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The cotranslational misfolding of the cystic fibrosis transmembrane conductance regulator (CFTR) plays a central role in the molecular basis of cystic fibrosis (CF). The misfolding of the most common CF variant (ΔF508) remodels both the translational regulation and quality control of CFTR. Nevertheless, it is unclear how the misassembly of the nascent polypeptide influences the activity of the translation machinery. In this work, we identify a structural motif within the CFTR transcript that stimulates efficient -1 ribosomal frameshifting and triggers the premature termination of translation. Though this motif does not appear to impact the wild-type CFTR interactome, silent mutations that disrupt this RNA structure alter how ΔF508 CFTR interacts with numerous translation and quality control proteins. Moreover, disrupting this RNA structure enhances both the expression and function of ΔF508 CFTR with no impact on wild-type. Finally, we show that disrupting this motif enhances the pharmacological rescue of ΔF508 by Trikafta, which implies ribosomal frameshifting antagonizes the effects of leading CF therapeutics. Together, our results reveal that ribosomal frameshifting selectively reduces the expression and assembly of a misfolded CFTR variant. These findings suggest cotranslational misfolding alters the processivity of translation and potentially the stability of the mRNA transcript through the dynamic modulation of ribosomal frameshifting.

Missense mutations that compromise the plasma membrane expression (PME) of integral membrane proteins are the root cause of numerous genetic diseases. Differentiation of this class of mutations from those that specifically modify the activity of the folded protein has proven useful for the development and targeting of precision therapeutics. Nevertheless, it remains challenging to predict the effects of mutations on the stability and/ or expression of membrane proteins. In this work, we utilize deep mutational scanning data to train a series of artificial neural networks to predict the PME of transmembrane domain variants of G protein-coupled receptors from structural and/ or evolutionary features. We show that our best-performing network, which we term the PME predictor, can recapitulate mutagenic trends within rhodopsin and can differentiate pathogenic transmembrane domain variants that cause it to misfold from those that compromise its signaling. This network also generates statistically significant predictions for the relative PME of transmembrane domain variants for another class A G protein-coupled receptor (β

Cystic fibrosis (CF) is a chronic genetic disease caused by mutations that compromise the expression and/ or function of the cystic fibrosis transmembrane conductance regulator chloride channel (CFTR). Most people with CF harbor a common misfolded CFTR variant (ΔF508), which can be rescued by combination therapies containing “corrector” compounds that restore its expression. Nevertheless, there are over 400 other CF variants that differ in their sensitivity to correctors for reasons that remain unclear. In this work, we utilize deep mutational scanning to quantitatively compare the effects of two FDA-approved correctors on the plasma membrane expression of 129 known CF variants, including 45 that are currently unclassified. Across 67 variants with attenuated expression, we find that VX-661-sensitive variants generally exhibit intermediate expression and feature mutations near its binding pocket in transmembrane domains (TMDs) 1, 2, 3, and 6. VX-445 also primarily rescues variants with intermediate expression but is instead uniquely effective towards mutations near its binding pocket in TMDs 10 & 11. Structural calculations suggest corrector binding provides similar stabilization to both sensitive and insensitive variants. These findings collectively suggest the mutation-specific effects of these compounds depend on the degree of variant destabilization and/ or the timing of cotranslational folding defects. Combining these correctors synergistically rescues variants with deficient and intermediate expression alike, presumably by doubling the total binding energy and suppressing defects at different stages of translation. These results provide an unprecedented overview of the properties of rare CFTR variants and establish new tools for CF pharmacology.

Over 100 mutations in the rhodopsin gene have been linked to a spectrum of retinopathies that include retinitis pigmentosa and congenital stationary night blindness. Though most of these variants exhibit a loss of function, the molecular defects caused by these underlying mutations vary considerably. In this work, we utilize deep mutational scanning to quantitatively compare the plasma membrane expression of 123 known pathogenic rhodopsin variants in the presence and absence of the stabilizing cofactor 9-cis-retinal. We identify 69 retinopathy variants, including 20 previously uncharacterized variants, that exhibit diminished plasma membrane expression in HEK293T cells. 67 of these apparent class II variants exhibit a measurable increase in expression in the presence of 9-cis-retinal. However, the magnitude of the response to this molecule varies considerably across this spectrum of mutations. Evaluation of the observed shifts in relation to thermodynamic estimates for the coupling between binding and folding suggests underlying differences in stability constrains the magnitude of their response to retinal. Nevertheless, estimates from computational modeling suggest many of the least sensitive variants also directly compromise binding. Finally, we evaluate the functional properties of three previous uncharacterized, retinal-sensitive variants (ΔN73, S131P, and R135G) and show that two retain residual function in vitro. Together, our results provide a comprehensive experimental characterization of the proteostatic properties of retinopathy variants and their response to retinal.

Osteosarcoma (OS) is an aggressive malignant bone neoplasm that occurs mostly in the appendicular skeleton of dogs and people. OS is classified based on the presence of malignant stroma and the formation of extracellular matrix into osteoblastic, chondroblastic and fibroblastic forms. This study investigated the correlation between the three histological subtypes of canine OS and clinical outcome. Additionally, we examined whether there was any difference in the immunolabelling of desmin, S100 and neuron-specific enolase (NSE) between the three histological subtypes. Formalin-fixed and paraffin wax-embedded tissues from 87 dogs with primary OS were available for this study. The survival times were correlated with appendicular OS subtypes in dogs that were treated surgically, received adjuvant chemotherapy and had no pulmonary metastasis at the time of diagnosis. Dogs with an appendicular fibroblastic OS had significantly prolonged mean average survival times (546 ± 105 days) in comparison with dogs having appendicular osteoblastic (257 ± 48 days) or appendicular chondroblastic (170 ± 28 days) OS (P = 0.003, Log Rank). The results also revealed that the appendicular chondroblastic subtype is a significant indicator for poor prognosis in dogs compared with the fibroblastic or osteoblastic subtypes (P = 0.006, Cox regression). Moreover, the findings indicated that there was no significant correlation between the localization of desmin, NSE or S100 and histological subtypes. Importantly, dogs with appendicular fibroblastic OS were found to have a better prognosis when compared with dogs with other subtypes. This may suggest that histological subtypes of appendicular OS have diverse behaviour and could be used to categorize patients for risk-based assessment.

More than 80 loss-of-function (LOF) mutations in the SLC6A8 creatine transporter (hCRT1) are responsible for cerebral creatine deficiency syndrome (CCDS), which gives rise to a spectrum of neurological defects, including intellectual disability, epilepsy, and autism spectrum disorder. To gain insight into the nature of the molecular defects caused by these mutations, we quantitatively profiled the cellular processing, trafficking, expression, and function of eight pathogenic CCDS variants in relation to the wild type (WT) and one neutral isoform. All eight CCDS variants exhibit measurable proteostatic deficiencies that likely contribute to the observed LOF. However, the magnitudes of their specific effects on the expression and trafficking of hCRT1 vary considerably, and we find that the LOF associated with two of these variants primarily arises from the disruption of the substrate-binding pocket. In conjunction with an analysis of structural models of the transporter, we use these data to suggest mechanistic classifications for these variants. To evaluate potential avenues for therapeutic intervention, we assessed the sensitivity of these variants to temperature and measured their response to the proteostasis regulator 4-phenylbutyrate (4-PBA). Only one of the tested variants (G132V) is sensitive to temperature, though its response to 4-PBA is negligible. Nevertheless, 4-PBA significantly enhances the activity of WT hCRT1 in HEK293T cells, which suggests it may be worth evaluating as a therapeutic for female intellectual disability patients carrying a single CCDS mutation. Together, these findings reveal that pathogenic SLC6A8 mutations cause a spectrum of molecular defects that should be taken into consideration in future efforts to develop CCDS therapeutics.

To describe the clinical characteristics, perioperative protocols, and outcomes in dogs diagnosed with ventricular fibrillation (VF) while undergoing pericardiectomy.Retrospective, multi-institutional study.Sixteen client-owned dogs.Cases were accrued through a listserve request posted to 3 subspecialty veterinary societies. Dogs were included if they developed VF during a pericardiectomy performed through an open or thoracoscopic approach. Data collected included signalment, history and physical examination, surgical approach, histopathology, treatment, and outcome.Indications for pericardiectomy included idiopathic chylothorax (n = 7), neoplasia (4), idiopathic pericardial effusion (4), and foreign body granuloma (1). Surgical approaches included thoracoscopy (12), intercostal thoracotomy (3) and median sternotomy (1). Electrosurgical devices were used to complete at least part of the pericardiectomy in 15 of 16 dogs. Ventricular fibrillation appeared to be initiated during electrosurgical use in 8/15 dogs. However, in 5/15 dogs it was not obviously associated with electrosurgical use. In 3/16 dogs the timing of initiation of VF was unclear. In 7/16 dogs, cardiac arrhythmias were noted prior to the development of VF. Fourteen of 16 dogs died from intraoperative VF.In most dogs ventricular fibrillation was a fatal complication of pericardiectomy. Ventricular fibrillation might be associated with the use of electrosurgical devices and cardiac manipulation during pericardiectomy although a causal link could not be established from the data in this study.Surgeons must be aware of the risk of VF during pericardial surgery. Electrosurgery might need to be used judiciously during pericardiectomy, particularly in dogs exhibiting cardiac arrythmias.

Missense mutations that compromise the plasma membrane expression (PME) of integral membrane proteins (MPs) are the root cause of numerous genetic diseases. Differentiation of this class of mutations from those that specifically modify the activity of the folded protein has proven useful for the development and targeting of precision therapeutics. Nevertheless, it remains challenging to predict the effects of mutations on the stability and/ or expression of MPs. In this work, we utilize deep mutational scanning data to train a series of artificial neural networks to predict the effects of mutations on the PME of the G-protein coupled receptor (GPCR) rhodopsin from structural and/ or evolutionary features. We show that our best performing network, which we term PMEpred, can differentiate pathogenic rhodopsin variants that induce misfolding from those that primarily compromise signaling. This network also generates statistically significant predictions for the effects of mutations on the PME of another GPCR (β2 adrenergic receptor) but not for an unrelated voltage-gated potassium channel (KCNQ1). Notably, our analyses of these networks suggest structural features alone are generally sufficient to recapitulate the observed mutagenic trends. Moreover, our findings imply that networks trained in this manner may be generalizable to proteins that share a common fold. Implications of our findings for the design of mechanistically specific genetic predictors are discussed.

CASE DESCRIPTION An 8-year-old spayed female Shih Tzu crossbreed dog (dog 1) and a 13-year-old neutered male Miniature Fox Terrier (dog 2) were evaluated for removal of neoplasms involving both the frontal lobe and olfactory bulb. CLINICAL FINDINGS Physical examination revealed decreased menace response and behavioral changes in both dogs. For dog 1, neuroanatomic localization of the lesion was the left forebrain region; for dog 2, neuroanatomic localization of the lesion was the right forebrain region. Both dogs underwent CT, and dog 1 also underwent MRI. Results of diagnostic imaging were consistent with frontal lobe and olfactory bulb neoplasia in both cases. Dog 1 had lysis of the frontal bone adjacent to the neoplasm. TREATMENT AND OUTCOME Both dogs underwent a transorbital craniectomy to permit surgical tumor removal. Dog 1 was discharged from the hospital 48 hours after surgery, at which time its mentation and cranial nerve examination findings were considered normal. Dog 2 developed neurologic deterioration after surgery but was ultimately discharged from the hospital after 72 hours, at which time its mentation appeared normal. CLINICAL RELEVANCE The transorbital approach to the cranium provided excellent access to facilitate removal of frontal lobe and olfactory bulb neoplasms in these 2 dogs.

OBJECTIVE To describe the signalment, clinical signs, biological behavior, and outcome for cats with apocrine gland anal sac adenocarcinoma (AGASACA) that underwent surgical excision. DESIGN Retrospective case series. ANIMALS 30 client-owned cats. PROCEDURES Databases of 13 Veterinary Society of Surgical Oncology member–affiliated institutions were searched for records of cats with a histologic diagnosis of AGASACA that underwent tumor excision. For each cat, information regarding signalment, clinical signs, diagnostic test results, treatment, and outcome was extracted from the medical record. The Kaplan-Meier method was used to determine median time to local recurrence (TLR), disease-free interval (DFI), and survival time. Cox regression was used to identify factors associated with TLR, DFI, and survival time. RESULTS Perineal ulceration or discharge was the most common clinical sign in affected cats. Eleven cats developed local recurrence at a median of 96 days after AGASACA excision. Incomplete tumor margins and a high nuclear pleomorphic score were risk factors for local recurrence. Nuclear pleomorphic score was negatively associated with DFI. Local recurrence and a high nuclear pleomorphic score were risk factors for death. Median DFI and survival time were 234 and 260 days, respectively. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that, in cats, perineal ulceration or discharge should raise suspicion of AGASACA and prompt rectal and anal sac examinations. Local recurrence was the most common life-limiting event in cats that underwent surgery for treatment of AGASACA, suggesting that wide margins should be obtained whenever possible during AGASACA excision. Efficacy of chemotherapy and radiation therapy for treatment of cats with AGASACA requires further investigation. (J Am Vet Med Assoc 2019;254:716–722)

Programmed ribosomal frameshifting (PRF) is a translational recoding mechanism that enables the synthesis of multiple polypeptides from a single transcript. During translation of the alphavirus structural polyprotein, the efficiency of −1PRF is coordinated by a ‘slippery’ sequence in the transcript, an adjacent RNA stem–loop, and a conformational transition in the nascent polypeptide chain. To characterize each of these effectors, we measured the effects of 4530 mutations on −1PRF by deep mutational scanning. While most mutations within the slip-site and stem–loop reduce the efficiency of −1PRF, the effects of mutations upstream of the slip-site are far more variable. We identify several regions where modifications of the amino acid sequence of the nascent polypeptide impact the efficiency of −1PRF. Molecular dynamics simulations of polyprotein biogenesis suggest the effects of these mutations primarily arise from their impacts on the mechanical forces that are generated by the translocon-mediated cotranslational folding of the nascent polypeptide chain. Finally, we provide evidence suggesting that the coupling between cotranslational folding and −1PRF depends on the translation kinetics upstream of the slip-site. These findings demonstrate how −1PRF is coordinated by features within both the transcript and nascent chain., Graphical Abstract Graphical AbstractNascent chain interactions that occur during translocon-mediated cotranslational folding generate mechanical forces that influence the decoding of a slippery sequence within the subgenomic RNA of the alphavirus structural polyprotein.

OBJECTIVE To investigate putative associations between oral melanoma size and variables of histologic grade such as mitotic index, nuclear atypia, junctional activity, ulceration, lymphatic invasion, and degree of pigmentation. SAMPLE 59 samples of oral melanomas from dogs sourced from 6 diagnostic laboratories within Australia. PROCEDURES The size of each melanoma was microscopically measured, and each sample was evaluated for variables of histologic grade including mitotic index, nuclear atypia, junctional activity, ulceration, lymphatic invasion, and degree of pigmentation by a veterinary pathologist. The association between tumor size and histologic outcomes was then statistically evaluated. RESULTS A significant relationship was identified between the size of oral melanomas and a single variable of histologic grade, lymphatic invasion, with larger tumors more likely to show lymphatic invasion. Further analysis revealed 2 applicable size thresholds for different clinical scenarios. Results indicated lymphatic invasion can confidently be ruled out for tumors < 6.5 mm in diameter (100% sensitivity) and ruled in for tumors ≥ 24.5 mm in diameter (100% specificity). CONCLUSIONS AND CLINICAL RELEVANCE An association was found for oral melanomas of dogs between tumor size and lymphatic invasion.

More than 80 loss-of-function (LOF) mutations in the

Sequence constraints associated with topological energetics restrict the evolution and mutational tolerance of membrane proteins., Membrane proteins must balance the sequence constraints associated with folding and function against the hydrophobicity required for solvation within the bilayer. We recently found the expression and maturation of rhodopsin are limited by the hydrophobicity of its seventh transmembrane domain (TM7), which contains polar residues that are essential for function. On the basis of these observations, we hypothesized that rhodopsin’s expression should be less tolerant of mutations in TM7 relative to those within hydrophobic TM domains. To test this hypothesis, we used deep mutational scanning to compare the effects of 808 missense mutations on the plasma membrane expression of rhodopsin in HEK293T cells. Our results confirm that a higher proportion of mutations within TM7 (37%) decrease rhodopsin’s plasma membrane expression relative to those within a hydrophobic TM domain (TM2, 25%). These results in conjunction with an evolutionary analysis suggest solvation energetics likely restricts the evolutionary sequence space of polar TM domains.

Membrane protein variants with diminished conformational stability often exhibit enhanced cellular expression at reduced growth temperatures. The expression of "temperature-sensitive" variants is also typically sensitive to corrector molecules that bind and stabilize the native conformation. There are many examples of temperature-sensitive rhodopsin variants, the misfolding of which is associated with the molecular basis of retinitis pigmentosa. In this work, we employ deep mutational scanning to compare the effects of reduced growth temperature and 9-cis-retinal, an investigational corrector, on the plasma membrane expression of 700 rhodopsin variants in HEK293T cells. We find that the change in expression at reduced growth temperatures correlates with the response to 9-cis-retinal among variants bearing mutations within a hydrophobic transmembrane domain (TM2). The most sensitive variants appear to disrupt a native helical kink within this transmembrane domain. By comparison, mutants that alter the structure of a polar transmembrane domain (TM7) exhibit weaker responses to temperature and retinal that are poorly correlated. Statistical analyses suggest that this observed insensitivity cannot be attributed to a single variable, but likely arises from the composite effects of mutations on the energetics of membrane integration, the stability of the native conformation, and the integrity of the retinal-binding pocket. Finally, we show that the characteristics of purified temperature- and retinal-sensitive variants suggest that the proteostatic effects of retinal may be manifested during translation and cotranslational folding. Together, our findings highlight several biophysical constraints that appear to influence the sensitivity of genetic variants to temperature and small-molecule correctors.

The substituted amphetamine, 3,4-methylenedioxy-methamphetamine (MDMA, ecstasy), is a widely used drug of abuse that induces non-exocytotic release of serotonin, dopamine, and norepinephrine through their cognate transporters as well as blocking the reuptake of neurotransmitter by the same transporters. The resulting dramatic increase in volume transmission and signal duration of neurotransmitters leads to psychotropic, stimulant, and entactogenic effects. The mechanism by which amphetamines drive reverse transport of the monoamines remains largely enigmatic, however, promising outcomes for the therapeutic utility of MDMA for post-traumatic stress disorder and the long-time use of the dopaminergic and noradrenergic-directed amphetamines in treatment of attention-deficit hyperactivity disorder and narcolepsy increases the importance of understanding this phenomenon. Previously, we identified functional differences between the human and Drosophila melanogaster serotonin transporters (hSERT and dSERT, respectively) revealing that MDMA is an effective substrate for hSERT but not dSERT even though serotonin is a potent substrate for both transporters. Chimeric dSERT/hSERT transporters revealed that the molecular components necessary for recognition of MDMA as a substrate was linked to regions of the protein flanking transmembrane domains (TM) V through IX. Here, we performed species-scanning mutagenesis of hSERT, dSERT and C. elegans SERT (ceSERT) along with biochemical and electrophysiological analysis and identified a single amino acid in TM10 (Glu394, hSERT; Asn484, dSERT, Asp517, ceSERT) that is primarily responsible for the differences in MDMA recognition. Our findings reveal that an acidic residue is necessary at this position for MDMA recognition as a substrate and serotonin releaser.

OBJECTIVE To evaluate adverse events and outcomes in dogs with appendicular osteosarcoma treated with limb amputation followed by a single SC infusion of carboplatin. ANIMALS 45 client-owned dogs with appendicular osteosarcoma treated with limb amputation and SC infusion of carboplatin between January 1, 2006, and January 15, 2017. PROCEDURES Medical records were reviewed, and data collected included signalment, tumor location, treatment, results of clinicopathologic analyses and diagnostic imaging, adverse effects of chemotherapy, metastasis-free interval, survival time, and communications with owners and referring veterinarians. Findings were evaluated with the Kaplan-Meier survival analysis and Mantel-Haenszel log-rank test. RESULTS 45 dogs were identified that met the inclusion criteria (12 of the 45 dogs had been reported in a previous case series). No dogs had pulmonary metastases detectable by CT or radiography before treatment. All dogs completed the protocol as planned. Median survival time (MST) was 196 days; metastasis-free interval was 197 days. Three of the 45 (7%) dogs required hospitalization for gastrointestinal signs related to chemotherapy. There were no chemotherapy-related deaths. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that although treatment with SC infusion of carboplatin was well tolerated, the MST for dogs in the present study was similar to reported MSTs in dogs with appendicular osteosarcoma treated with limb amputation alone and was in the lower range of historically reported survival times for dogs receiving IV adjunctive chemotherapy. Therefore, we could not recommend this protocol of SC infusion of carboplatin but recommended that protocols with IV administration of carboplatin be used instead.

Osteosarcoma (OS) originates from bone-forming mesenchymal cells and represents one of the primary bone tumours. It is the most common primary bone tumour in dogs and man. The characterization of an appropriate natural disease animal model to study human OS is essential to elucidate the pathogenesis of the disease. This study aimed to validate canine OS as a model for the human disease by evaluating immunohistochemically the expression of markers known to be important in human OS. The immunohistochemical panel included vimentin, alkaline phosphatase (ALP), desmin, S100, neuron-specific enolase (NSE), runt-related transcription factor 2 (Runx2) and bone morphogenetic protein 4 (BMP4). Immunohistochemistry was conducted on formalin-fixed, paraffin wax-embedded tissue sections from 59 dogs with confirmed primary OS. Vimentin, ALP, Runx2 and BMP4 were highly expressed by all tumours, while desmin, S100 and NSE were expressed variably. The findings were similar to those described previously for human OS and suggest that canine OS may represent a useful model for the study of the human disease.

Objective—To evaluate outcomes of radical excision of feline injection-site sarcomas (ISS) via assessment of local recurrence and metastasis rates, survival times, and complications associated with surgery. Design—Retrospective case series. Animals—91 cats with ISS. Procedures—Medical records of cats that had radical excision of ISS without adjunctive treatment were reviewed. Information extracted included sex, type of surgical procedure, histologic tumor grade, tumor diameter, time from tumor detection to definitive surgery, complications associated with surgery, whether tumors recurred locally or metastasized, and survival times. Diagnosis of ISS was histologically confirmed, and additional follow-up was performed. Results—Overall median survival time was 901 days. Thirteen of 91 (14%) cats had local tumor recurrence; 18 (20%) cats had evidence of metastasis after surgery. Median survival time of cats with and without recurrence was 499 and 1,461 days, respectively. Median survival time of cats with and without metastasis was 388 and 1,528 days, respectively. Tumor recurrence and metastasis were significantly associated with survival time, whereas other examined variables were not. Major complications occurred in 10 cats, including 7 with incisional dehiscence. Conclusions and Clinical Relevance—Radical excision of ISS resulted in a metastasis rate similar to rates reported previously; the local recurrence rate appeared to be substantially less than rates reported after less aggressive surgeries, with or without adjuvant treatment. Major complication rates were similar to rates reported previously after aggressive surgical resection of ISS. Radical excision may be a valuable means of attaining an improved outcome in the treatment of feline ISS.

A 6-year-old, spayed female Labrador retriever was presented 48 hours after an intestinal resection and anastomosis for management of a small intestinal foreign body. Abdominal ultrasound confirmed the presence of peritoneal effusion. Cytology of fluid collected by abdominocentesis revealed a large number of degenerate neutrophils with intracellular cocci. A diagnosis of septic peritonitis was made, presumably because of dehiscence of the anastomosis. Upon repeat exploratory celiotomy, the intestinal anastomosis (located 4 cm orad to the cecum) was found to be leaking intestinal contents into the abdomen. The distal ileum, cecum, and proximal colon were resected. An end-to-end, ileocolic anastomosis was performed and subsequently exteriorized into the subcutaneous space via a paramedian incision through the abdominal wall. The anastomosis was inspected daily for 4 days before it was returned to the abdomen and the subcutaneous defect was closed. Serial cytology of the peritoneal fluid, which was performed during this 4-day postoperative period, confirmed progressive resolution of peritonitis. The dog was discharged from the hospital 2 days following return of the anastomosis into the abdomen. Externalized intestinal anastomosis is used with good success in human medicine for repair of colonic injuries. In this case, externalization of the anastomosis permitted healing of the intestinal anastomosis in an environment isolated from the detrimental effects created by septic peritonitis. In addition, direct visualization of the anastomosis allowed assessment of healing. To our knowledge, this procedure has not been previously reported in companion animals.

Neurotransmitter transporters are responsible for removal of biogenic amine neurotransmitters after release into the synapse. These transporters are the targets for many clinically relevant drugs, such as antidepressants and psychostimulants. A high resolution crystal structure for the monoamine transporters has yet to be solved. We have developed a homology model for the serotonin transporter (SERT) based on the crystal structure of the leucine transporter (LeuT(Aa)) from Aquifex aeolicus. The objective of the present studies is to identify the structural determinants forming the entrance to the substrate permeation pathway based on predictions from the SERT homology model. Using the substituted cysteine accessibility method, we identified residues predicted to reside at the entrance to the substrate permeation pathway that were reactive with methanethiosulfonate (MTS) reagents. Of these residues, Gln(332) in transmembrane helix (TMH) VI was protected against MTS inactivation in the presence of serotonin. Surprisingly, the reactivity of Gln(332) to MTS reagents was enhanced in the presence of cocaine. Bifunctional MTS cross-linkers also were used to examine the distances between helices predicted to form the entrance into the substrate and ion permeation pathway. Our studies suggest that substrate and ligand binding may induce conformational shifts in TMH I and/or VI, providing new opportunities to refine existing homology models of SERT and related monoamine transporters.

Limb-salvage surgery and adjuvant chemotherapy are performed as a treatment of appendicular osteosarcoma in dogs. Approximately 50% of dogs that undergo limb-salvage surgery develop postoperative surgical wound infections. Postoperative surgical infections may affect survival in cancer patients. The purposes of this study were to examine the effect of surgical wound infection on survival, local recurrence, and metastasis in relation to other prognostic factors for dogs with spontaneous osteosarcoma treated with limb-salvage surgery. Forty-seven client-owned dogs with osteosarcoma of the distal radius were treated with limb-salvage surgery and adjuvant chemotherapy—either carboplatin or carboplatin and doxorubicin. Hazard ratios were estimated by using the Cox proportional hazard model, and survival functions were estimated by using the Kaplan-Meier product-limit life-table method. Of the 47 dogs in this study, 32 (68%) developed a postoperative wound infection. Infection, dog weight, and extent of the primary tumor (percentage of length) significantly affected survival, and infection and percentage of length significantly affected time to metastasis. None of the variables considered in this study affected local recurrence. Dogs that were diagnosed with an infection were less likely to die (hazard ratio, .446), and dogs with greater body weight and greater percentage length involvement were more likely to die (hazard ratios of 3.37 and 3.66, respectively). In dogs with osteosarcoma treated with limb-salvage surgery, infection has a positive influence on survival, as does a smaller initial length of radius involved and lower body weight.

Objective—To determine outcomes for dogs with soft tissue sarcomas in the distal aspects of the limbs that underwent second intention healing after wide excision (2-cm lateral surgical margins and a margin 1 fascial plane deep) of the tumors. Design—Retrospective case series. Animals—31 dogs with soft tissue sarcomas in the distal aspects of the limbs that underwent second intention healing following wide local excision of their tumors. Procedures—Tumors were excised with 2-cm lateral margins and a margin 1 fascial plane deep to tumors. Wounds healed by means of second intention. Time to healing, complications during healing, and information regarding tumor recurrence were recorded. Results—All tumors were excised with histologically tumor-free margins. Twenty-nine (93.5%) wounds healed completely by second intention (median time, 53 days). Two (6.5%) dogs required free skin graft procedures to facilitate healing. Complications during open wound management developed for 7 (22.6%) dogs. Long-term complications were detected for 8 (25.8%) dogs, including intermittent epidermal disruption (5/31 [16.1%]) and wound contracture (3/31 [9.7%]). All complications were managed conservatively. Local tumor recurrence was detected for 1 (3.2%) dog. Median follow-up time was 980 days (range, 380 to 2,356 days). No patients died because of tumor-related causes. Conclusions and Clinical Relevance—Results of this study indicated second intention healing of large wounds in the distal aspects of the limbs was complete and typically without complications for dogs that underwent wide excision of soft tissue sarcomas. Wide local excision of soft tissue sarcomas in the distal aspects of the limbs with 2-cm lateral margins and margins 1 fascial plane deep to the tumors provided excellent long-term local tumor control.

The current study evaluated the effect of low-temperature hydrogen peroxide gas plasma sterilization on the osteoinductive capability of human demineralized bone matrix using a rat model. Twelve athymic rats received three separate implants consisting of steam-sterilized demineralized bone matrix (negative control), sterile-harvest demineralized bone matrix (positive control), and gas-plasma-sterilized demineralized bone matrix. A demineralized bone matrix pellet from each sterilization group was placed individually into one of three separate soft tissue pockets created in the epaxial musculature of each rat. All 12 rats were euthanized 9 weeks after implantation. Each implantation site was removed along with 0.5-cm normal tissue around the implant. Histologic examination was done on each implant site to determine the presence or absence of new bone, cartilage, or bone marrow elements. All 12 sterile harvest demineralized bone matrix sites histologically contained new bone elements, whereas none of the negative control or gas plasma sterilized demineralized bone matrix sites contained any of these same elements. The results of this study indicate that demineralized bone matrix sterilized with low-temperature, gas-plasma sterilization loses its osteoinductive capacity in a manner similar to that of steam-sterilized demineralized bone matrix, making low-temperature, gas- plasma sterilization unsuitable as a method of secondary sterilization of demineralized bone matrix.

The biocompatibility, release and efficacy of gentamicin released from poly(lactic acid) (PLA) beads in an osteomyelitis model were evaluated. Microsphere particles of PLA containing gentamicin were compressed into 2.5 mm beads. Osteomyelitis was induced in the tibia by a gentamicin resistant Staphylococcus aureus in 32 Sprague-Dawley rats. Following debridement at 8 weeks, rats were randomized into four groups: 1) gentamicin-impregnated PLA bead implant, 2) PLA bead without gentamicin, 3) once-daily gentamicin injections (5 mg/kg) subcutaneously for 14 days, and 4) no treatment (control). Rats were sacrificed at 7 weeks, and the tibias were sampled for quantitative culture, histologic scoring and gentamicin concentration. Radiography and culture in 31 of the 32 rats confirmed osteomyelitis. A low level of gentamicin was measured in the serum of group 1 rats up to day 42. Gentamicin was not detectable in bone samples. There was no significant difference in radiographic scores, histologic scores or quantit...

The biocompatibility and levels of gentamicin released from a poly(lactic acid) (PLA) polymer system in the serum, milk and tissue of a food animal model were determined. Microsphere particles of PLA containing 6.7% (w/w) gentamicin were compressed into 5 mm beads onto suture. Three groups of three cull ewes each were implanted with a bead string of PLA/gentamicin in one caudal thigh (muscle) and PLA control beads in the opposite thigh for 2, 4 and 6 months. Milk and serum samples were obtained at regular intervals until sacrifice. Following sacrifice, implant sites, kidney, liver and skeletal muscle were sampled for gentamicin concentration and histologic evaluation. Serum gentamicin peaked by day 1 (up to 0.7 μg/mL) and decreased to near zero by day 16 in all groups. Milk levels were measurable (>0.01 μg/mL) only on 2 days, in one sheep. Kidney gentamicin levels approached 2 ppm, and liver gentamicin levels approached 1 ppm in the 6-month group. A mild to moderate foreign body reaction was seen histologically in all PLA/gentamicin and PLA control implantation sites. Gentamicin released from this implanted polymer system produced low serum and milk concentrations, but developed renal accumulation beyond tolerated levels (30 ppb). Polymer/antibiotic delivery systems show promise for the treatment of local infections in veterinary patients.

Soft tissue sarcomas (STS) are mesenchymal tumors arising from connective tissue elements and are grouped together based on a common biologic behavior. The most common histologic types include malignant peripheral nerve sheath tumors (schwannoma and neurofibrosarcoma) "hemangiopericytoma," fibrosarcoma, and malignant fibrous histiocytoma. These tumors are relatively slow growing yet locally invasive with a high rate of recurrence following conservative management. Appropriate preoperative planning and aggressive surgical resection often result in long-term remission or cure. Identification and evaluation of resection margins are paramount in appropriate case management. The addition of radiotherapy after surgical resection can aid in remission for incompletely resected masses. Systemic chemotherapy for STS should be considered for high-grade tumors with a moderate metastatic potential. Potential prognostic factors include grade, resection margins, size, location, histologic type, and previous treatment, with grade and margins being the most important. Tumor types classified as STS that differ slightly in their presentation or treatment, including synovial cell sarcoma, rhabdomyosarcoma, liposarcoma, and vaccine-associated STS in cats, are discussed. Soft tissue sarcomas can be a frustrating disease to treat, but adherence to solid surgical oncology principles can greatly increase the odds of good disease control.

A 4-month-old female kitten presented with chronic lower urinary tract signs and Escherichia coli cystitis, and was diagnosed with urinary bladder malakoplakia based upon histopathology. The kitten was treated with a prolonged antibiotic course and the malakoplakia resolved. Malakoplakia is a chronic granulomatous reaction characterized by the formation of Michaelis–Gutman bodies within von Hansemann macrophages. It is well described in humans, but has never been documented in a living veterinary patient. This case report describes the first successful treatment of malakoplakia in veterinary medicine.

Controlled cross circulation (CCC) was performed in six pairs of dogs for 45 minutes with aortic cross clamping and cardioplegia. Data were collected in donor dogs at 10 minute intervals three times before, three times during, and three times after CCC and included arterial blood pressure, pulmonary capillary wedge pressure (PCWP), central venous pressure (CVP), cardiac index (CI), heart rate (HR), blood gas analysis, temperature, maximum rate of rise of left ventricular pressure dP/dt max/End diastolic volume (EDV), blood volume (BV), complete blood count (CBC) and activated clotting times (ACT). Pulse pressure (PP), systemic vascular resistance (SVR), oxygen delivery (DO2), and left ventricular cardiac work (LVCW) were calculated. Arterial blood pressure, CVP, blood gas analysis, temperature, BV, CBC, and ACT were measured in recipient dogs. During CCC, donor hemodynamic changes resembled those observed in models of acute onset arteriovenous fistulas. Insidious BV shifts can occur despite the use of occlusive roller pumps. After CCC, donor hemodynamics resembled acute blood loss, characterized by decreases in mean arterial pressure (MAP), CVP, PCWP, and CI, and increases in SVR and dP/dt max/EDV. These changes were probably caused by pump imbalance and BV shift to the recipient dog.

Objective—To evaluate adverse effects and survival times in dogs with osteosarcoma that received a single SC infusion of carboplatin as adjunctive chemotherapeutic treatment following limb amputation or limb-sparing surgery. Design—Retrospective case series. Animals—17 client-owned dogs with spontaneously occurring osteosarcoma. Procedures—Medical records of dogs that underwent limb amputation or limb-sparing surgery followed by a single continuous SC infusion of carboplatin (total dose, 300 mg/m2 infused over 3, 5, or 7 days) were evaluated. Signalment, tumor location, type of surgery (amputation or limb-sparing), duration of carboplatin infusion, results of hematologic and serum biochemical analyses, and adverse effects were recorded. Kaplan-Meier survival analysis was performed. Results—Median survival time for all dogs was 365 days. Nine dogs had adverse bone marrow–related (hematologic) effects, 1 had adverse gastrointestinal effects, and 7 had infections at the surgical site. No significant differences were detected in survival times of dogs grouped according to tumor location, type of surgery, duration of carboplatin infusion, or development of postoperative infection. Conclusions and Clinical Relevance—Median survival time and adverse effects in dogs with osteosarcoma that received a single SC infusion of carboplatin over a 3-, 5-, or 7-day period as adjunctive treatment following limb amputation or limb-sparing surgery were comparable to those of previously reported chemotherapy protocols requiring IV drug administration over several weeks. Further investigation is needed to evaluate the efficacy of this protocol as adjunctive treatment for osteosarcoma and other tumors in dogs.

The serotonin transporter (SERT) regulates the serotonin concentration in the synapse and is a target of several antidepressant and psychostimulant drugs. Previous work suggested that the middle transmembrane helices (TMHs) of the biogenic amine transporters (TMHs) play a role in substrate and ion recognition. We focused our present studies on exploring the role of TMH VII in transporter function and ion recognition. Residues divergent between human SERT and Drosophila SERT (hSERT and dSERT, respectively) were identified and mutated in hSERT to the corresponding identity in dSERT. hSERT mutants V366S, M370L, S375A, and T381S exhibited a decrease in transport capacity. To further explore the role of these residues in the transport process, we generated cysteine mutants at multiple positions. Pretreatment with [2-(trimethylammonium)ethyl] methanethiosulfonate (MTSET) caused a decrease in transport of [(3)H]5-HT in the V366C and M370C mutants. The hSERT V366S, M370L, and M370C mutations also altered the sodium and chloride dependence for substrate transport. Interpretation of our results in the context of a homology model of SERT based on the crystal structure of the Aquifex aeolicus leucine transporter suggests flexibility in the conformation of TMH VII that impacts ion dependence and substrate transport.

Mouse embryonic fibroblast (MEF) cells prepared from transgenic mice overexpressing a cancer-specific and growth-related cell surface NADH oxidase with protein disulfide-thiol interchange activity grew at rates approximately twice those of wild-type embryonic fibroblast cells. Growth of transgenic MEF cells overexpressing tNOX was inhibited by low concentrations of the green tea catechin (-)-epigallocatechin-3-gallate (EGCg) or the synthetic isoflavene phenoxodiol. Both are putative tNOX-targeted inhibitors with anti-cancer activity. With both EGCg and phenoxodiol, growth inhibition was followed after about 48 h by apoptosis. Growth of wild-type mouse fibroblast cells from the same strain was unaffected by EGCg and phenoxodiol and neither compound induced apoptosis even at concentrations 100-1,000-fold higher than those that resulted in apoptotic death in the transgenic MEF cells. The findings validate earlier reports of evidence for tNOX presence as contributing to unregulated growth of cancer cells as well as the previous identification of the tNOX protein as the molecular target for the anti-cancer activities attributed to both EGCg and phenoxodiol. The expression of tNOX emerges as both necessary and sufficient to account for the cancer cell-specific growth inhibitions by both EGCg and phenoxodiol. J. Cell. Biochem. 101: 295-306, 2007. © 2006 Wiley-Liss, Inc.

Objective— To compare the accuracy of radiography, nuclear scintigraphy, and histopathology for determining the proximal extent of distal radius osteosarcoma in dogs. Study Design— Retrospective clinical study. Sample Population— Twenty client-owned dogs taken to Colorado State University with osteosarcoma of the distal radius. Methods— Medical records of 20 dogs with confirmed osteosarcoma that underwent a limb-salvage procedure were reviewed. Measurements were performed directly from the lateral view of each radius, from both the scintigram and the radiograph, to determine the length of the radius and the distance from the proximal extent of the tumor to the distal radiographic or scintographic extent of the radius. The ratio of distal radial involvement to total radius length was calculated. A similar ratio was also determined using the macroslide, which included the entire portion of bone that was excised during the limb-salvage procedure. All 3 methods of measurement were compared. Results— Nuclear scintigraphy significantly overestimated tumor length when compared with macroslide specimen measurements. Radiography also overestimated tumor length, but these results were not significantly different from macroslide specimen measurements. Conclusions— Both radiography and nuclear scintigraphy overestimate the extent of distal radiaus osteosarcoma in dogs when compared with histopathologic macroslides of the same lesions. Nuclear scintigraphy overestimated tumor extent to a larger degree than did radiography. Clinical Relevance— Although radiography is a more accurate method of measurement of the extent of distal radius osteosarcoma, because nuclear scintigraphy overestimates tumor length to a greater degree, scintigraphy may provide a larger margin of safety for determining the site of proximal osteotomy during a limb-salvage procedure. However, caution should be taken when utilizing scintigraphy, because this method may overestimate length of radius involved to such an extent as to cause the surgeon to believe that a patient is not a suitable limb-salvage candidate.

Twenty-four adrenocortical tumors were surgically removed from 21 dogs. Histopathological examination confirmed 18 carcinomas and six adenomas. Four dogs died in the perioperative period. Fifteen of the 17 dogs that survived the perioperative period had long-term resolution of their clinical signs. Two dogs with incompletely resected tumors were treated with mitotane to control their clinical signs. Overall median Kaplan-Meier life-table survival for dogs with carcinomas was 778 days (range, one to 1,593 days). Median survival for dogs with adenomas was not reached (range, 11 to 730 days). Histopathological diagnosis, histopathological cellular features, age of the dog, and tumor size were not prognostic of outcome.

Objective—To describe function and identify factors that affect outcome in dogs undergoing limb salvage surgery for osteosarcoma (OS) of the proximal humerus. Study Design—A retrospective study of dogs in which OS of the proximal humerus was treated with limb salvage surgery. Animals—17 client-owned dogs. Methods: Records were analyzed for functional outcome, recurrence, metastasis, and survival. Results—Outcome was good to excellent in 12% of dogs. Recurrence, metastasis, and survival were significantly affected by completeness of surgical margins. Double plating of the distal allograft-host junction significantly reduced frequency of biomechanical failure. Conclusions—Limb salvage surgery for OS of the proximal humerus did not result in acceptable function and was fraught with postoperative complications. Outcome was significantly affected by completeness of surgical margins. Clinical Relevance—Limb salvage surgery for OS of the proximal humerus in dogs cannot be recommended until improvement in functional outcome and reduction in postoperative complications can be achieved. The dependence of outcome on completeness of surgical margins supports aggressive en bloc resection and marking and evaluating surgical margins.

Thoracic surgical oncology involves surgical treatment of lesions of the thoracic wall, pulmonary parenchyma, or mediastinum (also including heart, esophagus, or trachea). The most common neoplasms of the thoracic wall are osteosarcoma and chondrosarcoma. Histopathologic type, the use of chemotherapy for osteosarcoma, and completeness of surgical margins are prognostic for survival. Relative to solitary pulmonary masses, carcinomas are most common, with histopathologic type, tumor size, tumor grade, and lymph node status prognostic for survival. Of the aforementioned variables, lymph node status is the most significant. Extensive preoperative workup, including bronchoscopy and transthoracic fine needle aspiration of solitary lung masses, is usually not recommended. Thymomas are the most common surgical mediastinal mass. Patients are frequently affected with paraneoplastic syndromes including myasthenia gravis, polymyositis, and nonthymic neoplasia. Patients without megaesophagus with surgically resectable masses have an excellent prognosis for survival. Provision of analgesia after surgery in thoracotomy patients is extremely important. Carefully selected analgesic agents in thoracotomy patients are far less damaging to cardiovascular status than is tachycardia from excessive pain. Given these and other guidelines, perioperative mortality in thoracotomy patients is minimal, and long-term survival in selected patients is excellent.

Fourteen dogs (11 females, three males) with extraskeletal osteosarcomas (EsOSAs) were identified. The median age was 11.5 years. The median body weight was 18 kg. The primary sites of the EsOSAs were the spleen (n=6), mammary gland (n=3), lung (n=2), and one each in the skin, axilla, and mesenteric root. The overall median survival time was 74 days. The only factor which was found to be prognostic for survival was the use of chemotherapy (p of 0.02). Cases which did not have chemotherapy were 3.62 times as likely to die a tumor-related death than cases which had chemotherapy.

Twenty-one patients underwent selective inferior thyroid arteriography prior to surgery, and solitary parathyroid adenomas were found in 15. Comparison of the subtracted angiogram with the 131I thyroid scan facilitated preoperative localization and sizing of 12 of these 15 adenomas. Only 4 of 6 adenomas were identified preoperatively in 3 patients with multiple adenomas : the remaining 3 patients had normal arteriograms and negative surgical explorations. These results indicate that selective inferior thyroid arteriography is highly successful in staining solitary parathyroid adenomas and point up the fact that accurate interpretation depends upon close correlation between the angiogram and the thyroid scan.

Regional geological investigations of nuclear power plant sites in Wisconsin have revealed conflicting information in published references to faults. Faults reported 45 years ago had been reinterpreted by subsequent authors, causing confusion as to the existence, location, and extent of these faults. Several of the changes appear to be due to drafting errors. The diagrammatic nature of the original maps has been largely ignored by investigators. The importance of correctly presenting and verifying structural data is stressed. Four faults (Madison, Waukesha, Two Rivers, and Peshtigo-Brussels) in southern and eastern Wisconsin serve as case histories.