Aberrant differentiation of second heart field mesoderm prefigures cellular defects in the outflow tract in response to loss of FGF8.

Development of the outflow tract of the heart requires specification, proliferation and deployment of a progenitor cell population from the second heart field to generate the myocardium at the arterial pole of the heart. Disruption of these processes leads to lethal defects in rotation and septation of the outflow tract. We previously showed that Fibroblast Growth Factor 8 (FGF8) directs a signaling cascade in the second heart field that regulates critical aspects of OFT morphogenesis. Here we show that in addition to the survival and proliferation cues previously described, FGF8 provides instructive and patterning information to OFT myocardial cells and their progenitors that prevents their aberrant differentiation along a working myocardial program.

Metabolic engineering of Caldicellulosiruptor bescii for 2,3-butanediol production from unpretreated lignocellulosic biomass and metabolic strategies for improving yields and titers.

The platform chemical 2,3-butanediol (2,3-BDO) is used to derive products, such as 1,3-butadiene and methyl ethyl ketone, for the chemical and fuel production industries. Efficient microbial 2,3-BDO production at industrial scales has not been achieved yet for various reasons, including product inhibition to host organisms, mixed stereospecificity in product formation, and dependence on expensive substrates (i.e., glucose). In this study, we explore engineering of a 2,3-BDO pathway in Caldicellulosiruptor bescii, an extremely thermophilic (optimal growth temperature = 78°C) and anaerobic bacterium that can break down crystalline cellulose and hemicellulose into fermentable C5 and C6 sugars. In addition, C. bescii grows on unpretreated plant biomass, such as switchgrass. Biosynthesis of 2,3-BDO involves three steps: two molecules of pyruvate are condensed into acetolactate; acetolactate is decarboxylated to acetoin, and finally, acetoin is reduced to 2,3-BDO. C. bescii natively produces acetoin; therefore, in order to complete the 2,3-BDO biosynthetic pathway, C. bescii was engineered to produce a secondary alcohol dehydrogenase (sADH) to catalyze the final step. Two previously characterized, thermostable sADH enzymes with high affinity for acetoin, one from a bacterium and one from an archaeon, were tested independently. When either sADH was present in C. bescii, the recombinant strains were able to produce up to 2.5-mM 2,3-BDO from crystalline cellulose and xylan and 0.2-mM 2,3-BDO directly from unpretreated switchgrass. This serves as the basis for higher yields and productivities, and to this end, limiting factors and potential genetic targets for further optimization were assessed using the genome-scale metabolic model of C. bescii.IMPORTANCELignocellulosic plant biomass as the substrate for microbial synthesis of 2,3-butanediol is one of the major keys toward cost-effective bio-based production of this chemical at an industrial scale. However, deconstruction of biomass to release the sugars for microbial growth currently requires expensive thermochemical and enzymatic pretreatments. In this study, the thermo-cellulolytic bacterium Caldicellulosiruptor bescii was successfully engineered to produce 2,3-butanediol from cellulose, xylan, and directly from unpretreated switchgrass. Genome-scale metabolic modeling of C. bescii was applied to adjust carbon and redox fluxes to maximize productivity of 2,3-butanediol, thereby revealing bottlenecks that require genetic modifications.

PPARγ and NOTCH Regulate Regional Identity in the Murine Cardiac Outflow Tract.

BACKGROUND: The arterial pole of the heart is a hotspot for life-threatening forms of congenital heart defects (CHDs). Development of this cardiac region occurs by addition of Second Heart Field (SHF) progenitor cells to the embryonic outflow tract (OFT) and subsequently the base of the ascending aorta and pulmonary trunk. Understanding the cellular and genetic mechanisms driving arterial pole morphogenesis is essential to provide further insights into the cause of CHDs. METHODS: A synergistic combination of bioinformatic analysis and mouse genetics as well as embryo and explant culture experiments were used to dissect the cross-regulatory transcriptional circuitry operating in future subaortic and subpulmonary OFT myocardium. RESULTS: Here, we show that the lipid sensor PPARγ (peroxisome proliferator-activated receptor gamma) is expressed in future subpulmonary myocardium in the inferior wall of the OFT and that PPARγ signaling-related genes display regionalized OFT expression regulated by the transcription factor TBX1 (T-box transcription factor 1). Modulating PPARγ activity in ex vivo cultured embryos treated with a PPARγ agonist or antagonist or deleting Pparγ in cardiac progenitor cells using Mesp1-Cre reveals that Pparγ is required for addition of future subpulmonary myocardium and normal arterial pole development. Additionally, the non-canonical DLK1 (delta-like noncanonical Notch ligand 1)/NOTCH (Notch receptor 1)/HES1 (Hes family bHLH transcription factor 1) pathway negatively regulates Pparγ in future subaortic myocardium in the superior OFT wall. CONCLUSIONS: Together these results identify Pparγ as a regulator of regional transcriptional identity in the developing heart, providing new insights into gene interactions involved in congenital heart defects.

Cardiac Development and Animal Models of Congenital Heart Defects.

The major events of cardiac development, including early heart formation, chamber morphogenesis and septation, and conduction system and coronary artery development, are briefly reviewed together with a short introduction to the animal species commonly used to study heart development and model congenital heart defects (CHDs).

Molecular Pathways and Animal Models of Tetralogy of Fallot and Double Outlet Right Ventricle.

Tetralogy of Fallot and double-outlet right ventricle are outflow tract (OFT) alignment defects situated on a continuous disease spectrum. A myriad of upstream causes can impact on ventriculoarterial alignment that can be summarized as defects in either i) OFT elongation during looping morphogenesis or ii) OFT remodeling during cardiac septation. Embryological processes underlying these two developmental steps include deployment of second heart field cardiac progenitor cells, establishment and transmission of embryonic left/right information driving OFT rotation and OFT cushion and valve morphogenesis. The formation and remodeling of pulmonary trunk infundibular myocardium is a critical component of both steps. Defects in myocardial, endocardial, or neural crest cell lineages can result in alignment defects, reflecting the complex intercellular signaling events that coordinate arterial pole development. Importantly, however, OFT alignment is mechanistically distinct from neural crest-driven OFT septation, although neural crest cells impact indirectly on alignment through their role in modulating signaling during SHF development. As yet poorly understood nongenetic causes of alignment defects that impact the above processes include hemodynamic changes, maternal exposure to environmental teratogens, and stochastic events. The heterogeneity of causes converging on alignment defects characterizes the OFT as a hotspot of congenital heart defects.

Cardiac Progenitor Cells of the First and Second Heart Fields.

The heart forms from the first and second heart fields, which contribute to distinct regions of the myocardium. This is supported by clonal analyses, which identify corresponding first and second cardiac cell lineages in the heart. Progenitor cells of the second heart field and its sub-domains are controlled by a gene regulatory network and signaling pathways, which determine their behavior. Multipotent cells in this field can also contribute cardiac endothelial and smooth muscle cells. Furthermore, the skeletal muscles of the head and neck are clonally related to myocardial cells that form the arterial and venous poles of the heart. These lineage relationships, together with the genes that regulate the heart fields, have major implications for congenital heart disease.

Fibroblast growth factor 10.

Fibroblast growth factor 10 (FGF10) is a major morphoregulatory factor that plays essential signaling roles during vertebrate multiorgan development and homeostasis. FGF10 is predominantly expressed in mesenchymal cells and signals though FGFR2b in adjacent epithelia to regulate branching morphogenesis, stem cell fate, tissue differentiation and proliferation, in addition to autocrine roles. Genetic loss of function analyses have revealed critical requirements for FGF10 signaling during limb, lung, digestive system, ectodermal, nervous system, craniofacial and cardiac development. Heterozygous FGF10 mutations have been identified in human genetic syndromes associated with craniofacial anomalies, including lacrimal and salivary gland aplasia. Elevated Fgf10 expression is associated with poor prognosis in a range of cancers. In addition to developmental and disease roles, FGF10 regulates homeostasis and repair of diverse adult tissues and has been identified as a target for regenerative medicine.

Bony encasement of the ulnar nerve secondary to heterotopic ossification of the elbow: an evaluation of long-term outcomes.

BACKGROUND: Ulnar neuropathy at the elbow caused by heterotopic ossification (HO) is a rare condition. This retrospective study aims to report on 32 consecutive cases of ulnar nerve encasement caused by elbow HO and evaluate long-term outcomes of operative management and a standardized postoperative rehabilitation regimen. METHODS: A retrospective case series was conducted on 32 elbows (27 patients) that underwent operative management of bony ulnar nerve encasement. All procedures were performed in the inpatient setting at an Academic Level 1 Trauma Center from September 1999 to July 2021 by one of 3 fellowship-trained shoulder and elbow. Postoperatively, all patients received formal physical therapy, HO prophylaxis (30 received indomethacin, 2 received radiation), and a structured continuous passive motion machine regimen. Patient demographics, age, gender, type of injury, history of tobacco use, and medical comorbidities were obtained to include in the analysis. Long-term follow-up examinations were performed to evaluate elbow flexion-extension arc of motion, Mayo Elbow Performance Score, and visual analog scale pain scores. RESULTS: Thirty-two elbows with complete bony ulnar nerve encasement secondary to HO were identified (14 from burns, 15 from trauma, 3 closed head injuries). Following surgery, the mean flexion-extension arc of motion improved significantly, increasing from 21° to 100° at long-term follow-up (average 8.7 years, range 2-17 years), with statistically significant improvements in preoperative vs. long-term postoperative elbow extension (P < .001), flexion (P < .001), and total arc of motion (P < .001). There was a statistically significant improvement in pre- vs. postprocedure ulnar nerve function, as demonstrated by a decrease in average McGowan grade (1.2-0.7; P = .002). Additionally, 63% of patients with preoperative ulnar neuropathy symptoms (20/32) had either complete resolution or subjective improvement after surgery. The mean time from injury to surgery was 518 days (range 65-943 days). Age, gender, time to surgery, and medical comorbidities were not associated with outcomes. The complication rate was 9% (3/32). Patients had an average flexion-extension arc of motion of 97° and average Mayo Elbow Performance Score of 80 ("good") at long-term follow-up. CONCLUSIONS: The combination of operative management, postoperative HO prophylaxis, and a regimented rehabilitation program has proven to be a durable solution for treating and ensuring good long-term functional outcomes for patients with elbow HO and bony ulnar nerve encasement. This treatment approach leads to superior range of motion, improved or resolved ulnar neuropathy, and good to excellent long-term functional outcomes.

Assessing the impact of environmental enrichment on behavior in understudied armadillo species: A case study.

The implementation of environmental enrichment (EE) can be effective in promoting zoo animal welfare by enhancing the performance of natural or species-typical behaviors. Research on the effects of EE is biased towards larger mammalian species, with less charismatic species being overlooked. Armadillos are one such overlooked example. A captive environment that results in inactivity, obesity, and associated poor health can negatively affect armadillo well-being. The aim of this study was to evaluate how the implementation of four physical object-based EEs could positively affect the behaviors of three armadillo species, housed in four similar exhibits. Behavioral data were collected both before (baseline) and during (treatment) EE periods, alongside of visitor number and environmental temperatures. The EE comprised of a plastic ball or a cardboard tube or a cardboard box, or a scatter-feed, and these were rotated each week of study until each exhibit had received them in turn. Despite the presence of different EE types, activity remained low throughout the study. However, results suggest that the plastic ball and cardboard box increased exploratory behaviors in the armadillos, but no overall increase in activity was noted during the scatter feed. Visitor presence had no effect on armadillo activity, and armadillos showed reduced activity with increasing environmental temperature. Overall, the use of physical object-based EE promoted beneficial natural behaviors in zoo-housed armadillos, but environmental conditions (i.e., temperature) also impacted armadillo activity, suggesting a complicated relationship between an enclosure's environmental variable and any behavioral husbandry measures.

Interplay between transcriptional regulators and VapBC toxin-antitoxin loci during thermal stress response in extremely thermoacidophilic archaea.

Thermoacidophilic archaea lack sigma factors and the large inventory of heat shock proteins (HSPs) widespread in bacterial genomes, suggesting other strategies for handling thermal stress are involved. Heat shock transcriptomes for the thermoacidophilic archaeon Saccharolobus (f. Sulfolobus) solfataricus 98/2 revealed genes that were highly responsive to thermal stress, including transcriptional regulators YtrASs (Ssol_2420) and FadRSs (Ssol_0314), as well as type II toxin-antitoxin (TA) loci VapBC6 (Ssol_2337, Ssol_2338) and VapBC22 (Ssol_0819, Ssol_0818). The role, if any, of type II TA loci during stress response in microorganisms, such as Escherichia coli, is controversial. But, when genes encoding YtrASs , FadRSs , VapC22, VapB6, and VapC6 were systematically mutated in Sa. solfataricus 98/2, significant up-regulation of the other genes within this set was observed, implicating an interconnected regulatory network during thermal stress response. VapBC6 and VapBC22 have close homologues in other Sulfolobales, as well as in other archaea (e.g. Pyrococcus furiosus and Archaeoglobus fulgidus), and their corresponding genes were also heat shock responsive. The interplay between VapBC TA loci and heat shock regulators in Sa solfataricus 98/2 not only indicates a cellular mechanism for heat shock response that differs from bacteria but one that could have common features within the thermophilic archaea.

Cardiopharyngeal mesoderm origins of musculoskeletal and connective tissues in the mammalian pharynx.

Cardiopharyngeal mesoderm (CPM) gives rise to muscles of the head and heart. Using genetic lineage analysis in mice, we show that CPM develops into a broad range of pharyngeal structures and cell types encompassing musculoskeletal and connective tissues. We demonstrate that CPM contributes to medial pharyngeal skeletal and connective tissues associated with both branchiomeric and somite-derived neck muscles. CPM and neural crest cells (NCC) make complementary mediolateral contributions to pharyngeal structures, in a distribution established in the early embryo. We further show that biallelic expression of the CPM regulatory gene Tbx1, haploinsufficient in 22q11.2 deletion syndrome patients, is required for the correct patterning of muscles with CPM-derived connective tissue. Our results suggest that CPM plays a patterning role during muscle development, similar to that of NCC during craniofacial myogenesis. The broad lineage contributions of CPM to pharyngeal structures provide new insights into congenital disorders and evolution of the mammalian pharynx.

Optimizing Strategies for Bio-Based Ethanol Production Using Genome-Scale Metabolic Modeling of the Hyperthermophilic Archaeon, Pyrococcus furiosus.

A genome-scale metabolic model, encompassing a total of 623 genes, 727 reactions, and 865 metabolites, was developed for Pyrococcus furiosus, an archaeon that grows optimally at 100°C by carbohydrate and peptide fermentation. The model uses subsystem-based genome annotation, along with extensive manual curation of 237 gene-reaction associations including those involved in central carbon metabolism, amino acid metabolism, and energy metabolism. The redox and energy balance of P. furiosus was investigated through random sampling of flux distributions in the model during growth on disaccharides. The core energy balance of the model was shown to depend on high acetate production and the coupling of a sodium-dependent ATP synthase and membrane-bound hydrogenase, which generates a sodium gradient in a ferredoxin-dependent manner, aligning with existing understanding of P. furiosus metabolism. The model was utilized to inform genetic engineering designs that favor the production of ethanol over acetate by implementing an NADPH and CO-dependent energy economy. The P. furiosus model is a powerful tool for understanding the relationship between generation of end products and redox/energy balance at a systems-level that will aid in the design of optimal engineering strategies for production of bio-based chemicals and fuels. IMPORTANCE The bio-based production of organic chemicals provides a sustainable alternative to fossil-based production in the face of today's climate challenges. In this work, we present a genome-scale metabolic reconstruction of Pyrococcus furiosus, a well-established platform organism that has been engineered to produce a variety of chemicals and fuels. The metabolic model was used to design optimal engineering strategies to produce ethanol. The redox and energy balance of P. furiosus was examined in detail, which provided useful insights that will guide future engineering designs.

Manipulating Fermentation Pathways in the Hyperthermophilic Archaeon Pyrococcus furiosus for Ethanol Production up to 95°C Driven by Carbon Monoxide Oxidation.

Genetic engineering of hyperthermophilic organisms for the production of fuels and other useful chemicals is an emerging biotechnological opportunity. In particular, for volatile organic compounds such as ethanol, fermentation at high temperatures could allow for straightforward separation by direct distillation. Currently, the upper growth temperature limit for native ethanol producers is 72°C in the bacterium Thermoanaerobacter ethanolicus JW200, and the highest temperature for heterologously-engineered bioethanol production was recently demonstrated at 85°C in the archaeon Pyrococcus furiosus. Here, we describe an engineered strain of P. furiosus that synthesizes ethanol at 95°C, utilizing a homologously-expressed native alcohol dehydrogenase, termed AdhF. Ethanol biosynthesis was compared at 75°C and 95°C with various engineered strains. At lower temperatures, the acetaldehyde substrate for AdhF is most likely produced from acetate by aldehyde ferredoxin oxidoreductase (AOR). At higher temperatures, the effect of AOR on ethanol production is negligible, suggesting that acetaldehyde is produced by pyruvate ferredoxin oxidoreductase (POR) via oxidative decarboxylation of pyruvate, a reaction known to occur only at higher temperatures. Heterologous expression of a carbon monoxide dehydrogenase complex in the AdhF overexpression strain enabled it to use CO as a source of energy, leading to increased ethanol production. A genome reconstruction model for P. furiosus was developed to guide metabolic engineering strategies and understand outcomes. This work opens the door to the potential for 'bioreactive distillation' since fermentation can be performed well above the normal boiling point of ethanol. IMPORTANCE Previously, the highest temperature for biological ethanol production was 85°C. Here, we have engineered ethanol production at 95°C by the hyperthermophilic archaeon Pyrococcus furiosus. Using mutant strains, we showed that ethanol production occurs by different pathways at 75°C and 95°C. In addition, by heterologous expression of a carbon monoxide dehydrogenase complex, ethanol production by this organism was driven by the oxidation of carbon monoxide. A genome reconstruction model for P. furiosus was developed to guide metabolic engineering strategies and understand outcomes.

Role of cell-substrate association during plant biomass solubilization by the extreme thermophile Caldicellulosiruptor bescii.

Caldicellulosiruptor species are proficient at solubilizing carbohydrates in lignocellulosic biomass through surface (S)-layer bound and secretomic glycoside hydrolases. Tapirins, surface-associated, non-catalytic binding proteins in Caldicellulosiruptor species, bind tightly to microcrystalline cellulose, and likely play a key role in natural environments for scavenging scarce carbohydrates in hot springs. However, the question arises: If tapirin concentration on Caldicellulosiruptor cell walls increased above native levels, would this offer any benefit to lignocellulose carbohydrate hydrolysis and, hence, biomass solubilization? This question was addressed by engineering the genes for tight-binding, non-native tapirins into C. bescii. The engineered C. bescii strains bound more tightly to microcrystalline cellulose (Avicel) and biomass compared to the parent. However, tapirin overexpression did not significantly improve solubilization or conversion for wheat straw or sugarcane bagasse. When incubated with poplar, the tapirin-engineered strains increased solubilization by 10% compared to the parent, and corresponding acetate production, a measure of carbohydrate fermentation intensity, was 28% higher for the Calkr_0826 expression strain and 18.5% higher for the Calhy_0908 expression strain. These results show that enhanced binding to the substrate, beyond the native capability, did not improve C. bescii solubilization of plant biomass, but in some cases may improve conversion of released lignocellulose carbohydrates to fermentation products.

Emergence of heart and branchiomeric muscles in cardiopharyngeal mesoderm.

Branchiomeric muscles of the head and neck originate in a population of cranial mesoderm termed cardiopharyngeal mesoderm that also contains progenitor cells contributing to growth of the embryonic heart. Retrospective lineage analysis has shown that branchiomeric muscles share a clonal origin with parts of the heart, indicating the presence of common heart and head muscle progenitor cells in the early embryo. Genetic lineage tracing and functional studies in the mouse, as well as in Ciona and zebrafish, together with recent experiments using single cell transcriptomics and multipotent stem cells, have provided further support for the existence of bipotent head and heart muscle progenitor cells. Current challenges concern defining where and when such common progenitor cells exist in mammalian embryos and how alternative myogenic derivatives emerge in cardiopharyngeal mesoderm. Addressing these questions will provide insights into mechanisms of cell fate acquisition and the evolution of vertebrate musculature, as well as clinical insights into the origins of muscle restricted myopathies and congenital defects affecting craniofacial and cardiac development.

Intravenous immunoglobulin treatment for refractory pyoderma gangrenosum.

BACKGROUND: Intravenous immunoglobulins (IVIG) have been increasingly used for various inflammatory dermatoses with success. Small case series and case reports suggest a role for IVIG in the management of refractory pyoderma gangrenosum (PG). OBJECTIVE: The objective was to study the characteristics of PG patients treated with IVIG and the efficacy and safety of IVIG for patients with refractory PG. METHODS: An analysis was performed of all patients with PG treated with IVIG from 2012 to 2022 at an Australian tertiary hospital seeing a high volume of PG patients. RESULTS: We identified 12 patients, 9 females and 3 males, with median age of 61 years (29-77) at IVIG commencement. All patients were taking systemic corticosteroid therapy prior to IVIG treatment, and all had been treated with a steroid-sparing agent-including ten patients who had been treated with a biologic agent. IVIG was used with corticosteroids in one patient, concurrently with a steroid-sparing agent in nine patients and with a biologic agent in eight patients. Eleven patients demonstrated treatment response to IVIG-six with excellent response and five with good response. Three patients had complete healing of their most active ulcer. One patient did not respond to IVIG. Nine patients were able to wean their prednisolone dose and one patient was able to cease prednisolone. Four adverse events were recorded, and only one patient had to cease treatment due to aseptic meningitis and headaches. CONCLUSION: Our experience suggests that IVIG may be an efficacious treatment for patients with refractory PG due to its pleiotropic and immunomodulatory effects, particularly for patients with malignancy or other systemic conditions where high-dose immunosuppressive agents are contraindicated.

The simultaneous occurrence of livedoid vasculopathy and lymphocytic thrombophilic arteritis in six cases.

Lymphocytic thrombophilic arteritis and livedoid vasculopathy may both present with livedo racemosa and ulceration. We present 6 cases with features of both conditions, raising the possibility that they are either closely linked or are part of a spectrum of the same condition.

Hospital transfer and delayed reduction of traumatic hip dislocations.

INTRODUCTION: Native hip dislocations are high energy injuries that cause substantial patient morbidity. Expedient reduction has been demonstrated to improve patient outcomes. The objective of our study was to compare complication rates in patients with native hip dislocations who presented directly to a level-one trauma center with those transferred from an outside hospital (OSH). Our hypothesis was that those transferred from an OSH would experience a delay in reduction and subsequently would experience higher rates of avascular necrosis (AVN), post-traumatic arthritis (PTA), and need for secondary surgery. MATERIAL AND METHODS: We conducted a retrospective chart review of all native hip dislocations from our level-one trauma center between January 2007 and December 2020. The initial query resulted 628 patients which was refined to 90 patients after excluding patients for incorrect diagnosis code or less than 6 months of follow-up. Our primary outcome was the development of AVN, PTA, and need for secondary surgery. Time from injury to reduction was recorded for all patients included. RESULTS: For every one hour of delay in time to reduction, there was a 3.4% increase in the risk of developing AVN (p = 0.004) and a 4.3% increase in risk for developing PTA (p = 0.01). The risk of requiring a secondary surgery increased 4.6% for each hour of delay in reduction (p = 0.03). The average time to reduction of transferred patients was higher compared to those who presented directly to our center (13.8 h vs 5.7 h); however, transfer status was not found to be an independent risk factor for the measured outcomes. CONCLUSIONS: Transfer status is not an independent risk factor for the development of AVN, PTA, or the need for a secondary surgery. However, transferred patients did experience an average delay of 8 h in time to reduction compared to those who presented directly to a trauma center. Of the 27 patients with a reduction delay greater than 12 h, 26 (96%) were transferred.

Recent Modifications of the Nuss Procedure: The Pursuit of Safety During the Minimally Invasive Repair of Pectus Excavatum.

OBJECTIVE: To review standardized Nuss correction of pectus excavatum and vacuum bell treatment over the last 10 years. SUMMARY OF BACKGROUND DATA: In 2010, we reported 21 years of the Nuss procedure in 1215 patients. METHODS: Over the last 10 years, 2008-2018, we evaluated 1885 pectus excavatum patients. Surgery was indicated for well-defined objective criteria. A consistent operation was performed by 8 surgeons in 1034 patients, median 15 years, (range 6-46); 996 were primary, and 38 redo operations. Surgical patients' mean computed tomography index was 5.46. Mitral valve prolapse was present in 5.4%, Marfan syndrome in 1.1% and scoliosis in 29%. Vacuum bell treatment was introduced for 218 patients who did not meet surgical criteria or were averse to surgery. RESULTS: At primary operation, 1 bar was placed in 49.8%; 2 bars, 49.4%; and 3 bars, 0.7%. There were no deaths. Cardiac perforation occurred in 1 patient who had undergone previous cardiac surgery. Paraplegia after epidural catheter occurred once. Reoperation for bar displacement occurred in 1.8%, hemothorax in 0.3%, and wound infection in 2.9%; 1.4% required surgical drainage. Allergy to stainless steel was identified in 13.7%. A good anatomic outcome was always achieved at bar removal. Recurrence requiring reoperation occurred in 3 primary surgical patients. Two patients developed carinate overcorrection requiring reoperation. Vacuum bell treatment produced better results in younger and less severe cases. CONCLUSIONS: A standardized Nuss procedure was performed by multiple surgeons in 1034 patients with good overall safety and results in primary repairs. Vacuum bell treatment is useful.

Fox Cluster determinants for iron biooxidation in the extremely thermoacidophilic Sulfolobaceae.

Within the extremely thermoacidophilic Sulfolobaceae, the capacity to oxidize iron varies considerably. While some species are prolific iron oxidizers (e.g. Metallosphaera sedula), other species do not oxidize iron at all (e.g. Sulfolobus acidocaldarius). Iron oxidation capacity maps to a genomic locus, referred to previously as the 'Fox Cluster', that encodes putative proteins that are mostly unique to the Sulfolobaceae. The role of putative proteins in the Fox Cluster has not been confirmed, but proteomic analysis here of iron-oxidizing membranes from M. sedula indicates that FoxA2 and FoxB (both cytochrome c oxidase-like subunits) and FoxC (CbsA/cytochrome b domain-containing) are essential. Furthermore, comparative genomics (locus organization and gene disruptions) and transcriptomics (polarity effects and differential expression) connect these genomic determinants with disparate iron biooxidation and respiration measurements among Sulfolobaceae species. While numerous homologous proteins can be identified for FoxA in genome databases (COX-like domains are prevalent across all domains of life), few homologues exist for FoxC or for most other Fox Cluster proteins. Phylogenetic reconstructions suggest this locus may have existed in early Sulfolobaceae, while the only other close homologues to the locus appear in the recently discovered candidate phylum Marsarchaota.