Hypoxia and intra-complex genetic suppressors rescue complex I mutants by a shared mechanism.

The electron transport chain (ETC) of mitochondria, bacteria, and archaea couples electron flow to proton pumping and is adapted to diverse oxygen environments. Remarkably, in mice, neurological disease due to ETC complex I dysfunction is rescued by hypoxia through unknown mechanisms. Here, we show that hypoxia rescue and hyperoxia sensitivity of complex I deficiency are evolutionarily conserved to C. elegans and are specific to mutants that compromise the electron-conducting matrix arm. We show that hypoxia rescue does not involve the hypoxia-inducible factor pathway or attenuation of reactive oxygen species. To discover the mechanism, we use C. elegans genetic screens to identify suppressor mutations in the complex I accessory subunit NDUFA6/nuo-3 that phenocopy hypoxia rescue. We show that NDUFA6/nuo-3(G60D) or hypoxia directly restores complex I forward activity, with downstream rescue of ETC flux and, in some cases, complex I levels. Additional screens identify residues within the ubiquinone binding pocket as being required for the rescue by NDUFA6/nuo-3(G60D) or hypoxia. This reveals oxygen-sensitive coupling between an accessory subunit and the quinone binding pocket of complex I that can restore forward activity in the same manner as hypoxia.

Genetic Screen for Cell Fitness in High or Low Oxygen Highlights Mitochondrial and Lipid Metabolism.

Human cells are able to sense and adapt to variations in oxygen levels. Historically, much research in this field has focused on hypoxia-inducible factor (HIF) signaling and reactive oxygen species (ROS). Here, we perform genome-wide CRISPR growth screens at 21%, 5%, and 1% oxygen to systematically identify gene knockouts with relative fitness defects in high oxygen (213 genes) or low oxygen (109 genes), most without known connection to HIF or ROS. Knockouts of many mitochondrial pathways thought to be essential, including complex I and enzymes in Fe-S biosynthesis, grow relatively well at low oxygen and thus are buffered by hypoxia. In contrast, in certain cell types, knockout of lipid biosynthetic and peroxisomal genes causes fitness defects only in low oxygen. Our resource nominates genetic diseases whose severity may be modulated by oxygen and links hundreds of genes to oxygen homeostasis.

Metabolic regulation of species-specific developmental rates.

Animals display substantial inter-species variation in the rate of embryonic development despite a broad conservation of the overall sequence of developmental events. Differences in biochemical reaction rates, including the rates of protein production and degradation, are thought to be responsible for species-specific rates of development1-3. However, the cause of differential biochemical reaction rates between species remains unknown. Here, using pluripotent stem cells, we have established an in vitro system that recapitulates the twofold difference in developmental rate between mouse and human embryos. This system provides a quantitative measure of developmental speed as revealed by the period of the segmentation clock, a molecular oscillator associated with the rhythmic production of vertebral precursors. Using this system, we show that mass-specific metabolic rates scale with the developmental rate and are therefore higher in mouse cells than in human cells. Reducing these metabolic rates by inhibiting the electron transport chain slowed down the segmentation clock by impairing the cellular NAD+/NADH redox balance and, further downstream, lowering the global rate of protein synthesis. Conversely, increasing the NAD+/NADH ratio in human cells by overexpression of the Lactobacillus brevis NADH oxidase LbNOX increased the translation rate and accelerated the segmentation clock. These findings represent a starting point for the manipulation of developmental rate, with multiple translational applications including accelerating the differentiation of human pluripotent stem cells for disease modelling and cell-based therapies.

Loss of LUC7L2 and U1 snRNP subunits shifts energy metabolism from glycolysis to OXPHOS.

Oxidative phosphorylation (OXPHOS) and glycolysis are the two major pathways for ATP production. The reliance on each varies across tissues and cell states, and can influence susceptibility to disease. At present, the full set of molecular mechanisms governing the relative expression and balance of these two pathways is unknown. Here, we focus on genes whose loss leads to an increase in OXPHOS activity. Unexpectedly, this class of genes is enriched for components of the pre-mRNA splicing machinery, in particular for subunits of the U1 snRNP. Among them, we show that LUC7L2 represses OXPHOS and promotes glycolysis by multiple mechanisms, including (1) splicing of the glycolytic enzyme PFKM to suppress glycogen synthesis, (2) splicing of the cystine/glutamate antiporter SLC7A11 (xCT) to suppress glutamate oxidation, and (3) secondary repression of mitochondrial respiratory supercomplex formation. Our results connect LUC7L2 expression and, more generally, the U1 snRNP to cellular energy metabolism.

Hepatic NADH reductive stress underlies common variation in metabolic traits.

The cellular NADH/NAD+ ratio is fundamental to biochemistry, but the extent to which it reflects versus drives metabolic physiology in vivo is poorly understood. Here we report the in vivo application of Lactobacillus brevis (Lb)NOX1, a bacterial water-forming NADH oxidase, to assess the metabolic consequences of directly lowering the hepatic cytosolic NADH/NAD+ ratio in mice. By combining this genetic tool with metabolomics, we identify circulating α-hydroxybutyrate levels as a robust marker of an elevated hepatic cytosolic NADH/NAD+ ratio, also known as reductive stress. In humans, elevations in circulating α-hydroxybutyrate levels have previously been associated with impaired glucose tolerance2, insulin resistance3 and mitochondrial disease4, and are associated with a common genetic variant in GCKR5, which has previously been associated with many seemingly disparate metabolic traits. Using LbNOX, we demonstrate that NADH reductive stress mediates the effects of GCKR variation on many metabolic traits, including circulating triglyceride levels, glucose tolerance and FGF21 levels. Our work identifies an elevated hepatic NADH/NAD+ ratio as a latent metabolic parameter that is shaped by human genetic variation and contributes causally to key metabolic traits and diseases. Moreover, it underscores the utility of genetic tools such as LbNOX to empower studies of 'causal metabolism'.

Circulating N-lactoyl-amino acids and N-formyl-methionine reflect mitochondrial dysfunction and predict mortality in septic shock.

INTRODUCTION: Sepsis is a highly morbid condition characterized by multi-organ dysfunction resulting from dysregulated inflammation in response to acute infection. Mitochondrial dysfunction may contribute to sepsis pathogenesis, but quantifying mitochondrial dysfunction remains challenging. OBJECTIVE: To assess the extent to which circulating markers of mitochondrial dysfunction are increased in septic shock, and their relationship to severity and mortality. METHODS: We performed both full-scan and targeted (known markers of genetic mitochondrial disease) metabolomics on plasma to determine markers of mitochondrial dysfunction which distinguish subjects with septic shock (n = 42) from cardiogenic shock without infection (n = 19), bacteremia without sepsis (n = 18), and ambulatory controls (n = 19) - the latter three being conditions in which mitochondrial function, proxied by peripheral oxygen consumption, is presumed intact. RESULTS: Nine metabolites were significantly increased in septic shock compared to all three comparator groups. This list includes N-formyl-L-methionine (f-Met), a marker of dysregulated mitochondrial protein translation, and N-lactoyl-phenylalanine (lac-Phe), representative of the N-lactoyl-amino acids (lac-AAs), which are elevated in plasma of patients with monogenic mitochondrial disease. Compared to lactate, the clinical biomarker used to define septic shock, there was greater separation between survivors and non-survivors of septic shock for both f-Met and the lac-AAs measured within 24 h of ICU admission. Additionally, tryptophan was the one metabolite significantly decreased in septic shock compared to all other groups, while its breakdown product kynurenate was one of the 9 significantly increased. CONCLUSION: Future studies which validate the measurement of lac-AAs and f-Met in conjunction with lactate could define a sepsis subtype characterized by mitochondrial dysfunction.

Fascial plane mapping for superficial tumor resection in dogs. Part III: Hindlimb and pelvis.

OBJECTIVES: To categorize the fascial planes and the intersections of these fascial planes in the hindlimb of the dog to facilitate preoperative planning for superficial cancers. STUDY DESIGN: Qualitative anatomical study. SAMPLE POPULATION: Four male and five female mixed breed dogs, weighing ~15-35 kg. METHODS: Skin and subcutaneous fat were removed, and fascial planes were explored to determine borders and quality. Fascia was categorized as type I (discrete sheets), type II (adhered to thin muscles), type III (adhered to thick muscles), or type IV (associated with periosteum). Digital modification of specimen photographs was performed to map tissues. RESULTS: Differences in dogs were noted due to either size or sex but were sufficiently minor to allow mapping. Fasciae of the hindlimb were predominantly type II or III, with type I fascia primarily at the lateral gluteal region, fascia lata, and lateral crus. Type IV fascia was seen at the iliac wing, ischium, patella, tibial tuberosity, medial tibia, distal crus, and pes. Fascia for surgical use was thin or absent at the ischiorectal fossa, femoral triangle, extensor mechanism, medial and distal crus, and pes. Intersections and tissues at the ventral perineum may also pose challenges for maintenance of a deep margin. CONCLUSION: Fascial types and integrity of the hindlimb varied with location, with the perineum, cranial stifle, and distal limb presenting the greatest challenges. CLINICAL SIGNIFICANCE: These images may be used to guide both therapeutic decision-making and intraoperative excision of superficial tumors of the hindlimb and pelvis.

A natural fusion of flavodiiron, rubredoxin, and rubredoxin oxidoreductase domains is a self-sufficient water-forming oxidase of Trichomonas vaginalis.

Microaerophilic pathogens such as Giardia lamblia, Entamoeba histolytica, and Trichomonas vaginalis have robust oxygen consumption systems to detoxify oxygen and maintain intracellular redox balance. This oxygen consumption results from H2O-forming NADH oxidase (NOX) activity of two distinct flavin-containing systems: H2O-forming NOXes and multicomponent flavodiiron proteins (FDPs). Neither system is membrane bound, and both recycle NADH into oxidized NAD+ while simultaneously removing O2 from the local environment. However, little is known about the specific contributions of these systems in T. vaginalis. In this study, we use bioinformatics and biochemical analyses to show that T. vaginalis lacks a NOX-like enzyme and instead harbors three paralogous genes (FDPF1-3), each encoding a natural fusion product between the N-terminal FDP, central rubredoxin (Rb), and C-terminal NADH:Rb oxidoreductase domains. Unlike a "stand-alone" FDP that lacks Rb and oxidoreductase domains, this natural fusion protein with fully populated flavin redox centers directly accepts reducing equivalents of NADH to catalyze the four-electron reduction of oxygen to water within a single polypeptide with an extremely high turnover. Furthermore, using single-particle cryo-EM, we present structural insights into the spatial organization of the FDP core within this multidomain fusion protein. Together, these results contribute to our understanding of systems that allow protozoan parasites to maintain optimal redox balance and survive transient exposure to oxic conditions.

MitoCarta3.0: an updated mitochondrial proteome now with sub-organelle localization and pathway annotations.

The mammalian mitochondrial proteome is under dual genomic control, with 99% of proteins encoded by the nuclear genome and 13 originating from the mitochondrial DNA (mtDNA). We previously developed MitoCarta, a catalogue of over 1000 genes encoding the mammalian mitochondrial proteome. This catalogue was compiled using a Bayesian integration of multiple sequence features and experimental datasets, notably protein mass spectrometry of mitochondria isolated from fourteen murine tissues. Here, we introduce MitoCarta3.0. Beginning with the MitoCarta2.0 inventory, we performed manual review to remove 100 genes and introduce 78 additional genes, arriving at an updated inventory of 1136 human genes. We now include manually curated annotations of sub-mitochondrial localization (matrix, inner membrane, intermembrane space, outer membrane) as well as assignment to 149 hierarchical 'MitoPathways' spanning seven broad functional categories relevant to mitochondria. MitoCarta3.0, including sub-mitochondrial localization and MitoPathway annotations, is freely available at http://www.broadinstitute.org/mitocarta and should serve as a continued community resource for mitochondrial biology and medicine.

Buccal transposition flap for closure of maxillary lip defects in 5 dogs.

OBJECTIVE: To describe the technique and outcomes of the closure of maxillary lip defects using a buccal transposition flap and to identify potential routes of vascular supply to the flap. ANIMALS: Five dogs treated clinically and 1 cadaveric dog head. STUDY DESIGN: Short case series and cadaveric study. METHODS: A left maxillary labial defect and a buccal transposition flap were created on a cadaver head. Iodinated contrast was injected into the left common carotid artery and computed tomography was performed to assess the vascular supply. Medical records were reviewed for all dogs that underwent tumor excision with maxillary lip resection, reconstructed with a buccal transposition flap. RESULTS: The buccal transposition flap was perfused by branches of the angularis oris artery and superior labial artery. Five dogs were included in this study. All flaps survived. Three dogs developed postoperative complications, including oronasal fistula (n = 2) and partial flap dehiscence (n = 1). The cosmetic and functional outcomes were considered satisfactory in all cases. CONCLUSION: Buccal transposition flaps for the closure of large maxillary lip defects provided adequate functional and cosmetic outcomes. The buccal transposition flap had vascular contributions from the angularis oris artery and the superior labial artery.

Fascial plane mapping for superficial tumor resection in dogs. Part I: Neck and trunk.

OBJECTIVE: To provide qualitative fascial categories and classify the intersections of various fascial planes of the trunk of the dog to facilitate preoperative planning for superficial cancers. STUDY DESIGN: Qualitative anatomical study. SAMPLE POPULATION: Two male and three female mixed breed canine cadavers weighing approximately 15 to 35 kg. METHODS: The skin and subcutaneous fat were excised. Fascial planes were incised and elevated to allow exploration of their quality and borders. Fascia was categorized as type I (discrete sheets), type II (tightly adhered to thin muscles), type III (tightly adhered to thick muscles), or type IV (associated with periosteum). Photographs of specimens were digitally modified with overlays to map tissue types. RESULTS: Differences between cadavers were largely associated with muscle mass or sex, with only minor anatomical differences and enough subjective similarity among specimens to allow mapping. The fasciae of the neck and trunk were predominantly type I or type II, with type III fascia at the shoulder and type IV fascia at the scapular spine, 13th rib, dorsal spinous processes, and the wing of the ilium. CONCLUSION: The superficial fasciae of the canine trunk were consistent among the dogs evaluated and can be classified as four broad fascial types. The population used was small, and individual variation should be considered when using these images in a clinical setting. CLINICAL SIGNIFICANCE: The images and categorization of fascia and transitions between fascial layers detailed here provide a visual and written reference for surgeons to facilitate preoperative planning and excision of superficial cancers.

Fascial plane mapping for superficial tumor resection in dogs. Part II: Forelimb.

OBJECTIVE: To detail the qualitative fascial categories and fascial intersections of the forelimb of the dog to facilitate preoperative planning for superficial cancers. STUDY DESIGN: Qualitative anatomical study. SAMPLE POPULATION: Three male and four female mixed breed canine cadavers weighing approximately 20-35 kg. METHODS: The skin and subcutaneous fat were excised. Fascial planes were incised and elevated to allow exploration of their quality and borders. Fascia was categorized as type I (discrete sheets), type II (tightly adhered to thin muscles), type III (tightly adhered to thick muscles), or type IV (associated with periosteum). Photographs of specimens were digitally modified with overlays to map tissue types. RESULTS: Differences between the cadavers used were largely based on muscle mass and sex, with minimal other subjective differences affecting fascial mapping. The fasciae of the forelimb were largely type II or type III, with type I fascia at the antebrachium and type IV fascia at the olecranon, scapular spine, and accessory carpal bone. Fascial integrity was often questionable or lacking distal to the distal quarter of the antebrachium. CONCLUSION: The fascial types and integrity of the forelimb varied with anatomic location with thin or absent fascia for surgical use at the elbow, carpus, and manus. CLINICAL SIGNIFICANCE: This study provides information for preoperative planning and excision of superficial tumors of the forelimb. Knowledge of the potential limitations of fascia to provide a deep margin may influence selection of treatment modalities.

The development of ventricular fibrillation as a complication of pericardiectomy in 16 dogs.

OBJECTIVE: To describe the clinical characteristics, perioperative protocols, and outcomes in dogs diagnosed with ventricular fibrillation (VF) while undergoing pericardiectomy. STUDY DESIGN: Retrospective, multi-institutional study. ANIMALS: Sixteen client-owned dogs. METHODS: 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. RESULTS: 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. CONCLUSION: 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. CLINICAL SIGNIFICANCE: 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.

Complications associated with iliosacral lymphadenectomy in dogs with metastatic apocrine gland anal sac adenocarcinoma.

Objective: To report intraoperative and immediate postoperative complications associated with removal of metastatic iliosacral lymph nodes in dogs with apocrine gland anal sac adenocarcinoma. Animals: There were 136 client-owned dogs in the study. Procedure: Retrospective multi-institutional study. The database of collaborating institutions was searched for dogs with metastatic apocrine gland anal sac adenocarcinoma that underwent lymphadenectomy for removal of one or more iliosacral lymph nodes. Information of signalment, hematological abnormalities, abdominal computed tomography or ultrasound findings, number and size of enlarged lymph nodes, intraoperative and postoperative complications, treatment and outcome were collected. Results: The overall complication rate associated with metastatic iliosacral lymphadenectomy was 26.1%. The only intraoperative complication recorded was hemorrhage and was reported in 24 (17.6%) surgeries, 11 (45.8%) of which received a blood transfusion. Postoperative complications were reported in 10.4% of surgeries, and included edema formation (n = 4, 2.6%), unilateral or bilateral paraparesis (n = 4, 2.6%), hypotension (n = 3, 2.0%), surgical site infection (n = 2, 1.3%), abdominal incision dehiscence (n = 1, 0.6%), urinary incontinence (n = 1, 0.6%), and death (n = 1, 0.6%). The size of the iliosacral lymph nodes was significantly associated with a greater risk of complications, hemorrhage, and the need of transfusion during lymphadenectomy for metastatic apocrine gland anal sac adenocarcinoma. Conclusion: Complications associated with iliosacral lymphadenectomy for metastatic apocrine gland anal sac adenocarcinoma are relatively common and mostly relate to hemorrhage. These complications are significantly associated with the size of the extirpated metastatic lymph nodes. Clinical relevance: This retrospective study provides information for the clinician regarding the potential surgical complications for extirpation of metastatic iliosacral lymph nodes. These complications, although not common, can be severe and should be discussed with owners before surgery.

Objectif: Rapporter les complications peropératoires et postopératoires immédiates associées à l'ablation des ganglions lymphatiques ilio-sacrés métastatiques chez les chiens atteints d'un adénocarcinome des glandes apocrines des sacs anaux. Animaux: Il y avait 136 chiens appartenant à des clients dans l'étude. Procédure: Étude multi-institutionnelle rétrospective. La base de données des institutions collaboratrices a été recherchée pour les chiens atteints d'un adénocarcinome métastatique des glandes apocrines des sacs anaux qui ont subi une lymphadénectomie pour l'ablation d'un ou plusieurs ganglions lymphatiques ilio-sacrés. Des informations sur le signalement, les anomalies hématologiques, les résultats de la tomodensitométrie abdominale ou de l'échographie, le nombre et la taille des ganglions élargis, les complications peropératoires et postopératoires, le traitement et les résultats ont été recueillis. Résultats: Le taux global de complications associées à la lymphadénectomie ilio-sacrée métastatique était de 26,1 %. La seule complication peropératoire enregistrée était une hémorragie et a été rapportée dans 24 (17,6 %) chirurgies, dont 11 (45,8 %) ont reçu une transfusion sanguine. Des complications postopératoires ont été signalées dans 10,4 % des interventions chirurgicales et comprenaient la formation d'oedème (n = 4, 2,6 %), la paraparésie unilatérale ou bilatérale (n = 4, 2,6 %), l'hypotension (n = 3, 2,0 %), l'infection du site opératoire (n = 2, 1,3 %), la déhiscence de l'incision abdominale (n = 1, 0,6 %), l'incontinence urinaire (n = 1, 0,6 %) et le décès (n = 1, 0,6 %). La taille des ganglions ilio-sacrés était significativement associée à un risque accru de complications, d'hémorragie et à la nécessité d'une transfusion lors d'une lymphadénectomie pour un adénocarcinome métastatique des glandes apocrines des sacs anaux. Conclusion: Les complications associées à la lymphadénectomie ilio-sacrée pour l'adénocarcinome métastatique des glandes apocrines des sacs anaux sont relativement fréquentes et concernent principalement l'hémorragie. Ces complications sont significativement associées à la taille des ganglions lymphatiques métastatiques retirés. Pertinence clinique: Cette étude rétrospective fournit des informations au clinicien concernant les complications chirurgicales potentielles pour le retrait des ganglions lymphatiques ilio-sacrés métastatiques. Ces complications, bien que rares, peuvent être graves et doivent être discutées avec les propriétaires avant la chirurgie.(Traduit par Dr Serge Messier).

Influence of intraoperative closed glove exchange on glove contamination during clean soft tissue surgeries.

OBJECTIVES: To determine the influence of intraoperative glove exchange on glove contamination during clean soft tissue surgery. STUDY DESIGN: Prospective clinical study. SAMPLE POPULATION: Two hundred pairs of gloves and gowns from 50 clean soft tissue surgeries. METHODS: Gloves and gown cuffs were cultured from the primary surgeon and first assistant using a standardized protocol. Cultures were taken from outer surface of both gown cuffs prior to surgery and after gloves were removed at the end of surgery; gloves were cultured prior to surgery, at end of surgery and after a new pair was donned after closed glove exchange. Cultures were evaluated for colony-forming units after 72 h of inoculation. RESULTS: Bacterial contamination was documented in 41 of the 50 surgeries (82%). The most common species cultured was Streptocococcus spp. There was no difference (p = .719) in the bacterial contamination rate of gown cuffs prior to surgery (10%; 20/200) compared to after surgery (9.5%; 19/200). The bacterial contamination rate for gloves was 10.5% (21/200) prior to surgery, 19.5% (39/196) after surgery, and 11% (22/200) after regloving. Gloves cultured following surgery were significantly more contaminated than gloves cultured preoperatively (p = .010) or gloves cultured following regloving (p = .018). CONCLUSION: Glove exchange did not increase bacterial contamination of gloves during the clean soft tissue surgeries tested here. CLINICAL SIGNIFICANCE: The outside of the gown cuff does not seem to represent a major source of contamination during clean procedures. This study does not provide evidence to support a change in current practices for intraoperative closed glove exchange.

Metastasis to ipsilateral medial retropharyngeal and deep cervical lymph nodes in 22 dogs with thyroid carcinoma.

OBJECTIVE: To determine the rate of nodal metastasis to the medial retropharyngeal (MRP) and deep cervical lymph nodes in dogs surgically treated for thyroid carcinoma. STUDY DESIGN: Retrospective study. ANIMALS: Twenty-two client-owned dogs. METHODS: Medical records between July 2015 and October 2019 at the Universities of Missouri and Florida were reviewed. Dogs that underwent thyroidectomy with concurrent elective MRP lymphadenectomy ± deep cervical lymphadenectomy were included. Tumor site, preoperative staging, and histopathological findings were recorded. RESULTS: Twenty-two dogs with 26 total thyroid carcinomas were included. Primary tumors were lateralized in 19 dogs, bilateral in two dogs, and bilateral and midline ectopic in one dog. All dogs underwent ipsilateral MRP resection, including bilateral resection in dogs with bilateral tumors. Three contralateral MRP lymph nodes were excised from dogs with unilateral carcinomas. Four deep cervical lymph nodes and one superficial cervical lymph node were excised. Metastases were identified in 14 lymph nodes in 10 of 22 (45%) dogs. All four excised deep cervical lymph nodes and one contralateral MRP lymph node were identified as metastatic. Size of deposit could be classified in 13 of 14 metastatic lymph nodes. Macrometastasis was detected in seven lymph nodes, micrometastasis was detected in one node, and isolated tumor cells were detected in five lymph nodes. CONCLUSION: Regional metastasis was common within the lymph nodes sampled in this population of dogs with thyroid carcinoma. CLINICAL SIGNIFICANCE: These results provide evidence to justify further exploration of a larger population to verify the rate of regional metastasis and determine the prognostic impact of nodal metastasis.

Staged mandibular lip flap for closure of a large palatal defect after maxillectomy in a dog.

OBJECTIVE: To report closure of an oronasal defect secondary to maxillectomy with a staged mandibular lip flap. STUDY DESIGN: Case report ANIMALS: One 9-year-old female spayed golden retriever. METHODS: A combined dorsolateral and intraoral approach was used to perform a central maxillectomy to excise a 2.4- × 2- × 2.7-cm oral osteosarcoma with 1-cm margins. A buccal mucosal flap was used to close the palatal defect but the site subsequently dehisced. A staged mandibular lip flap was performed to close the defect. An incision was made on the mandible at the intersection of the buccal mucosa and gingiva from the mandibular canine to the level of the commissure. A second incision was made 3 cm ventral to the lip margin. The flap pedicle was based at the commissure. The flap was rotated to cover the palatal defect from rostral to the canine tooth to the fourth premolar. A second procedure was performed 4 weeks after flap placement to desquamate the haired skin and transect the flap pedicle. RESULTS: Partial dehiscence at the caudal aspect of the flap occurred after the first revision. The defect was closed after pedicle transection on day 41, with acceptable cosmesis. The dog was eating canned food with no evidence of discomfort 159 days after the maxillectomy. Recurrence was noted on day 270 postoperatively. CONCLUSION: Closure of a large palatal defect with a staged mandibular lip flap led to good cosmesis and function.

Survival time of juvenile dogs with oral squamous cell carcinoma treated with surgery alone: A Veterinary Society of Surgical Oncology retrospective study.

OBJECTIVE: To report the signalment, staging, surgical treatment, and survival time of juvenile dogs treated surgically for oral squamous cell carcinoma (OSCC). STUDY DESIGN: Retrospective study. ANIMALS OR SAMPLE POPULATION: Twenty-five dogs, <2 years of age with OSCC treated with surgery. METHODS: Cases were solicited from the Veterinary Society of Surgical Oncology. Data retrieved included sex, breed, age, weight, clinical signs, tumor location, preoperative diagnostics and staging, histopathological diagnosis with margin evaluation, disease-free interval, and date and cause of death. A minimum follow-up time of 3 months was required for inclusion. RESULTS: Eighteen dogs were <12 months of age, and seven were <24 months. Various breeds were represented, with a mean body weight of 22.3 ± 14.4 kg. No dogs had evidence of metastatic disease prior to surgery. All dogs underwent partial maxillectomy or mandibulectomy. Histological margins were complete in 24 dogs and incomplete in one. No dogs had evidence of metastatic disease or tumor recurrence. The median follow-up time was 1556 days (92 to 4234 days). All dogs were alive at the last follow-up except for one documented death, due to dilated cardiomyopathy. Median disease-specific survival time was not reached. CONCLUSION: The prognosis after wide surgical excision of OSCC in juvenile dogs was excellent. CLINICAL SIGNIFICANCE: OSCC in juvenile dogs can be effectively treated with surgery alone.

Top-down characterization of endogenous protein complexes with native proteomics.

Protein complexes exhibit great diversity in protein membership, post-translational modifications and noncovalent cofactors, enabling them to function as the actuators of many important biological processes. The exposition of these molecular features using current methods lacks either throughput or molecular specificity, ultimately limiting the use of protein complexes as direct analytical targets in a wide range of applications. Here, we apply native proteomics, enabled by a multistage tandem MS approach, to characterize 125 intact endogenous complexes and 217 distinct proteoforms derived from mouse heart and human cancer cell lines in discovery mode. The native conditions preserved soluble protein-protein interactions, high-stoichiometry noncovalent cofactors, covalent modifications to cysteines, and, remarkably, superoxide ligands bound to the metal cofactor of superoxide dismutase 2. These data enable precise compositional analysis of protein complexes as they exist in the cell and demonstrate a new approach that uses MS as a bridge to structural biology.