large, fluid-filled cystic structure extending cranially and dorsally to the prostate. Computed tomography scan images revealed that the fluid-filled cavity resembled a uterus, with both horns entering the scrotum through the inguinal canal adjacent to the testes. On cytogenetic analysis, the dog was found to have a homozygote mutation on AMHRII consistent with persistent Müllerian duct syndrome (PMDS). A gonadohysterectomy was performed, and surgical and histologic findings confirmed the presence of a uterus, oviducts, vagina, and testes in this dog. Additionally, an intraoperative fluoroscopy exam revealed a communication between the uterus and the bladder via an enlarged utricle, explaining the hematuria and urine in the reproductive tract (urometra). To our knowledge, this is the first clinical report of a phenotypically intact male dog with PMDS and urometra due to an enlarged prostatic utricle. This case illustrates a combination of a disorder of sex and urogenital sinus development.

Identification of a Class of WNK Isoform-Specific Inhibitors Through High-Throughput Screening.

Introduction: WNK [with no lysine (K)] kinases are serine/threonine kinases associated with familial hyperkalemic hypertension (FHHt). WNKs are therapeutic targets for blood pressure regulation, stroke and several cancers including triple negative breast cancer and glioblastoma. Here, we searched for and characterized novel WNK kinase inhibitors. Methods: We used a ~210,000-compound library in a high-throughput screen, re-acquisition and assay, commercial specificity screens and crystallography to identify WNK-isoform-selective inhibitors. Results: We identified five classes of compounds that inhibit the kinase activity of WNK1: quinoline compounds, halo-sulfones, cyclopropane-containing thiazoles, piperazine-containing compounds, and nitrophenol-derived compounds. The compounds are strongly pan-WNK selective, inhibiting all four WNK isoforms. A class of quinoline compounds was identified that further shows selectivity among the WNK isoforms, being more potent toward WNK3 than WNK1. The crystal structure of the quinoline-derived SW120619 bound to the kinase domain of WNK3 reveals active site binding, and comparison to the WNK1 structure reveals the potential origin of isoform specificity. Discussion: The newly discovered classes of compounds may be starting points for generating pharmacological tools and potential drugs treating hypertension and cancer.

ABORTION AND NEONATAL MORTALITY DUE TO TOXOPLASMA GONDII IN BIGHORN SHEEP (OVIS CANADENSIS).

Low lamb recruitment can be an obstacle to bighorn sheep (Ovis canadensis) conservation and restoration. Causes of abortion and neonate loss in bighorn sheep, which may affect recruitment, are poorly understood. Toxoplasma gondii is a major cause of abortion and stillbirth in domestic small ruminants worldwide, but no reports exist documenting abortion or neonatal death in bighorn sheep attributable to toxoplasmosis. Between March 2019 and May 2021, eight fetal and neonatal bighorn lamb cadavers from four western US states (Idaho, Montana, Nebraska, and Washington) were submitted to the Washington Animal Disease Diagnostic Laboratory for postmortem examination, histologic examination, and ancillary testing to determine the cause of abortion or neonatal death. Necrotizing encephalitis characteristic of toxoplasmosis was identified histologically in six of eight cases, and T. gondii infection was confirmed by PCR in five cases with characteristic lesions. Other lesions attributable to toxoplasmosis were pneumonia (3/5 cases) and myocarditis (2/5 cases). Protozoal cysts were identified histologically within brain, lung, heart, skeletal muscle, adipose tissue, or a combination of samples in all five sheep with PCR-confirmed T. gondii infections. Seroprevalence of T. gondii ranged from 40-81% of adult females sampled in the Washington population in October and November 2018-2021, confirming high rates of exposure before detection of Toxoplasma abortions in this study. Of 1,149 bighorn sheep postmortem samples submitted to Washington Animal Disease Diagnostic Laboratory between January 2000 and May 2021, 21 of which were from fetuses or neonates, a single case of chronic toxoplasmosis was diagnosed in one adult ewe. Recent identification of Toxoplasma abortions in bighorn sheep suggests that toxoplasmosis is an underappreciated cause of reproductive loss. Abortions and neonatal mortalities should be investigated through postmortem and histologic examination, particularly in herds that are chronically small, demographically stagnant, or exhibit reproductive rates lower than expected.

Synthesis and Structural Characterization of Novel Trihalo-sulfone Inhibitors of WNK1.

With No lysine (K) [WNK] kinases are structurally unique serine/threonine protein kinases that have therapeutic potential for blood pressure regulation and cancer. A novel class of trihalo-sulfone compounds was identified by high-throughput screening. Trihalo-sulfone 1 emerged as an effective inhibitor of WNK1 with an IC50 value of 1.6 µM. Herein, we define chemical features necessary for inhibition of WNK1 using chemical synthesis and X-ray crystallography. Analogues that probed the role of specific functional groups to the inhibitory activity were synthesized. X-ray structures of trihalo-sulfone 1 and a second trihalo-sulfone 23 bound to WNK1 revealed active site binding to two of the three previously defined canonical inhibitor binding pockets as well as a novel binding site for the trihalo-sulfone moiety. The elucidation of these novel interaction sites may allow for the strategic design of even more selective and potent WNK inhibitors.

WNK1 collaborates with TGF-ß in endothelial cell junction turnover and angiogenesis.

Angiogenesis is essential for growth of new blood vessels, remodeling existing vessels, and repair of damaged vessels, and these require reorganization of endothelial cell-cell junctions through a partial endothelial-mesenchymal transition. Homozygous disruption of the gene encoding the protein kinase WNK1 results in lethality in mice near embryonic day (E) 12 due to impaired angiogenesis. This angiogenesis defect can be rescued by endothelial-specific expression of an activated form of the WNK1 substrate kinase OSR1. We show that inhibition of WNK1 kinase activity not only prevents sprouting of endothelial cells from aortic slices but also vessel extension in inhibitor-treated embryos ex vivo. Mutations affecting TGF-ß signaling also result in abnormal vascular development beginning by E10 and, ultimately, embryonic lethality. Previously, we demonstrated cross-talk of WNK1 with TGF-ß-regulated SMAD signaling, and OSR1 was identified as a component of the TGF-ß interactome. However, molecular events jointly regulated by TGF-ß and WNK1/OSR1 have not been delineated. Here, we show that inhibition of WNK1 promotes TGF-ß-dependent degradation of the tyrosine kinase receptor AXL, which is involved in TGF-ß-mediated cell migration and angiogenesis. We also show that interaction between OSR1 and occludin, a protein associated with endothelial tight junctions, is an essential step to enable tight junction turnover. Furthermore, we show that these phenomena are WNK1 dependent, and sensitive to TGF-ß. These findings demonstrate intimate connections between WNK1/OSR1 and multiple TGF-ß-sensitive molecules controlling angiogenesis and suggest that WNK1 may modulate many TGF-ß-regulated functions.

WNK1 Enhances Migration and Invasion in Breast Cancer Models.

Metastasis is the major cause of mortality in patients with breast cancer. Many signaling pathways have been linked to cancer invasiveness, but blockade of few protein components has succeeded in reducing metastasis. Thus, identification of proteins contributing to invasion that are manipulable by small molecules may be valuable in inhibiting spread of the disease. The protein kinase with no lysine (K) 1 (WNK1) has been suggested to induce migration of cells representing a range of cancer types. Analyses of mouse models and patient data have implicated WNK1 as one of a handful of genes uniquely linked to invasive breast cancer. Here, we present evidence that inhibition of WNK1 slows breast cancer metastasis. We show that depletion or inhibition of WNK1 reduces migration of several breast cancer cell lines in wound healing assays and decreases invasion in collagen matrices. Furthermore, WNK1 depletion suppresses expression of AXL, a tyrosine kinase implicated in metastasis. Finally, we demonstrate that WNK inhibition in mice attenuates tumor progression and metastatic burden. These data showing reduced migration, invasion, and metastasis upon WNK1 depletion in multiple breast cancer models suggest that WNK1 contributes to the metastatic phenotype, and that WNK1 inhibition may offer a therapeutic avenue for attenuating progression of invasive breast cancers.

Limited inhibition of multiple nodes in a driver network blocks metastasis.

Metastasis suppression by high-dose, multi-drug targeting is unsuccessful due to network heterogeneity and compensatory network activation. Here, we show that targeting driver network signaling capacity by limited inhibition of core pathways is a more effective anti-metastatic strategy. This principle underlies the action of a physiological metastasis suppressor, Raf Kinase Inhibitory Protein (RKIP), that moderately decreases stress-regulated MAP kinase network activity, reducing output to transcription factors such as pro-metastastic BACH1 and motility-related target genes. We developed a low-dose four-drug mimic that blocks metastatic colonization in mouse breast cancer models and increases survival. Experiments and network flow modeling show limited inhibition of multiple pathways is required to overcome variation in MAPK network topology and suppress signaling output across heterogeneous tumor cells. Restricting inhibition of individual kinases dissipates surplus signal, preventing threshold activation of compensatory kinase networks. This low-dose multi-drug approach to decrease signaling capacity of driver networks represents a transformative, clinically relevant strategy for anti-metastatic treatment.

Functional divergence caused by mutations in an energetic hotspot in ERK2.

The most frequent extracellular signal-regulated kinase 2 (ERK2) mutation occurring in cancers is E322K (E-K). ERK2 E-K reverses a buried charge in the ERK2 common docking (CD) site, a region that binds activators, inhibitors, and substrates. Little is known about the cellular consequences associated with this mutation, other than apparent increases in tumor resistance to pathway inhibitors. ERK2 E-K, like the mutation of the preceding aspartate (ERK2 D321N [D-N]) known as the sevenmaker mutation, causes increased activity in cells and evades inactivation by dual-specificity phosphatases. As opposed to findings in cancer cells, in developmental assays in Drosophila, only ERK2 D-N displays a significant gain of function, revealing mutation-specific phenotypes. The crystal structure of ERK2 D-N is indistinguishable from that of wild-type protein, yet this mutant displays increased thermal stability. In contrast, the crystal structure of ERK2 E-K reveals profound structural changes, including disorder in the CD site and exposure of the activation loop phosphorylation sites, which likely account for the decreased thermal stability of the protein. These contiguous mutations in the CD site of ERK2 are both required for docking interactions but lead to unpredictably different functional outcomes. Our results suggest that the CD site is in an energetically strained configuration, and this helps drive conformational changes at distal sites on ERK2 during docking interactions.

Effect of Opioid vs Nonopioid Medications on Pain-Related Function in Patients With Chronic Back Pain or Hip or Knee Osteoarthritis Pain: The SPACE Randomized Clinical Trial.

Importance: Limited evidence is available regarding long-term outcomes of opioids compared with nonopioid medications for chronic pain. Objective: To compare opioid vs nonopioid medications over 12 months on pain-related function, pain intensity, and adverse effects. Design, Setting, and Participants: Pragmatic, 12-month, randomized trial with masked outcome assessment. Patients were recruited from Veterans Affairs primary care clinics from June 2013 through December 2015; follow-up was completed December 2016. Eligible patients had moderate to severe chronic back pain or hip or knee osteoarthritis pain despite analgesic use. Of 265 patients enrolled, 25 withdrew prior to randomization and 240 were randomized. Interventions: Both interventions (opioid and nonopioid medication therapy) followed a treat-to-target strategy aiming for improved pain and function. Each intervention had its own prescribing strategy that included multiple medication options in 3 steps. In the opioid group, the first step was immediate-release morphine, oxycodone, or hydrocodone/acetaminophen. For the nonopioid group, the first step was acetaminophen (paracetamol) or a nonsteroidal anti-inflammatory drug. Medications were changed, added, or adjusted within the assigned treatment group according to individual patient response. Main Outcomes and Measures: The primary outcome was pain-related function (Brief Pain Inventory [BPI] interference scale) over 12 months and the main secondary outcome was pain intensity (BPI severity scale). For both BPI scales (range, 0-10; higher scores = worse function or pain intensity), a 1-point improvement was clinically important. The primary adverse outcome was medication-related symptoms (patient-reported checklist; range, 0-19). Results: Among 240 randomized patients (mean age, 58.3 years; women, 32 [13.0%]), 234 (97.5%) completed the trial. Groups did not significantly differ on pain-related function over 12 months (overall P = .58); mean 12-month BPI interference was 3.4 for the opioid group and 3.3 for the nonopioid group (difference, 0.1 [95% CI, -0.5 to 0.7]). Pain intensity was significantly better in the nonopioid group over 12 months (overall P = .03); mean 12-month BPI severity was 4.0 for the opioid group and 3.5 for the nonopioid group (difference, 0.5 [95% CI, 0.0 to 1.0]). Adverse medication-related symptoms were significantly more common in the opioid group over 12 months (overall P = .03); mean medication-related symptoms at 12 months were 1.8 in the opioid group and 0.9 in the nonopioid group (difference, 0.9 [95% CI, 0.3 to 1.5]). Conclusions and Relevance: Treatment with opioids was not superior to treatment with nonopioid medications for improving pain-related function over 12 months. Results do not support initiation of opioid therapy for moderate to severe chronic back pain or hip or knee osteoarthritis pain. Trial Registration: clinicaltrials.gov Identifier: NCT01583985.

How activating mutations affect MEK1 regulation and function.

The MEK1 kinase directly phosphorylates ERK2, after the activation loop of MEK1 is itself phosphorylated by Raf. Studies over the past decade have revealed a large number of disease-related mutations in the MEK1 gene that lead to tumorigenesis and abnormal development. Several of these mutations result in MEK1 constitutive activity, but how they affect MEK1 regulation and function remains largely unknown. Here, we address these questions focusing on two pathogenic variants of the Phe-53 residue, which maps to the well-characterized negative regulatory region of MEK1. We found that these variants are phosphorylated by Raf faster than the wild-type enzyme, and this phosphorylation further increases their enzymatic activity. However, the maximal activities of fully phosphorylated wild-type and mutant enzymes are indistinguishable. On the basis of available structural information, we propose that the activating substitutions destabilize the inactive conformation of MEK1, resulting in its constitutive activity and making it more prone to Raf-mediated phosphorylation. Experiments in zebrafish revealed that the effects of activating variants on embryonic development reflect the joint control of the negative regulatory region and activating phosphorylation. Our results underscore the complexity of the effects of activating mutations on signaling systems, even at the level of a single protein.

Discovery of novel TAOK2 inhibitor scaffolds from high-throughput screening.

The MAP3K (Mitogen Activated Protein Kinase Kinase Kinase) TAOK2 (Thousand-And-One Kinase 2) is an activator of p38 MAP kinase cascade that is up-regulated in response to environmental stresses. A synthetic lethal screen performed using a NSCLC (non-small cell lung cancer) cell line, and a second screen identifying potential modulators of autophagy have implicated TAOK2 as a potential cancer therapeutic target. Using a 200,000 compound high throughput screen, we identified three specific small molecule compounds that inhibit the kinase activity of TAOK2. These compounds also showed inhibition of autophagy. Based on SAR (structure-activity relationship) studies, we have predicted the modifications on the reactive groups for the three compounds.

Lesbian, Gay, Bisexual, and Transgender Patient Care: Medical Students' Preparedness and Comfort.

UNLABELLED: Phenomenon: Lesbian, gay, bisexual, and transgender (LGBT) individuals face significant barriers in accessing appropriate and comprehensive medical care. Medical students' level of preparedness and comfort caring for LGBT patients is unknown. APPROACH: An online questionnaire (2009-2010) was distributed to students (n = 9,522) at 176 allopathic and osteopathic medical schools in Canada and the United States, followed by focus groups (2010) with students (n = 35) at five medical schools. The objective of this study was to characterize LGBT-related medical curricula, to determine medical students' assessments of their institutions' LGBT-related curricular content, and to evaluate their comfort and preparedness in caring for LGBT patients. FINDINGS: Of 9,522 survey respondents, 4,262 from 170 schools were included in the final analysis. Most medical students (2,866/4,262; 67.3%) evaluated their LGBT-related curriculum as "fair" or worse. Students most often felt prepared addressing human immunodeficiency virus (HIV; 3,254/4,147; 78.5%) and non-HIV sexually transmitted infections (2,851/4,136; 68.9%). They felt least prepared discussing sex reassignment surgery (1,061/4,070; 26.1%) and gender transitioning (1,141/4,068; 28.0%). Medical education helped 62.6% (2,669/4,262) of students feel "more prepared" and 46.3% (1,972/4,262) of students feel "more comfortable" to care for LGBT patients. Four focus group sessions with 29 students were transcribed and analyzed. Qualitative analysis suggested students have significant concerns in addressing certain aspects of LGBT health, specifically with transgender patients. Insights: Medical students thought LGBT-specific curricula could be improved, consistent with the findings from a survey of deans of medical education. They felt comfortable, but not fully prepared, to care for LGBT patients. Increasing curricular coverage of LGBT-related topics is indicated with emphasis on exposing students to LGBT patients in clinical settings.

Map2k4 functions as a tumor suppressor in lung adenocarcinoma and inhibits tumor cell invasion by decreasing peroxisome proliferator-activated receptor γ2 expression.

MAP2K4 encodes a dual-specificity kinase (mitogen-activated protein kinase kinase 4, or MKK4) that is mutated in a variety of human malignancies, but the biochemical properties of the mutant kinases and their roles in tumorigenesis have not been fully elucidated. Here we showed that 8 out of 11 cancer-associated MAP2K4 mutations reduce MKK4 protein stability or impair its kinase activity. On the basis of findings from bioinformatic studies on human cancer cell lines with homozygous MAP2K4 loss, we posited that MKK4 functions as a tumor suppressor in lung adenocarcinomas that develop in mice owing to expression of mutant Kras and Tp53. Conditional Map2k4 inactivation in the bronchial epithelium of mice had no discernible effect alone but increased the multiplicity and accelerated the growth of incipient lung neoplasias induced by oncogenic Kras. MKK4 suppressed the invasion and metastasis of Kras-Tp53-mutant lung adenocarcinoma cells. MKK4 deficiency increased peroxisomal proliferator-activated receptor γ2 (PPARγ2) expression through noncanonical MKK4 substrates, and PPARγ2 enhanced tumor cell invasion. We conclude that Map2k4 functions as a tumor suppressor in lung adenocarcinoma and inhibits tumor cell invasion by decreasing PPARγ2 levels.

Unique MAP Kinase binding sites.

Map kinases are drug targets for autoimmune disease, cancer, and apoptosis-related diseases. Drug discovery efforts have developed MAP kinase inhibitors directed toward the ATP binding site and neighboring "DFG-out" site, both of which are targets for inhibitors of other protein kinases. On the other hand, MAP kinases have unique substrate and small molecule binding sites that could serve as inhibition sites. The substrate and processing enzyme D-motif binding site is present in all MAP kinases, and has many features of a good small molecule binding site. Further, the MAP kinase p38alpha has a binding site near its C-terminus discovered in crystallographic studies. Finally, the MAP kinases ERK2 and p38alpha have a second substrate binding site, the FXFP binding site that is exposed in active ERK2 and the D-motif peptide induced conformation of MAP kinases. Crystallographic evidence of these latter two binding sites is presented.

Clostridium difficile lacks detectable superantigen activity.

Clostridium difficile colitis causes striking leukocytosis. We examined the possibility that toxins A or B, or other nontoxin products of C. difficile, act as superantigens, thereby stimulating leukocytosis. Our results failed to show major histocompatibility complex class II-dependent T lymphocyte proliferation, the hallmark of superantigen activity. Elevated white blood cell counts in C. difficile colitis are probably due to increased generation of cytokines such as interleukin-6 (IL-6) or IL-8.

Crystal structure of the MAP3K TAO2 kinase domain bound by an inhibitor staurosporine.

Mitogen-activated protein kinase (MAPK) signal transduction pathways are ubiquitous in eukaryotic cells, which transfer signals from the cell surface to the nucleus, controlling multiple cellular programs. MAPKs are activated by MAPK kinases [MAP2Ks or MAP/extracellular signal-regulated kinase (ERK) kinases (MEK)], which in turn are activated by MAPK kinase kinases (MAP3Ks). TAO2 is a MAP3K level kinase that activates the MAP2Ks MEK3 and MEK6 to activate p38 MAPKs. Because p38 MAPKs are key regulators of expression of inflammatory cytokines, they appear to be involved in human diseases such as asthma and autoimmunity. As an upstream activator of p38s, TAO2 represents a potential drug target. Here we report the crystal structure of active TAO2 kinase domain in complex with staurosporine, a broad-range protein kinase inhibitor that inhibits TAO2 with an IC50 of 3 mM. The structure reveals that staurosporine occupies the position where the adenosine of ATP binds in TAO2, and the binding of the inhibitor mimics many features of ATP binding. Both polar and nonpolar interactions contribute to the enzyme-inhibitor recognition. Staurosporine induces conformational changes in TAO2 residues that surround the inhibitor molecule, but causes very limited global changes in the kinase. The structure provides atomic details for TAO2-staurosporine interactions, and explains the relatively low potency of staurosporine against TAO2. The structure presented here should aid in the design of inhibitors specific to TAO2 and related kinases.

Docking interactions induce exposure of activation loop in the MAP kinase ERK2.

MAP kinases bind activating kinases, phosphatases, and substrates through docking interactions. Here, we report a 1.9 A crystallographic analysis of inactive ERK2 bound to a "D motif" docking peptide (pepHePTP) derived from hematopoietic tyrosine phosphatase, a negative regulator of ERK2. In this complex, the complete D motif interaction defined by mutagenic analysis is observed, including extensive electrostatic interactions with the "CD" site of the kinase. Large conformational changes occur in the activation loop where the dual phosphorylation sites, which are buried in the inactive form of ERK2, become exposed to solvent in the complex. Similar conformational changes occur in a complex between ERK2 and a MEK2 (MAP/ERK kinase-2)-derived D motif peptide (pepMEK2). D motif peptides are known to bind homologous loci in the MAP kinases p38alpha and JNK1, also inducing conformational changes in these enzymes. However, the binding interactions and conformational changes are unique to each, thus contributing to specificity among MAP kinases.

Long-term follow-up of basal cell carcinomas treated with perilesional interferon alfa 2b as monotherapy.

BACKGROUND: Interferon alfa-2b (IFN) may be used to treat basal cell carcinoma (BCC) as an alternative to surgical or destructive methods. OBJECTIVE: The purpose of this study is to determine the long-term effectiveness of IFN treatment for BCC. METHODS: Fifty patients with 98 biopsy-proven primary superficial and nodular BCCs were treated perilesionally and intradermally with injections of IFN between 1985 and 1992. RESULTS: Clinical cures were noted in 95 of 98 BCCs (51 nodular and 44 superficial), with a mean follow-up period of 10.5 years (9 months to 18.5 years). Of these, 35 of the 50 patients, which would include 68 of the 98 tumors, were followed up for a minimum of 10 years, with an average follow-up of 13.5 years. The 3 lesions requiring further treatment were nodular type BCC. One of these lesions showed no response to treatment, whereas the other two responded, then recurred at 4 and 154 months. Of the 68 tumors followed up for a minimum of 10 years, cure rates were 96%. Overall data analysis using Kaplan-Meier estimates showed 98% success rates at years 5 and 10, and a 96% success rate at year 15. LIMITATIONS: Since 31% of patients could not be evaluated for more than 10-year follow-up, it is possible that the long-term cure rate is lower than that found in those available for evaluation. CONCLUSIONS: Treatment of superficial and nodular BCCs with perilesional IFN is an acceptable treatment that may provide benefits over other treatment modalities depending on patients' individual needs. In addition, on the basis of this study, results of IFN treatment for BCC are comparable to most other methods of tumor destruction.