The respiratory tract is constantly exposed to the external environment, and therefore, must be equipped to respond to and eliminate pathogens. Viral clearance and resolution of infection requires a complex, multi-faceted response initiated by resident respiratory tract cells and innate immune cells and ultimately resolved by adaptive immune cells. Although an effective immune response to eliminate viral pathogens is essential, a prolonged or exaggerated response can damage the respiratory tract. Immune-mediated pulmonary damage is manifested clinically in a variety of ways depending on location and extent of injury. Thus, the antiviral immune response represents a balancing act between the elimination of virus and immune-mediated pulmonary injury. In this review, we highlight major components of the host response to acute viral infection and their role in contributing to mitigating respiratory damage. We also briefly describe common clinical manifestations of respiratory viral infection and morphological correlates. The continuing threat posed by pandemic influenza as well as the emergence of novel respiratory viruses also capable of producing severe acute lung injury such as SARS-CoV, MERS-CoV, and enterovirus D68, highlights the need for an understanding of the immune mechanisms that contribute to virus elimination and immune-mediated injury. The cells that line the respiratory tract are continually exposed to the external environment, making the lungs a particularly vulnerable site for infection. Respiratory infections represent a major disease and economic burden worldwide. According to the CDC, influenza virus infection and associated complications are one of the top ten causes of death and result in millions of hospitalizations, costing over $10 billion each year in the USA [A limited survey of respiratory viral infections reveals that respiratory viruses with distinct virion and genome structures, unique entry receptors, and modes of replication, cause similar clinical syndromes and sequelae Table . These cA virus must replicate and orchestrate the assembly of virion constituents to produce progeny virus and propagate itself. This often happens at the expense of the infected cell. A radical but ultimately effective response to stop virus replication is for the infected cell to self-destruct via apoptosis although some viruses have evolved strategies to circumvent this . CytopatNearly all body cells have mechanisms to detect viruses  by pattern recognition receptors (PRRs) which recognize pathogen-associated molecular patterns (PAMPs) or molecules associated with viral and microbial pathogens but not typically found in host cells. PRRs that are important for detection of viruses include Toll-like receptors (TLRs), retinoic acid-inducible gene I (RIG-I), NOD-like receptors (NLRs), and other cytosolic virus sensors. Although these receptors are expressed in many types of cells, their activation in respiratory epithelial cells, typically among the first cell types to be infected, is critical in limiting virus spread and alerting the immune system to respond to the infection. Activation of PRRs in these cells by virus infection triggers production and release of type I and III interferons (IFNs) and other proinflammatory mediators  which initiate the host innate and adaptive immune response. Thus, the degree of PRR activation throughout the respiratory tract ultimately influences the degree of immune cell recruitment and proinflammatory mediator release and subsequently, any immunopathology ensued.Many different types of cellular sensors can detect viruses and induce the expression of the type I IFNs—IFN-α and IFN-β. Type I IFNs bind to the ubiquitously expressed IFNAR (IFN-α/β receptor), activating the JAK/STAT pathway . The typIn addition to their intrinsic antiviral activity, type I IFNs promote the initiation of the adaptive immune response by mediating dendritic cell (DC) activation, enhancing effector functions of lymphocytes and macrophages, and stimulating the humoral (antibody) response to neutralize virus. Type I IFNs have a significant impact on DCs by (1) stimulating the differentiation and maturation of monocytes into DCs, (2) promoting migration of DCs to lymph nodes through induction of chemokine receptors, 3) enhancing antigen (Ag) presentation to CD4 T cells and cross-presentation to CD8 T cells, and (4) upregulating costimulatory molecules requisite for naïve T cell activation and effector T cell generation [ enhancinType I IFNs directly enhance the functional activities of lymphocytes. They stimulate the production and secretion of IFN-γ (type II IFN) which in turn activates macrophages and phagocytosis, enhances Ag presentation by DCs, and directly limits viral replication . Type I Unlike IFNAR which is expressed in nearly all body cells, expression of the receptor for type III IFNs—IFNLR or interferon lambda receptor—and IFN-λ production is limited in the lungs primarily to epithelial cells . Like tyAs a consequence of their widespread effects on host immune responses, IFNs indirectly facilitate much of the inflammation and injury in the lungs during acute virus infection.Cytokines, chemokines, and other factors discussed in this section are also secreted by many types of immune cells (discussed later), and therefore will cause similar pathological or protective effects.Airway epithelial cells secrete a host of cytokines, chemokines, antimicrobial peptides and other factors in response to viral infection. Cytokines beyond the IFNs produced by airway epithelium include interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), granulocyte colony stimulating factor (G-CSF), and granulocyte macrophage-CSF (GM-CSF). IL-6 and TNF-α are potent proinflammatory cytokines that modulate many types of immune cells. IL-6 facilitates the transition from the innate to adaptive immune response by driving down neutrophil activity while concurrently promoting the recruitment, differentiation, and activity of monocytes and T cells . High ILG-CSF and GM-CSF both induce differentiation of myeloid lineage cells. G-CSF stimulates the production, differentiation, proliferation, and survival of neutrophils thereby mobilizing one of the first responders—neutrophils . GM-CSF Chemokines produced by respiratory epithelium stimulate the migration of both innate and adaptive immune cells to the lungs. IL-8/CXCL8 recruits neutrophils to the lungs, stimulates degranulation, and the subsequent release of cytotoxic and inflammatory mediators and may support neutrophil survival . ElevateIP-10/CXCL10 stimulates the chemotaxis of T cells, NK cells, and monocytes in addition to promoting monocyte and DC activation in concert with other cytokines. CXCL10/IP-10 may be protective in some cases since neutralization of IP-10 worsens disease caused by respiratory viral infection . In otheCCL5/RANTES recruits several types of leukocytes: T cells, NK cells, monocytes, DCs, and granulocytes. In addition to its chemotactic properties, CCL5 can also activate T cells, monocytes, NK cells, and DCs promoting a diverse range of immune responses important in controlling virus infection and generating protective immunity . In respRespiratory epithelial cells secrete numerous broad-spectrum antimicrobial/antiviral products that directly inhibit respiratory pathogens and in some cases, modulate immune responses. The most abundant antimicrobial factors in the lungs are lysozyme, lactoferrin, and secretory leukocyte proteinase inhibitor (SLPI) . LactofeAirway epithelial cells produce substantial amounts of β-defensins—cationic peptides that exert both antimicrobial and antiviral properties . β-defenRespiratory epithelial cells constitutively produce nitric oxide (NO) which is further increased by exposure to proinflammatory cytokines and respiratory viruses . NO and Alveolar type II epithelial cells constitutively produce and secrete four surfactant proteins . SP-B and SP-C contribute to the properties of surfactant which reduces the surface tension in the alveoli; thus, SP-B and SP-C serve a critical role in pulmonary function but have limited antiviral activity . HydrophAlthough respiratory epithelial cells are the primary targets of most respiratory viruses, infection of endothelium by virus can occur but is highly dependent on the viral pathogen and frequently is species-specific. For example, human RSV productively infects pulmonary vascular endothelium leading to endothelial cell activation but also cell death . In contRelease of proinflammatory cytokines, NO, and ROS from epithelium and leukocytes can enhance endothelial permeability and disrupt the integrity of the endothelial-epithelial barrier . Damage Regardless of infectivity of endothelium, endothelial cells play an essential role in leukocyte migration and can modulate the immune response, particularly early innate immune responses to virus infection. Extravasation of immune cells into the lungs from the circulation  is highly dependent on interactions between endothelium and leukocytes. Selectins and adhesion molecules requisite for extravasation are upregulated on endothelium in response to cytokines and growth factors secreted by respiratory epithelium and other neighboring cells . ActivatAlveolar macrophages—the tissue-resident macrophages of the lungs—are localized to the airspaces within alveoli, uniquely positioning them to respond to threats to the lower airways. During homeostasis, alveolar macrophages regularly encounter innocuous antigens. To prevent inappropriate inflammatory responses to these benign antigens, the lung environment maintains alveolar macrophages in a suppressive state. Negative regulation of alveolar macrophages is accomplished through expression of IL-10, alphaV/beta6 integrin, GM-CSF, CD200 receptor, and pulmonary surfactants by alveolar epithelium . Upon reAmong the first immune cells to arrive to the site of infection, neutrophils excel in eliminating infected cells and clearing microbial pathogens, dead cells, and debris. Neutrophils are generally not productively infected by respiratory viruses but do phagocytose virions, viral particles, and apoptotic bodies containing virus . Once phSufficient neutrophil recruitment and activity control virus dissemination and mitigate severe disease. However, an excessive neutrophil response can be detrimental to the host by causing uncontrolled inflammation and indiscriminate damage to respiratory epithelium . ROS, hyIn addition to their phagocytic and cytotoxic activities, neutrophils can mediate both innate and adaptive immune processes. Neutrophils secrete chemokines that can recruit more neutrophils to the site of inflammation; elevated levels of these chemokines have been associated with more severe lung pathology during infection , 20, 43.Although historically the role of neutrophils in the immune response has been centered on inflammatory and innate responses, novel roles for neutrophils in shaping the adaptive immune response have emerged. Some neutrophils can express B cell-activating cytokines that support B cell activity and may function as “B-cell helper” neutrophils . NeutropNatural killer (NK) cells are part of a family of innate lymphoid cells (ILC) that includes ILC1, −2 and −3. The role of ILC (other than NK cells) during respiratory virus infection is unclear although ILC2 have been reported to facilitate repair after lung injury . NK cellActivation of NK cells triggers cytotoxic activity which eliminates virus-infected cells. NK cells kill infected cells by releasing cytotoxic granules, engaging death receptors, or utilizing antibody-dependent cell-mediated cytotoxicity (ADCC)—a process in which antibodies bind viral proteins expressed on the surface of infected cells marking them for elimination , 48. AllNatural killer T cells (NKTs) represent a heterogeneous population of T cells that possess properties of both NK and T cells. NKTs may be classified as either type I (or invariant NKT cells) or type II NKT cells depending on the type of TCR expressed . The besIn contrast to αβ T cells, γδ T cells express TCRs of limited diversity composed of γδ chains instead of αβ chains and predominantly reside within mucosal or non-lymphoid tissues such as the skin, lungs, and intestine . BecauseDendritic cells (DCs) represent a diverse population of cells that serve as fundamental orchestrators of the immune response. DCs activate T cells, secrete various cytokines and chemokines, and promote protective adaptive immunity. Some DC subsets preferentially reside in either lymphoid or non-lymphoid tissue; while other subsets can migrate between these tissues. Under normal, non-inflamed conditions, respiratory DC populations can be found below and within conducting airway epithelium as well as in the lower airways within the walls of alveoli . Thus, tIn the lungs, CD103+ DCs are mostly associated with the mucosal epithelium of the conducting airways and function primarily as APCs, activating both CD8 and CD4 T cell proliferation and differentiation through antigen presentation , 61. CD1pDCs are potent producers of type I IFNs during respiratory viral infection, and thus play a role in the initial sensing of viral pathogens and initiating inflammatory and innate immune responses. Unexpectedly, in response to influenza virus, the absence of pDCs had no effect on viral clearance or disease severity . AlthougA large fraction of mo-DCs initially arrive in the lungs as “monocytes” which then differentiate into DCs in response to type I IFNs and direct infection with virus , 60. TheInflammatory monocytes are recruited from the circulation to the lungs following viral infection. They produce type I IFNs and chemokines and can differentiate into DCs (as discussed above) or macrophages which promote viral clearance, cytotoxic activity, and T cell activation . PotentiAlthough the innate immune response plays a significant role in initiating both antiviral responses and adaptive immune responses, ultimately the adaptive immune response is responsible for complete viral clearance by halting viral replication to prevent the generation of new virions and eliminating infectious virions. Because adaptive immune cells are dependent on early innate immune responses, this does not, however, mitigate the role of innate immune response to virus infection.B cells contribute to virus clearance predominantly through production of virus-specific antibodies which can (1) neutralize, opsonize, and inactivate virions or (2) initiate killing of infected cells. Preventing the spread of infectious virions from infected cells to neighboring cells is essential to controlling virus dissemination. Neutralizing antibodies produced by B cells effectively prevent free virions from invading uninfected cells by blocking surface proteins on the virus that bind to host receptors for cellular entry. Viruses coated in antibodies are also tagged for inactivation by complement proteins or elimination by phagocytic immune cells such as macrophages and neutrophils. Antibodies can bind to viral proteins expressed on the surface of infected cells, triggering the complement cascade and antibody-mediated cell-mediated cytotoxicity (ADCC)—processes that ultimately eliminate infected cells. Although essential to viral clearance, these cytotoxic processes if extensive can compromise respiratory function through the loss of airway epithelium, disruption of the epithelial-endothelial barrier, and accumulation of apoptotic bodies and cellular debris in the airways.FH) cells play a critical role in facilitating an effective B cell response during infection. Adoptively transferred CD4 T cells protect athymic mice  from succumbing to influenza infection. However, when CD4 T cells are adoptively transferred into SCID (severe combined immunodeficiency) mice that lack functional T and B cells, CD4 T cells are no longer sufficient for protection, and depletion of CD4 T cells during SARS-CoV infection coincided with a reduction in antibody production [FH cells promote the formation of germinal centers in secondary lymphoid tissues where virus-specific B cells mature, proliferate, undergo antibody class switching, and differentiate into either antibody-producing plasma cells or long-term memory cells. TFH cell numbers have been shown to correlate with the production of influenza-specific IgG and IgM antibodies after vaccination in healthy young adults [A subset of subset of CD4 T cells called follicular helper T , do not recover from influenza infection due to the inability to clear virus. SARS-CoV are relatively new pulmonary viruses whose pathogeneses are still being elucidated; however, research indicates that CD8 T cells are essential for virus clearance. Adoptive transfer of SARS-CoV-activated T cells into SCID mice enhanced survival and reduced pulmonary virus titers . Thus, tWhile essential, adaptive immune-mediated clearance of virus-infected cells can compromise the respiratory tract, and if unregulated, will cause catastrophic injury to the host tissue. This has been demonstrated in multiple models of adaptive immune deficient mice. Despite not being able to clear virus, RAG1 (recombination-activation gene-1) knockout mice that lack both functional T and B cells have delayed morbidity and mortality after high dose inoculation with influenza virus . FurtherNormal respiratory function is dependent on the preservation of pulmonary architecture and the endothelial-epithelial barrier between the airways and circulation. During viral infection, pulmonary architecture is frequently compromised primarily by the host immune response and to a lesser extent directly by virus replication in most cases. The extent of structural alteration, functional compromise and subsequently, clinical manifestations of infection are dependent on many factors but most prominently on the degree of virus dissemination throughout the respiratory tract.Often the URT is the initial site of viral replication since many respiratory viruses are inhaled or transferred by contact to the nasal mucosa. In healthy patients without pre-existing conditions, infections with common strains of rhinovirus, coronavirus, and adenovirus are typically limited to the upper airways. Symptomatic viral infection of the URT  Table  reflectsViruses capable of producing more severe infections such as influenza, proceed from the URT to the LRT where they disseminate to the bronchi and bronchioles and if not controlled, alveoli. Patient monitoring during respiratory viral infection corroborates this, as upper respiratory illness precedes any LRT (lower respiratory tract) symptoms. Among respiratory viruses, influenza viruses, SARS-CoV, and MERS-CoV are commonly capable of spreading into the bronchi and bronchioles in otherwise healthy individuals. However, in infants and young children, RSV frequently infects small conducting airways and is a leading cause of pediatric hospitalization worldwide . EnterovInfection of the bronchi and bronchioles leads to similar cellular changes and localized inflammation as in URT infections. However, because of the progressively decreasing diameter of LRT conducting airways, inflammation and injury in this compartment—e.g., desquamation of airway epithelial cells in response to RSV or airway epithelial cell hyperplasia during influenza infection—results in more severe compromise of respiratory function. The absence of ciliated epithelial cells in the LRT limits the expulsion of mucus, proteinaceous fluid, bacteria, and cellular fragments from these airways. This is compounded by enhanced epithelial mucus secretion, vasculature leak, edema, mucosal inflammation, and epithelial sloughing. The influx of inflammatory cells in response to signals from infected epithelium and tissue resident innate immune cells increases edema, vascular congestion, and tissue swelling further limiting air passage through smaller airways. The buildup of edematous fluid, mucus and fibrin plugs in small bronchi and bronchioles can restrict airflow to terminal airways further preventing efficient gas exchange. Increased pressure within the blood vessels leaks more fluid into the airspaces. As all these factors build up in the already restricted bronchi and bronchioles, they begin to clot and form plugs that prevent gas exchange. This manifests clinically as wheezing and shortness of breath. In serious cases, this can lead to difficulty in breathing which requires oxygen therapy. Typically with supportive care, bronchitis and bronchiolitis will resolve in a few days without lasting effects.While viruses such as RSV and Enterovirus D68 are capable of reaching the terminal airways, influenza viruses and the SARS-CoV and MERS-CoV coronaviruses more frequently reach the terminal airways, and thus are more likely to disrupt pulmonary function and cause pneumonia . The terNot surprisingly, the immunopathology of respiratory virus infection reflects a complex interplay involving direct effects by a given virus and other viral factors but also the response of resident respiratory cells and recruited innate and adaptive immune cells to the lungs. Studies carried out over the last several decades suggest that in most—but not all—instances, the host response to respiratory virus infection (rather than the direct effects of virus replication) dictates the type and extent of injury incurred.


Agrin, a multidomain proteoglycan and neurotrypsin, a neuronal serine protease, are important for forming (neuromuscular) synapses. Proteolytical activity of neurotrypsin produces a C-terminal fragment of agrin, termed CAF, of approximately 22 kDA molecular size which also circulates in blood. The presence of CAF in urine suggests either glomerular filtration or secretion into urine. Blood levels of CAF have been identified as a potential novel marker of kidney function. Here we describe that several nephron segments in the mouse kidney express agrin and neutrotrypsin in addition to the localization of both protein in the glomerulum. Agrin mRNA and protein was detected in almost all nephron segments and mRNA abundance was highest in the inner medullary collecting duct. Neurotrypsin mRNA was mostly detected in the thick ascending limb of the loop of Henle, the distal convoluted tubule, and the inner medullary collecting duct. Moreover, we show that the proximal tubule absorbs injected recombinant CAF by a process shared with receptor-mediated and fluid phase endocytosis. Co-injection of CAF with recombinant human transferrin, a substrate of the receptor-mediated endocytic pathway as well as with FITC-labelled dextran (10 kDa), a marker of fluid phase endocytosis, showed partial colocalization of CAF with both markers. Further colocalization of CAF with the lysosomal marker cathepsin B suggested degradation of CAF by the lysosome in the proximal tubule. Thus, the murine kidney expresses agrin and neurotrypsin in nephron segments beyond the glomerulum. CAF is filtered by the glomerulum and is reabsorbed by endocytosis by the proximal tubule. Thus, impaired kidney function could impair glomerular clearance of CAF and thereby increase circulating CAF levels. Agrin is a large proteoglycan of approximately 250 kDa that is highly expressed in brain and in the neuromuscular junction where it serves, among other functions, the positioning of acetylcholine receptors and synaptogenesis ,2. MoreoAgrin is cleaved by neurotrypsin, a serine protease, generating a 110 kDa fragment (CAF110) by cleavage at the alpha site, whereas cleavage at the beta site produces the 22 kDa C-terminal fragment (CAF22) . In humaSmall proteins are partly filtered in the renal glomerulum and can be either found in urine or are mostly reabsorbed by various endocytic mechanisms including receptor-mediated endocytosis and fluid-phase endocytosis. Both processes mainly occur in the proximal tubule. Receptor-mediated endocytosis involves the endocytic receptors megalin and cubilin binding a large variety of low molecular weight proteins including transferrin or cathepsin B ,16,17,18Thus, we aimed to examine whether the murine kidney expresses neurotrypsin and produces the small 22 kDa agrin fragment (CAF) and could thereby contribute to circulating or urinary CAF22 levels. Second, we investigated whether CAF22 could be filtered by the glomerulum and reabsorbed by the proximal tubule. Our results demonstrate that agrin and neurotrypsin are both present in murine kidney and that their mRNA expression profile along the nephron partly overlaps. Agrin protein is detected, as expected, in the glomerulum and in the basement membrane of all nephron segments and is paralleled by strong staining for the CAF22 fragment produced by neurotrypsin as indicated by its absence from the kidneys of neurotrpysin KO mice. Moreover, we provide evidence that injected CAF22 accumulates in the endocytic pathway of the proximal tubule and overlaps highly with markers of receptor-mediated endocytosis suggesting that CAF22 may be filtered in the glomerulum, reabsorbed by the proximal tubule via receptor-mediated endocytosis and degraded in lysosomes.-/-) were used. The generation and genotyping of neurotrypsin KO mice has been previously described , where Ct represents the cycle number at the threshold 0.06.To generate complementary DNA (cDNA), each RNA sample was diluted to 10 ng/μl and was used as a template for reverse transcription using the TaqMan Reverse Transcription Kit . Quantitative real-time (qRT-PCR) was performed on the ABI PRISM 7700 Sequence Detection System (Applied Biosystems). Primers for all genes of interest were designed using Primer Express from Applied Biosystems . Probes Determination of recombinant CAF22 from mouse body fluids by Western blot analysis was performed as described in Hettwer et al., 2013 [As reporter molecule, streptavidin-poly-HRP conjugate (PIERCE) was used. For detection, Chem Glow West substrate (Alpha Innotech) was applied and the chemiluminescence was recorded with a Stella imaging system .CAF22 variants were produced and purified as described  using cohttp://rsb.info.nih.gov/ij/).C57B1/6J mice were anesthetized with ketamine: xylazin and perfused through the left ventricle with phosphate-buffered saline (PBS) followed by paraformaldehyde-lysine-periodate (PLP) fixative . KidneysExpression of agrin and neurotrypsin mRNA was tested in various in mouse organs by semi-quantitative RT-PCR . mRNA ofWe examined further the relative mRNA expression of agrin and neurotrypsin in hand-dissected mouse nephron segments. Agrin mRNA was detected in the glomerulum, and all nephron segments. The highest levels of agrin mRNA were found in the inner medullary collecting duct . In cont-/- that partly colocalized with agrin in the basement membrane of the glomerulum and the renal tubules  to block lysosomal degradation. Plasma, urine and kidneys were collected 20 or 60 min after CAF injection. Immunoblotting of plasma samples for CAF22 readily detected CAF22 confirming successful injection into the vascular system  from the basolateral to the apical/luminal side of cells is unlikely to occur. We cannot entirely rule out that human recombinant CAF22 might behave differently from endogenous murine CAF22. However, the fact that endogenous CAF22 was detected in a similar subapical localization as the injected human recombinant CAF22 suggests similar handling by the kidney.More importantly, our data support the hypothesis that CAF22 circulating in blood is cleared, at least in part, from circulation by glomerular filtration. We injected recombinant human CAF22 i.v. into NTnsferrin ,16,27. MStaining of CAF22 in proximal tubules is reminiscent of other low molecular weight proteins that are reabsorbed from urine by the proximal tubule ,16,27. TIn summary, we provide evidence that CAF22 is cleared from circulation by glomerular filtration. Filtered CAF22 may then be reabsorbed by the proximal tubule by receptor-mediated and fluid-phase mediated endocytosis and targeted to lysosomes for degradation. These findings may imply that impaired renal function may rapidly increase circulating CAF22 levels and thereby facilitate the detection of reduced kidney function by elevated CAF22 serum levels. It may further suggest that increased urinary CAF22 levels may be another biomarker for reduced proximal tubular function similar to other low molecular weight proteins such as β2 microglobulin. However, this is due to further examinations.S1 TablePrimers and probes used for semi-quantitative RT-PCR.(DOC)Click here for additional data file.


Viridiplantae plastids participates in adapting the photosynthetic response to environmental stress, suggesting its involvement in the transition to terrestrial habitats. Although relatively rare, the loss or pseudogenization of plastid NDH genes is widely distributed across diverse lineages of photoautotrophic seed plants and mutants/transgenics lacking NDH function demonstrate little difference from wild type under non-stressed conditions. This study analyzes large transcriptomic and genomic datasets to evaluate the persistence and loss of NDH expression across plants.Key innovations have facilitated novel niche utilization, such as the movement of the algal predecessors of land plants into terrestrial habitats where drastic fluctuations in light intensity, ultraviolet radiation and water limitation required a number of adaptations. The NDH (NADH dehydrogenase-like) complex of Nuclear expression profiles showed accretion of the NDH gene complement at key transitions in land plant evolution, such as the transition to land and at the base of the angiosperm lineage. While detection of transcripts for a selection of non-NDH, photosynthesis related proteins was independent of the state of NDH, coordinate, lineage-specific loss of plastid NDH genes and expression of nuclear-encoded NDH subunits was documented in Pinaceae, gnetophytes, Orchidaceae and Geraniales confirming the independent and complete loss of NDH in these diverse seed plant taxa.The broad phylogenetic distribution of NDH loss and the subtle phenotypes of mutants suggest that the NDH complex is of limited biological significance in contemporary plants. While NDH activity appears dispensable under favorable conditions, there were likely sufficiently frequent episodes of abiotic stress affecting terrestrial habitats to allow the retention of NDH activity. These findings reveal genetic factors influencing plant/environment interactions in a changing climate through 450 million years of land plant evolution.The online version of this article (doi:10.1186/s12870-015-0484-7) contains supplementary material, which is available to authorized users. Key innovations have facilitated novel niche utilization, such as the movement of the algal predecessors of land plants into terrestrial habitats  where fl2 concentration or drought stress [During the light reactions of photosynthesis in cyanobacteria, algae and plants, photons excite pigment/chlorophyll molecules in photosystem II (PSII) at the stromal face of the chloroplast thylakoid membrane. This light energy, in the form of water derived electrons, is ultimately relayed to photosystem I (PSI) via the intermediate carriers of the electron transport chain to drive proton translocation across the membrane and into the thylakoid lumen. The resulting proton gradient is coupled to ATP synthesis via ATP Synthase with the concomitant reduction of NADP to yield NADPH by ferredoxin-NADP reductase. This process is referred to as linear electron flow and/or transport (LEF). While LEF is the primary pathway for conversion of photons into storable energy, CEF occurs exclusively around photosystem I and contributes to generation of the proton gradient. However the electrons that reduce the plastoquinone (PQ) in CEF are recycled directly from ferredoxin (Fd) allowing for the generation of ATP without the production of NADPH. The resulting high pH differential across the thylakoid membrane induces non-photochemical quenching (NPQ) allowing the dissipation of excess electrons under potentially unfavorable growth conditions including fluctuating, high intensity light, low COt stress ,7.Klebsormidium flaccidum genome, a streptophyte alga, revealed genes encoding both a functional NDH, with subunit genes in the plastid and nuclear genomes, and components of the nuclear encoded PGR5-dependent  pathway of CEF [Two independent pathways of CEF have been characterized across Streptophyta, the lineage that includes charophyte algae and land plants ,8. The gy of CEF .While both pathways clearly originated in the ancestor of plastids, and both rely on Fd-dependent reduction of PQ, the PGR5-dependent pathway is the main contributor to the pH differential and ultimately ATP generation in CEF . This main vivo [Cyclic electron flow is essential to efficient photosynthesis and complete inhibition of CEF severely affects LEF in vivo ; howeverin vivo -13. Phenin vivo -14, pronin vivo ,10,15.2 limitation, extended exposure to low temperatures and high or low light intensities revealed mildly deleterious phenotypes. Oryza sativa crr6 (required for NDH complex biogenesis) mutants lacked the post illumination increase in chlorophyll fluorescence that is a hallmark of Fd-dependent PQ reduction, and exhibited mild inhibition of all photosynthetic parameters measured and a diminution in plant biomass when grown at 20°C [ndhB in Marchantia polymorpha resulted in a PQ pool that was significantly more reduced at low light intensities relative to the wild type [Nicotiana tabacum inactivated for plastid encoded NDH-B showed a reduction in transient chlorophyll fluorescence following actinic illumination, but otherwise performed normally [Under favorable growth conditions disruption of NDH has little effect. However CO at 20°C . The disild type . Nicotianormally . Repeatenormally .Like many plastid-localized, multi-subunit complexes, both plastid- and nuclear-encoded proteins assemble to form the NDH complex . AlthougBased on phenotypes of mutants/transgenics, NDH function appears to be dispensable under favorable growth conditions; however, the plastid-encoded genes for NDH subunits are conserved across the phylogeny of Streptophyta suggesting a strong selective advantage in retention of NDH function . This stArabidopsis NDH-related coding sequences and the results of the survey mapped in a phylogenetic context. Queries included constituents of each of the four major subunits containing nuclear proteins, the Lhca (light harvesting complex associated) proteins involved in tethering NDH to PSI during supercomplex formation, assembly and accessory proteins, ndhF transcription factor Sig4 and proteins involved in maturation and editing of NDH transcripts in plastids  and five in the nucleus (ndhL-S) [ndhF transcription factor Sig4, and PPR proteins involved in processing of plastid NDH polycistrons and RNA editing  . Additiog Figure . The co-Erodium (Geraniaceae). It appears that another species in the Geraniales, Melianthus villosus, is undergoing NDH loss as four of its plastome-encoded NDH genes are pseudogenes [Najas Vallisneria, Thalassia) with two further losses likely among the marine ‘seagrasses’ in the order Alismatales  [Despite near ubiquity, a number of exceptional cases exist among photosynthetic species where the NDH genes have been pseudogenized or are entirely missing from plastid genomes Figure A. Among udogenes . Plastidhibolis) ,24.FigurArabidopsis reference sequences may have also allowed some transcripts to go undetected due to high levels of divergence between the reference and the taxa examined.Of the non-NDH plastome genes that have been lost or pseudogenized, several have been detected in the nuclear genome where their expression is regulated by nuclear factors and subsequent products targeted back to plastids. Although several groups have suggested that plastid NDH pseudogenes may be functionally represented by nuclear sequences -22,25, nlhca5 , however the transcript was not detected using the present data mining strategy. Of course these species are known to lack NDH and likely their lhca5 sequences are not similar enough to be detected using Arabidopsis queries. Even in more closely related groups, i.e. gymnosperms and angiosperms, there is a margin for error although calls of presence or absence are expected to be more reliable between more recently diverged groups. The lhca5 transcript was detected in seven of 11 gymnosperm species that are competent for NDH CEF  lacks a cyanobacterial homolog and is nearly ubiquitous across the species surveyed, apparently arising at the node leading to Zygnematophycaeae, the proposed sister lineage to land plants Figure . Althougectively ,38. Giveectively ,39, the rbcL and matK, and reflects the relationships identified in Lee et al. [Gnetum and Ephedra and a single detectable pseudogene in Welswitchia [PnsL5 and, at least for the Pinaceae species, ndhS  -45. Accoe et al. , placingswitchia ,48 suggeswitchia . If indeg others .Masdevallia outside the clade of higher epidendroids that it groups with here [sig4 transcripts were detected among the orchid species that retain NDH function as well as in Amborella  of dhD gene  suggestiplastome . This cle Figure D.These data allow insight into the distribution and timing of NDH gene loss across seed plants and suggest at least limited dispensability of NDH function. In no case were functional nuclear copies of plastome NDH sequences identified but rather the degradation and loss of nuclear-encoded interacting proteins was revealed. The broad phylogenetic distribution of NDH loss and the subtle phenotypes of mutants suggest a high propensity for gene loss and may be indicative of its limited biological significance in contemporary plants . On an eThe timing of changes in the NDH gene complement supports the hypothesis that the NDH system, among other adaptations, has been involved in the transition to terrestrial habitats and possibly other key innovations throughout 450 million years of land plant evolution. Given the present outlook on climate change a deeper understanding of the genetic factors influencing the adaptation of land plants to novel conditions will inform management programs.Tissue samples for species of Geraniales were harvested from the living collection at University of Texas at Austin. All specimens have been vouchered and deposited in the UT Plant Resources Center (TEX-LL). Voucher numbers are listed in Additional file Apostasia odorata was collected from Yunnan, China. Cypripedium formosanum was obtained from highland experimental farm, National Taiwan University in Mei-feng, Taiwan. Three commercial Orchidaceae species were purchased from a local grower in Taiwan, two Epidendroideae  and one Orchidoideae (Goodyera fumata). These three species were grown in the greenhouses at Academia Sinica, Taipei, and National Chung Hsing University, Taichung, Taiwan.Pelargonium x hortorum was isolated and cDNA libraries constructed following the protocols described in Zhang et al. [A. odorata, C. formosanum, M. picturata, G. fumata, E. pusilla and H. longidenticulata) were constructed using the Illumina TruSeq™ Stranded mRNA HT, insert size was 300 bp. The libraries were sequenced on Illumina NextSeq™ 500 paired-end system using a NextSeq™ 500 Mid output kit . Sequence data of Geraniales and the five species of orchid were preprocessed and assembled as described in Zhang et al. [http://sourceforge.net/projects/trinityrnaseq/) using the parameters “--JM 100G --full_cleanup --min_contig_length 200 --CPU 24” on a 24-core 3.33 GHz linux work station with 1 TB memory at the Texas Advanced Computing Center .Total RNA from newly emerged leaves of 26 species in Geraniales and four tissues  of g et al. . Transcrg et al. . TranscrArabidopsis were downloaded from TAIR [Arabidopsis transcriptome using BLASTX with default settings, and the gene was considered present if the top hit was the corresponding reference gene. If the top hit was not the corresponding gene, manual inspection of these candidate genes was performed to resolve potential chimeric assembly problems and the candidate genes were confirmed again with BLASTX. All newly generated sequences have been submitted to NCBI GenBank, accession numbers are in Additional file The protein sequences of reference genes of rom TAIR ; accessiAcorus and 26 species of Geraniales plus Arabidopsis were generated by RAxML with parameters “raxmlHPC-PTHREADS-SSE3 -f a -x 12345 -p 12345 -T 12 -m GTRGAMMAI -N 100” using 12 plastid genes , and bootstrap values were generated using RAxML with 100 replicates and the above settings. The gymnosperm phylogeny of 11 species plus Psilotum was generated by the same parameters except that two sequences, matK and rbcL were employed. For Figure The phylogenies of 11 species of orchids plus Divergence time estimates were derived from previous studies of gymnosperms ,56, Orchhttp://dx.doi.org/10.5061/dryad.h9m07). Sequence data are available in the Short Read Archive at GenBank, http://www.ncbi.nlm.nih.gov/sra; accession numbers are listed in Additional files Phylogenetic datasets are available in Dryad Digital Repository (


Background: Structural brain abnormalities in schizophrenia have been repeatedly demonstrated in magnetic resonance imaging (MRI) studies, but it remains unclear whether these are static or progressive in nature. While longitudinal MRI studies have been traditionally used to assess the issue of progression of brain abnormalities in schizophrenia, information from cross-sectional neuroimaging studies directly comparing first-episode and chronic schizophrenia patients to healthy controls may also be useful to further clarify this issue. With the recent interest in multisite mega-analyses combining structural MRI data from multiple centers aiming at increased statistical power, the present multisite voxel-based morphometry (VBM) study was carried out to examine patterns of brain structural changes according to the different stages of illness and to ascertain which (if any) of such structural abnormalities would be specifically correlated to potential clinical moderators, including cumulative exposure to antipsychotics, age of onset, illness duration and overall illness severity.Methods: We gathered a large sample of schizophrenia patients  and controls (151) from four previous morphometric MRI studies (1.5 T) carried out in the same geographical region of Brazil. Image processing and analyses were conducted using Statistical Parametric Mapping (SPM8) software with the diffeomorphic anatomical registration through exponentiated Lie algebra (DARTEL) algorithm. Group effects on regional gray matter (GM) volumes were investigated through whole-brain voxel-wise comparisons using General Linear Model Analysis of Co-variance (ANCOVA), always including total GM volume, scan protocol, age and gender as nuisance variables. Finally, correlation analyses were performed between the aforementioned clinical moderators and regional and global brain volumes.Results: First-episode schizophrenia subjects displayed subtle volumetric deficits relative to controls in a circumscribed brain regional network identified only in small volume-corrected (SVC) analyses , including the insula, temporolimbic structures and striatum. Chronic schizophrenia patients, on the other hand, demonstrated an extensive pattern of regional GM volume decreases relative to controls, involving bilateral superior, inferior and orbital frontal cortices, right middle frontal cortex, bilateral anterior cingulate cortices, bilateral insulae and right superior and middle temporal cortices . GM volumes in several of those brain regions were directly correlated with age of disease onset on SVC analyses for conjoined (first-episode and chronic) schizophrenia groups. There were also widespread foci of significant negative correlation between duration of illness and relative GM volumes, but such findings remained significant only for the right dorsolateral prefrontal cortex after accounting for the influence of age of disease onset. Finally, significant negative correlations were detected between life-time cumulative exposure to antipsychotics and total GM and white matter volumes in schizophrenia patients, but no significant relationship was found between indices of antipsychotic usage and relative GM volume in any specific brain region.Conclusion: The above data indicate that brain changes associated with the diagnosis of schizophrenia are more widespread in chronic schizophrenia compared to first-episode patients. Our findings also suggest that relative GM volume deficits may be greater in (presumably more severe) cases with earlier age of onset, as well as varying as a function of illness duration in specific frontal brain regions. Finally, our results highlight the potentially complex effects of the continued use of antipsychotic drugs on structural brain abnormalities in schizophrenia, as we found that cumulative doses of antipsychotics affected brain volumes globally rather than selectively on frontal-temporal regions. •Structural brain changes are more widespread in chronic than first-episode schizophrenia.•Regional GM deficits may be greater in cases with earlier age of onset.•Illness duration seems to impact in some specific frontal structural brain changes.•Antipsychotics seem to affect brain volumes globally rather than regionally. HoweverAlthough longitudinal MRI studies have been traditionally used to address the question of progression of brain abnormalities in schizophrenia, information from cross-sectional neuroimaging studies may also provide useful contributions to further clarify this issue . Cross-sRecently, there has been growing interest in multisite mega-analyses combining structural MRI data from multiple centers through a uniform procedure of preprocessing and analysis, aiming at increased sensitivity and statistical power to detect group differences and investigate within-group correlations with clinical variables . Three oIn the present cross-sectional, multisite VBM study, we sought to replicate findings of brain structural changes in schizophrenia reported in previous large-sized VBM investigations  by compa22.1The present investigation gathered a large sample of schizophrenia patients and controls from four previous morphometric MRI studies carried out in medical institutions of two cities located in the same geographical region of Brazil (São Paulo State), namely the University of São Paulo Medical School in São Paulo City, and the University of São Paulo Medical School in Ribeirão Preto City. Such MRI studies had been carried out to address different research questions and their separate results have been published elsewhere . All parChronic schizophrenia patients were recruited at local outpatient clinics at the Institute of Psychiatry of University of São Paulo  and the Inclusion criteria for schizophrenia subjects were: diagnosis of schizophrenia according to DSM-IV criteria on the basis of interviews with the Structured Clinical Interview for DSM-IV Axis I Disorders for all studies ; and an In three of the MRI studies , healthyExclusion criteria specific for the control group were personal history of other Axis I disorders (as determined by interviews using the SCID)  or psychDetailed sociodemographic and clinical data were retrieved from schizophrenia patients of each site. Although different scales for psychopathology severity were originally published in each study , scores We also collected data on antipsychotics usage . Given that many schizophrenia patients are maintained on antipsychotic treatment for long periods, it is useful to quantitatively assess cumulative medication exposure, especially because recent studies have highlighted the potential association between structural brain changes and use of antipsychotics in schizophrenia . In orde2.2http://www.fil.ion.ucl.ac.uk/spm/software/spm8/) run in MATLAB . Briefly, following manual reorientation in order to place the anterior commissure at the origin of the three-dimensional Montreal Neurological Institute (MNI) coordinate system, T1-W images were segmented into tissue classes of GM, white matter (WM), and cerebrospinal fluid (CSF) by using the standard unified segmentation model in SPM8  implemented for SPM8 on the native space gray, white, and cerebrospinal fluid segmentations for each subject. Similarly, to compute total intracranial volume (TIV) using SPM's unified segmentation and spatial normalization procedure, probabilistic tissue class images can be integrated, with the estimated TIV simply being the sum of GM, WM and CSF volumes , and smoothed . In order to compensate for the effects of spatial normalization, GM and WM normalized images are modulated by multiplying each voxel value by its relative volume before and after warping, which allows obtaining relative volume for each voxel; the maps so produced are referred to as images of GM and WM volumes in order to distinguish them from the images of “concentration” or “density” that result if the modulation stage is omitted . Therefo volumes , as here volumes . However volumes . For ins volumes , these e volumes . It is e volumes .Finally, in order to ensure that all images were segmented adequately, subject outlier detection was performed within and across the four sites using a Pearson correlation, by comparing the degree to which subjects were related to the average smoothed GM map. Outliers were defined as subjects deviating more than 2% from the mean GM volumes, according to criteria defined elsewhere ; no outl2.3t test and categorical variables through Pearson's χ2 test.Group differences in demographic characteristics were assessed with IBM SPSS Statistics, version 21 . The significance threshold was set at 0.05, two-tailed; continuous variables were analyzed through the Regarding the VBM analyses, group effects on regional GM volume were investigated through whole-brain voxel-wise comparisons of the preprocessed GM images of schizophrenia patients and controls by using General Linear Model (GLM) Analysis of Co-variance (ANCOVA), which always included total GM volume, scan protocol, age and gender as nuisance variables (hereafter defined as “standard covariance”). Moreover, within-group correlations were performed between regional brain volumes and potential clinical moderators , life-tIn order to give a general overview of the main VBM findings of the study, regardless of as to whether they were significant in the whole-brain exploratory analyses  or in the (more flexible) SVC-based analyses, all figures were constructed based on statistical parametric maps thresholded at p < 0.001 (uncorrected for multiple comparisons) with a minimum cluster extent of 10 voxels, as employed elsewhere .33.1Sociodemographic and clinical data are listed in 3.2Patients and controls did not differ in terms of total GM or WM volumes, TBV and TIV . PatientBivariate correlation analyses revealed significant negative correlations between life-time cumulative exposure to antipsychotics and global volumes for both the conjoined group  and for the chronic schizophrenia group . When these analyses for antipsychotic exposure were repeated in the chronic schizophrenia group with covariance for disease duration, both the negative correlations with total GM  and TBV  retained statistical significance, while the negative correlations with total WM  and TIV  remained as significant trends. Repeated analyses for the conjoined four groups with covariance for disease duration indicated trend significance for the negative correlation between total GM and antipsychotic exposure .For the conjoined group only, a significant positive correlation was found between CSF volumes and duration of disease . However, this finding lost its statistical significance after covariance for antipsychotic exposure and age of disease onset .No significant correlations between severity of disease and global volumes were found for any group.3.3The VBM analysis of main effect of diagnosis, under the strict threshold of p < 0.05 (FWE-corrected) and minimum cluster extent of 20 voxels over the whole brain, revealed a pattern of cortical volume deficits restricted to the bilateral insulae, right striatum and left inferior frontal gyrus when contrasting all schizophrenia patients versus healthy controls , Table 4There were no regional GM volume deficits in the first-episode schizophrenia sample relative to controls at FWE-corrected analyses over the whole brain. Small volume-corrected analyses revealed reduced GM volumes in first-episode schizophrenia patients in the following brain regions predicted a priori to show abnormalities: bilateral insulae, right amygdala, right striatum and right hippocampus/parahippocampal gyrus . The voxThe sample of chronic schizophrenia patients presented a more extensive pattern of regional GM volume decreases relative to controls a, involvIn comparison to first-episode patients, chronic schizophrenia patients showed extended GM decreases b within At the p < 0.05 (FWE-corrected) statistical threshold over the whole brain, there were no significant findings of regional GM volume increases detectable in the between-group comparisons above.Regarding the different methods of covariation for total GM, although subtle variations in the magnitude of VBM findings were found in comparison to our standard approach, the neuroanatomical pattern of regional changes was maintained, suggesting that results of this study were not substantially affected by the method of global covariation.3.4When we repeated the above comparison between chronic and first-episode schizophrenia patients including age of onset as a confounding covariate, we detected a similar pattern of regional GM volume deficits in chronic patients relative to first-episode subjects. There were extensive GM decreases within the bilateral superior and middle temporal cortices, right superior and inferior frontal cortices, left precentral and postcentral cortices, and right occipital structures ; other brain regions involved were the right thalamus, bilateral inferior parietal cortices and left superior parietal cortex  . Small vIn the investigation of within-group correlations between regional brain volumes and age of onset, we found  positive correlations involving the bilateral insulae and anterior cingulate and dorsolateral prefrontal cortices, the right hippocampus/parahippocampal gyrus and right superior temporal gyrus for the conjoined four groups, after accounting for the effects of life-time cumulative exposure to antipsychotics. When additionally accounting for the effects of illness severity , the foci of positive correlation in the left anterior cingulate cortex, bilateral dorsolateral prefrontal cortices, left insula, right hippocampus/parahippocampal gyrus and right superior temporal gyrus all remained statistically significant. The voxel extent of these findings and their corresponding Z-scores are provided in 3.5No significant positive or negative correlations between other potential clinical moderators  and regional brain structural changes were found for any of the patient subgroups when analyses were performed under the strict threshold of p < 0.05, FWE-corrected over the whole brain.In regard to small volume-corrected analyses, one cluster of significant negative correlation was detected in the first-episode schizophrenia group between duration of disease and GM volume of the left superior temporal gyrus . The voxel extent of this finding and its corresponding Z-score are provided in In the conjoined four groups, small volume-corrected analyses revealed several foci of significant negative correlation between duration of illness and GM volumes in the bilateral insulae, dorsolateral prefrontal cortices and hippocampi/parahippocampal gyri, after accounting for effects of lifetime cumulative exposure to antipsychotics . There w4This large cross-sectional VBM multisite mega-analysis compared groups of schizophrenia patients (n = 161) and healthy controls (n = 151) recruited in the same geographical region. While first-episode schizophrenia subjects displayed subtle and highly circumscribed GM volume reductions in the brain relative to controls, chronic schizophrenia patients had more significant and widespread cortical GM deficits. In some brain foci, relative regional GM volumes were directly correlated with age of disease onset. Also, global brain volumes in schizophrenia were inversely related to the degree of exposure to antipsychotic drugs. While the results of our between-group comparisons provide replication to the findings of a number of previous MRI studies of schizophrenia , the rel4.1The comparison of first-episode schizophrenia subjects against controls identified regional GM volume deficits in the former group in a circumscribed network of brain regions including the insula, temporolimbic structures  and striatum. Volume reductions in these brain regions have been often reported in previous VBM investigations of schizophrenia, despite some degree of variation across separate studies and meta-analyses . In our The involvement of the insula in the pathophysiology of schizophrenia has been sometimes seen simply as an “extension” of primary frontal and temporal lobe pathology . The relThe insula plays a critical role in emotional processing, integration of sensory  input with the limbic system, pain perception, as well as being uniquely involved in interoception and neural representations of the self . Since tWhile none of the large multi-site cross-sectional VBM investigations of schizophrenia to date assessed first-episode schizophrenia patients separately, a number of single-site MRI studies have evaluated relatively large first-episode schizophrenia samples . In the The first-episode schizophrenia sample included in the present study is the same evaluated previously by our group in a VBM investigation using a less optimized (SPM 2) version of the image processing software employed herein . The fac4.2Whether schizophrenia is characterized or not by progressive structural brain changes is of importance to further clarify the pathophysiology of the disorder. The neurodevelopmental hypothesis proposes that schizophrenia is a consequence of a disruption in early brain development, and that an interaction between early brain insults and later environmental factors would trigger the emergence of psychotic behavior and other overt symptoms of schizophrenia from early adulthood onwards . EvidencIn concordance with our initial hypothesis, we detected a more widespread pattern of structural GM changes in chronic than first-episode schizophrenia patients in comparison to controls. Such pattern of results might be taken to support the notion that brain changes associated with the diagnosis of schizophrenia progress over the course of the illness. Our findings of extensive GM deficits among chronic schizophrenia patients are consistent with the results of previous meta-analyses that performed indirect comparisons between groups of first-episode and chronic schizophrenia patients . Our finThe convergent finding of large fronto-temporolimbic GM volumetric deficits in our analyses and the aforementioned studies of chronic schizophrenia subjects  is consiOne issue when interpreting results of widespread brain volume deficits in samples of chronic schizophrenia patients is the extent to which such findings may vary specifically as a function of greater illness severity, rather than reflecting the continuation of schizophrenia over time. Cases of schizophrenia that are more severe could conceivably display, at the time of disease onset, greater brain abnormalities that may not necessarily progress over time . In our The notion that greater GM volume deficits in schizophrenia depends on clinical severity rather than simply reflecting illness chronicity is also supported by a number of recent longitudinal MRI studies of schizophrenia which have suggested that significant progression of brain volume deficits in schizophrenia over time relative to controls may be present predominantly in a subset of patients with more severe illness and worse prognosis . Include4.3The continued use of antipsychotic medication has been previously highlighted as a potentially contributing factor influencing on the progression of GM volume deficits in chronic schizophrenia patients . This poIn contrast with the previous multisite VBM-based mega-analyses of schizophrenia , we wereIt should be noted that we found no significant correlations between indices of antipsychotic exposure and relative regional GM volume deficits in schizophrenia patients. Therefore, we cannot attribute the findings of regional GM volume deficits located in fronto-temporal cortices in chronic schizophrenia subjects to the continued exposure to antipsychotic drugs . In lighThe combined findings of our study regarding medication use, with effects on global rather than regional brain volumes in schizophrenia, are pioneer for a cross-sectional VBM mega-analysis. Nevertheless, this remains an open question, yet to be scrutinized in further mega-analyses of cross-sectional and longitudinal MRI studies.4.4Our study has several strengths. With our multisite study design, we gained statistical power by pooling together large samples of schizophrenia patients and demographically matched controls. The potentially confounding effects of heterogeneous image acquisition protocols were addressed by using a uniform image preprocessing pipeline and by entering site/scanning protocol in the GLM used for the statistical analysis. Previous technical reports have demonstrated that results of analyses performed by pooling together MRI datasets from multiple centers are not substantially confounded by scanner differences when such methodological approach is applied . SimilarOn the other hand, we acknowledge that our study is not without limitations. First, while attempting to assess the influence of various clinical moderators on GM volumes, we were unable to address the role of some other potentially relevant confounders evaluated in the previous neuroimaging literature on schizophrenia, such as changes in patterns of antipsychotic usage, treatment adherence, duration of untreated psychosis and substance abuse . Also, w4.5In conclusion, the results of this multi-site VBM study of schizophrenia carried out in Brazil reinforce the notion that structural brain changes associated with the diagnosis of schizophrenia are more widespread in chronic schizophrenia compared to first-episode patients, mainly affecting fronto-temporal regions and the insula. Our VBM findings of significant correlations also suggest that relative GM volume deficits may be greater in (presumably more severe) cases with earlier age of onset, as well as varying as a function of illness duration in specific frontal brain regions. Finally, our results highlight the potentially complex effects of the continued use of antipsychotic drugs on structural brain abnormalities in schizophrenia, as we found that cumulative doses of antipsychotics affected brain volumes globally rather than selectively on frontal-temporal regions.U.S.T. was supported by a PhD grant from Foundation for the Support of Research in the State of São Paulo , Brazil (process 11/18631-1). JASC is partly funded by a Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq - Brazil) Productivity fellowship. G.F.B. is partly funded by CNPq, Brazil. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.


Intensive care unit (ICU) admission may be required in cancer patients for management of acute illnessess associated with underlying malignancy or complications of cancer therapy. The higher mortality rates found in this population, in comparison to non-cancer critically ill patients, may be attributable to confounding bias due to a higher incidence of multiple organic dysfunction in those patiens with malignant neoplasms; however data regarding this hypothesis are scarce.To compare the crude and propensity score matched mortality rates between cancer and non-cancer adult patients admited to a mixed medical-surgical ICU.We conducted a retrospective analysis of a comprehensive longitudinal ICU database from a tertiary referral hospital in Southern Brazil. All adult patients who were admitted to the ICU from January 2001 to December 2008 were evaluated. Crude and propensity score adjusted all cause 30-day mortality rates of critically ill cancer patients were compared with those of critically ill patients without cancer.A total of 4221 patients were evaluated. The overral population 30-day mortality was 12.2 %. In a Cox regression analysis, crude mortality rates were higher among cancer patients . However, after adjusting for propensity score, the 30-day mortality rates of cancer and non-cancer patients were similar .The present study showed that the higher crude 30-day mortality found in ICU cancer patients, when comparable to non-cancer patients, was a result of confounding. The propensity score matched analysis showed no evidence of excess of mortality in critically ill patients due to cancer diagnosis.


At the Amazon estuary, the oldest logging frontier in the Amazon, no studies have comprehensively explored the potential long-term population and yield consequences of multiple timber harvests over time. Matrix population modeling is one way to simulate long-term impacts of tree harvests, but this approach has often ignored common impacts of tree harvests including incidental damage, changes in post-harvest demography, shifts in the distribution of merchantable trees, and shifts in stand composition. We designed a matrix-based forest management model that incorporates these harvest-related impacts so resulting simulations reflect forest stand dynamics under repeated timber harvests as well as the realities of local smallholder timber management systems. Using a wide range of values for management criteria , we projected the long-term population dynamics and yields of hundreds of timber management regimes in the Amazon estuary, where small-scale, unmechanized logging is an important economic activity. These results were then compared to find optimal stand-level and species-specific sustainable timber management (STM) regimes using a set of timber yield and population growth indicators. Prospects for STM in Amazonian tidal floodplain forests are better than for many other tropical forests. However, generally high stock recovery rates between harvests are due to the comparatively high projected mean annualized yields from fast-growing species that effectively counterbalance the projected yield declines from other species. For Amazonian tidal floodplain forests, national management guidelines provide neither the highest yields nor the highest sustained population growth for species under management. Our research shows that management guidelines specific to a region’s ecological settings can be further refined to consider differences in species demographic responses to repeated harvests. In principle, such fine-tuned management guidelines could make management more attractive, thus bridging the currently prevalent gap between tropical timber management practice and regulation. One underlying tenet of sustainable forest management (SFM) is that forests should provide for multiple needs continuously into the future . For remAt present, hundreds of small-scale, family-run timber operations in the Amazon estuary are supplied by many more thousands of smallholders that rely on timber as a source of income ,11. ThesMost research on the impacts of timber harvest/management on tropical forests have focused on either harvest damage, post-harvest effects on growth, recruitment and mortality independently –19. HencWe used a matrix-based management simulation model to evaluate the prospects for sustained timber production in the tidal floodplain forests of the Amazon estuary. Matrix population models are often used to simulate long-term impacts of forest harvest and management . However2 Mazagão watershed at the Western side of the Amazon estuary  characterized by freshwater tidal fluctuations of 2–3 m. This area has a long history of small-scale timber use . After each simulated harvest, we applied the ratio to all populations based on the number of trees harvested. Because we found no clear size-related patterns in residual stand mortality, the residual stand mortality ratio used in the model is indiscriminate of merchantable and unmerchantable trees and size class.All trees surviving a harvest are modeled using matrices that include post-harvest demographic effects. First, we attempted to define the length of post-harvest effects on growth using standard least square regression of post-harvest diameter increments against years since logging. This analysis revealed no clear impact of time since harvest on growth across harvested plots, suggesting post-harvest effects last longer than the amount of time between harvest and monitoring in our harvested plots. Based on limited literature indicating that post-harvest effects on growth generally do not last longer than 10 years ,34, we cBesides using harvested plot data to modify unharvested plot matrices to project population dynamics post-harvest, we computed treatment differences in individual/ environmental factors that could influence demography including flooding and light regimes  to exploEvery management simulation included a projection period of 120 years. Population annual growth rates (λ) under simulated harvest regimes were calculated empirically from model projections.The model also calculated the necessary compensatory recruitment needed to be planted yearly to stabilize population size at pre-harvest levels.3 ha-1 yr-1 . Each of these management regimes was applied for the duration of the 120 yr projection period to a hypothetical 1ha forest stand that represents the average size distribution for each species across all surveyed plots.Each of the 1440 management regimes we simulated represents combinations of possible management criteria: cutting cycles of 10–40 years; MCDs of 30–70 cm DBH; harvest intensity of 0.5–0.9 of stand volume; minimum species density (MD) of 0.03 and 1 trees/ha per harvest species; high and low seed-tree quality ; and simulations with or without a harvest volume limit of a 1 ma-1 yr-1 . The ran5 AY), annualized yield of third harvest (H3 AY), mean annualized yield (mean AY) during the 120 years excluding the first harvest, and the summation of the ranked mean annualized yield and the ranked λH (ranked AY and λH). First harvests were excluded from mean AY because annualized yields are not computable.To evaluate which management regimes produced the best long-term yield and least long-term population impacts, all management regime simulation outcomes were rated according to 4 different STM indicators: annualized yield of fifth harvest  and no consistent treatment response for the adult size classes (>20 DBH). Hence, we classified species post-harvest growth effects into juvenile and adult values. Analysis of individual tree / environmental factors that could influence demography revealed that a larger proportion of 5–20 cm DBH trees were observed under high light conditions in harvested plots compared to unharvested plots . Only Mora paraensis showed a recruitment boost into the 5 cm DBH class after logging. Post-harvest increases in mortality were not apparent for any focal species and were hence not included in the management simulation model. Lastly, post-harvest diameter growth was not clearly related to maximum unharvested forest growth, precluding the use of maximum growth as post-harvest growth [Nearly all species showed a positive diameter growth response after logging. Surprisingly, M. paraensis and C. spruceanum (species with sufficient data to parameterize matrices for harvested and unharvested treatments independently) showed that post-harvest λ increases are large for M. paraensis  but not for C. spruceanum . The LTRE analysis using these independent harvested/ unharvested matrices shows that the increases in M. paraensis λ are mostly due to increases in growth of the smaller size classes and increases in fertility by the middle size classes, despite larger responses observed in the larger size classes  for subsequent harvests. Similarly, Callycophyllum spruceanum yields 9% of the first harvest volume but by the fifth harvest yields 4% of total harvest volume. On the other hand, yields of historically harvested C. guianensis and V. surinamensis increase from 2% (0.5 m3) during the first harvest to 23% (7 m3) during the fifth harvest for the two species combined.Model outputs show that under the management requirements of Brazilian law, on average 50.9 mV. surinamensis and P. filipes). In contrast, species with low λs have a low ability to recover between harvests and therefore have merchantable proportions that drop after each harvest . The remaining species with λs slightly above 1 stabilized at pre-harvest merchantable proportions of approximately 0.6–0.7.The exclusion of volume-based harvest limits generally increased the rate of decline of merchantable proportion in modeled projections. For instance, without volume-based harvest limits the 30 yr Brazilian legal management regime led to a decrease in the merchantable proportion for all harvested species, but with degree of recovery related to λs . The rap3 with a MCD of 30 cm DBH, 0.9 harvest intensity, and no harvest volume restrictions) but also resulted in very low future harvest volumes (6.2 m3 per ha at fifth harvest under a 30-yr cutting cycle). The optimal management regimes for the four STM indicators varied in terms of most management criteria except for the common prescription of short 10-year cutting cycles and, surprisingly, no volume limits per harvests (3 AY indicator) included MCDs of 50 cm DBH or greater. Most of the top ranked AY and λH management regimes required a MCD of 60 cm DBH and volume harvest limits. These STM regimes showed little indication of yield decreases over time and required little additional recruitment to compensate for population growth decreases. All optimal management regimes for the other three STM indicators showed signs of declining yields and higher compensatory recruitment needs. The Brazilian legal management regimes resulted in lower mean AY than any of the optimal STM regimes while still requiring similar numbers of compensatory recruitment, indicating a sub-optimal resource utilization.The most intensive management regime resulted in the largest first harvests . Higher MCD has a small positive effect on mean AY of M. paraensis, but strong effects on all other species. However, this relationship was curvilinear with mean AY peaking around a MCD of 50–60 for most species. Higher MD decreases the mean AY of the least common species , with its negative effects increasing with species scarcity. Most species-specific λH increase with higher MCD and the inclusion of harvest volume limits but also suffer mild decreases with increasing harvest intensity. MD has a positive effect on λH for the three least common species.Stand mean AY decreases with longer cutting cycles and the inclusion of harvest volume limits, but peaks at a MCD of 50 cm . While oC. spruceanum (a light-demanding species) may be due to relatively small canopy openings that benefited its shade tolerant competitors [C. spruceanum because damage assessment from harvest monitoring and wider observations from recently logged areas showed minimal damage affecting species indiscriminately.Results from monitoring of harvesting operations show that demographic consequences of selective harvesting were small and likely a result of some damage from non-mechanized operations in relatively vine-free forests. The observed weak harvest effects are in agreement with upland forest studies that show tree fall gaps have small and short-lived demographic effects ,19. Crowpetitors . Loggingbut see [While other studies have found increased post-harvest mortality persisting years after initial harvests , mortalbut see , the lonMora paraensis produced a strong increase in recruitment into the smallest measured size class (>5 cm DBH) after logging for that species alone. Because harvested plots varied from 1 to 6 years since last extraction, it is possible that the post-harvest recruits of other species had not yet reached measurement size. Nevertheless field observations also show there were nearly no C. spruceanum saplings present in logged and natural gaps, indicating the common extent of logging disturbance is likely insufficient for the recruitment of this and other light-demanding species. Low intensity harvests have been found elsewhere to be insufficient to boost regeneration of light-demanding species [Widespread advanced regeneration of  species .These re species ,17,43.M. paraensis (the most abundant species) as an example, the integration of demographic effects of logging using life table response experiments (LTRE) shows that demographic rates most affected by logging may not be the most important for determining post-harvest tree population dynamics. These conclusions are important because harvest evaluations commonly consider growth, survival, and recruitment effects separately, and may misrepresent changes in population dynamics resulting from observed demographic effects [Using  effects . WithoutInga species and palms; [Residual stand damage was relatively low compared to other studies –47, withd palms; ).Most evaluations of stock recovery rates in tropical forests are below 50% based on estimated yields following a first harvest, casting doubt over the feasibility of long-term STM ,48,49. HV. surinamensis and C. guianensis that effectively counterbalance the projected yield declines from other species. Given the historically high volumes of C. guianensis and V. surinamensis extracted in the past, the projected recovery of these two species may be explained by the differences between common extractive practices and simulated forested management. As described elsewhere, smallholders often practice re-entry logging where tree populations are harvested down to small tree diameters [C. guianensis and V. surinamensis.In this study, recovery rates between harvests are high for most simulated management regimes due to the comparatively high projected mean AYs  that areiameters ,52. In fC. spruceanum and L. mahuba, not only exhibited limitations in population recovery but had continuously declining proportions of merchantable trees after each harvest cycle. Fortunately, the number of recruits needed to maintain population stability was low. This need for additional recruits is particularly relevant to the Amazon estuary because, at the smallholder scale of management, additional costs related to managing timber species regeneration are low due to low opportunity costs [Callycophyllum spruceanum's lack of post-harvest recruitment, despite its ample regeneration in large forest clearings and secondary forests [While our results point to increases in the proportion of unmerchantable trees due to consecutive harvests, model results also suggest that fast population growth and related fast diameter growth reduce the possibility of forests of unmerchantable trees. Nevertheless slow-growing species exhibit slow recovery after logging and face continuously decreasing proportion of merchantable trees. For instance, while most study species under simulated management reached a new lower proportion of merchantable trees under intensive harvests, the two species that showed signs of population decline, ty costs . Local ity costs . Callyco forests  and comm forests  indicate forests . In fact forests ,57. The Admittedly, future research could refine our management simulation model with better estimates of post-harvest effects given our relatively small plot area in recently harvested areas. However, it is extremely challenging to collect information from informal, unplanned  micro-scale operations. The collection of the data presented was only possible after multiple years of engagement with the local community, which is an effort that hardly could be scaled up with ease. Yet, currently there are no comparable studies of the long-term impact of timber management available to help refine the broad federal management regulations enforced in the region today.For Amazonian tidal floodplain forests, general country-level management guidelines provide neither the highest yields nor the highest sustained population growth for species under management. In fact, the variability and complexity of population responses to logging observed here and elsewhere suggest limited prospects for simple and effective one-size-fits-all management prescriptions ,58. Due H indicator holding the cutting cycle length and harvest volume limits constant at the optimal stand values results in easily implementable species-specific management regimes Click here for additional data file.S2 File(PDF)Click here for additional data file.


U tests were conducted to evaluate the hypotheses that infants in the intervention condition would consume more calories and have a positive change in weight-to-age z-score (WAZ) and shorter length of stay (LOS) following the first and second surgeries compared to infants in the control condition. After the protocol, the change in WAZ during the interstage increased by virtually one standard deviation from 0.05 to 0.91. Median LOS dropped 32% after the first surgery and 43% after the second surgery. Since first- and second-stage palliative surgeries occur within the same year of life, this represents savings of $500,000 to $800,000 per year in a 10-infant model. The standardized feeding approach improved growth in single ventricle infants while concurrently lowering hospital costs.Congenital heart disease is identified as the most common birth defect with single ventricle physiology carrying the highest mortality. Staged surgical palliation is required for treatment, with mortality historically as high as 22% in the four- to six-month period from the first- to second-stage surgical palliation, known as the interstage. A standardized postoperative feeding approach was implemented through an evidence-based protocol, parent engagement, and interprofessional team rounds. Five infants with single ventricle physiology preprotocol were compared with five infants who received the standardized feeding approach. Mann-Whitney  Congenital heart disease is the most common birth defect, occurring in nine out of every 1000 live births, with congenital anomalies being the leading cause of death in infants below one year of age [Typically, three surgical palliations are required in the first few years of life to achieve survival for children born with a single functional ventricle. The goal of the first surgery is to establish a reliable pathway for systemic circulation, control pulmonary circulation, and limit ventricular workload. Depending on the native anatomy, variable approaches are required for the first-stage surgery often involving complete ventricular outflow tract reconstruction and a systemic to pulmonary artery shunt. Usually, the first surgery is performed in the first few days of life. The second palliation classically is performed at four to six months of age and is the first step in separating the systemic circulation from the pulmonary circulation. At this operation, the superior vena cava is anastomosed to the pulmonary artery, and the pulmonary artery is disconnected from the heart. Pulmonary blood flow occurs without being pumped by a ventricle and depends on unobstructed pulmonary arteries and healthy pulmonary microvasculature. This operation, known as the bidirectional Glenn, also decreases workload on the functional single ventricle. The third operation directs the inferior vena cava and hepatic venous return around the heart to the pulmonary arteries. This results in what has come to be known as Fontan circulation, where systemic circulation and pulmonary circulation occur in series and ventricular workload is reduced to support only the systemic circulation. The third-stage palliative surgery occurs around two to four years. With Fontan circulation, all caval blood flow is directed to the pulmonary system while the functional single ventricle is responsible for systemic circulation. The highest mortality, up to 22%, remains in the four- to six-month period from first-stage until second-stage surgical palliation, known as the interstage [Recent advances in these staged palliative procedures have improved survival and according to Lowry  seventy  standardization, itself, has emerged as the element that decreases provider variability and improves growth objectives in these vulnerable infants [z-scores (WAZ) indicate improved growth. WAZ is the method of central tendency describing body weight mean, frequency distribution, and standard deviation based on the World Health Organization standard for infant gender and age (http://www.who.int/).For years, healthcare professionals considered infants with single ventricle physiology in the interstage incapable of somatic growth; however, improved growth has been realized with standardized feeding approaches , 5–11. AMost studies report infants with single ventricle physiology as having a high incidence of failure to thrive which is associated with increased mortality at second-stage palliation, along with increased postoperative complications, poorer long-term neurological outcomes, and longer lengths of hospital stay , 7. MultThe purpose of this quality improvement project was to implement an evidence-based standardized feeding approach, as recommended by the JCCHD-NPCQIC, for infants with single ventricle physiology with the goals of standardizing postoperative feeding practices, increasing interstage growth, reducing length of stay, and ultimately improving long-term neurodevelopmental outcomes in this vulnerable population. A standardized postoperative feeding protocol, which included the formation of an interprofessional feeding team, was initiated to reduce provider-dependent variability in feeding practices through translational science. The primary outcome objective in implementing a standardized evidence-based feeding approach was improved WAZ from home discharge following first-stage surgery until admission for second-stage palliation. The institutional review board waived this project as quality improvement.Do WAZ scores differ for infants with single ventricle physiology receiving a standardized feeding approach versus the standard of care at completion of the interstage?Does caloric consumption at postoperative days 5–7 and 7–10 differ for infants with single ventricle physiology receiving a standardized feeding approach versus the standard of care following first-stage surgical palliation?Do median lengths of stay following first- and second-stage surgical palliations differ for infants receiving a standardized feeding approach versus the standard of care?Project questions included the following:Hemodynamic stability with acceptable vital signs, reasonable urine output (≥2 mL/kg/hour), and good perfusion.Minimal or no acidosis, base deficit not worse than −4.Receiving no or low vasoactive support for at least 12 hours; may be on milrinone or epinephrine ≤0.03 mcg/kg/min.Infant's chest closed.Gestational age ≥35 weeks.Absence of congenital intestinal malformations or history of NEC.Absence of chylothorax.The taskforce also established recommendations for nutrition initiation, rate of nutrition advancement, and required feeding team consultation. The pediatric cardiac feeding team was responsible for operationalizing the taskforce-developed protocol of a standardized approach to reduce healthcare provider variability.The quality improvement project was twofold: (1) formulate an evidence-based standardized feeding approach as an interprofessional congenital heart team and (2) implement the agreed upon protocol through a pediatric cardiac feeding team that would provide interstage continuity in both the inpatient and outpatient settings. An interprofessional feeding taskforce was formed with representation from pediatric cardiology, pediatric cardiothoracic surgery, pediatric critical care intensivists, neonatology, nursing leadership, a registered dietitian, speech therapist, and occupational therapist. The taskforce developed a mutually agreed upon evidence-based standardized feeding approach with inclusion criteria as follows:The pediatric cardiac feeding team consisted of a registered dietitian, a speech therapist, an occupational or physical therapist, and a nurse practitioner. The feeding team rounded on infants who met inclusion criteria and made recommendations to the pediatric intensivists in the postoperative period during interprofessional rounds. The feeding team monitored nutritional status and growth throughout the interstage during hospitalization and later outpatient in collaboration with pediatric cardiology through the Single Ventricle Clinic.Goals of the standardized feeding included optimizing calories through nutritional fortification. The primary nutrition goal was to obtain and maintain consumption of at least 120 calories per kilogram per day  by postoperative day (POD) 14 and throughout the interstage with expected weight gain of 20–30 grams per day (g/d). Staged goals of caloric consumption were set at 60 Kcal/kg/d by POD 3–5, 80 Kcal/kg/d by POD 5–7, and 100 Kcal/kg/d by POD 7–10.Families of vulnerable children are important stakeholders in improving health outcomes. Parental engagement was secured through predischarge teaching by the feeding team and outpatient follow-up. Parents were educated on the goals for growth and equipped with home scales for daily weights and a binder in which to record the data. The nurse practitioner and pediatric cardiology team made weekly phone calls to parents ensuring nutrition and growth indicators were being achieved and nutritional adjustments were implemented. The infant was followed up closely through the Single Ventricle Clinic as recommended in the literature . Data wen = 5) or the intervention standardized interprofessional feeding approach condition (n = 5). The design has inherent bias in comparing outcomes over time but it is important to mention that no other major changes in standard of care were implemented concurrently. All data were entered and analyzed using IBM SPSS version 22. Mann-Whitney U tests were conducted to evaluate the expectation that infants in the intervention condition would consume more calories at 5–7 and 7–10 days postoperatively and have a higher WAZ and shorter length of stays following first and second surgeries, on average, than infants in the preintervention condition. The a priori alpha level was set at 0.10 for all statistical tests. A less conservative alpha was used due to the exploratory nature of the investigation.The study utilized a cohort control group design. Participants were analyzed by the preintervention standard of care (control) condition .Median postoperative length of stay following first-stage palliation dropped 32% to 42 days . The resThe scarcity of infants with single ventricle physiology creates unique challenges in process improvement execution and evaluation. As such, a primary limitation is the difficulty in obtaining large sample sizes for rare health conditions such as single ventricle physiology. The small sample size in this intervention adversely influences statistical power to correctly identify significant differences between groups. A significant effect size was demonstrated but these results should be interpreted with caution due to the small sample and variables outside of the investigators control. In addition, a larger sample might provide data suitable for parametric statistical analysis.In this quality improvement project, infants following palliation for single ventricle physiology receiving a standardized feeding protocol were compared with a historical cohort. Both the intervention and the historical cohort included parent engagement with home weight monitoring to track growth indicators; only the intervention group received the benefit of the feeding team and the postoperative standardized feeding approach. Home weight monitoring has been associated with improved weight gain during the single ventricle interstage . InfantsCost savings are realized through decreased hospital length of stay following first-stage and second-stage surgical palliation as confirmed in the literature . Median In addition, the length of stay following second-stage palliation of the bidirectional Glenn was noted to decline from 7 to 4 days after implementation of a standardized feeding approach. This three-day decrease in hospital length of stay represents a potential cost saving of $40,000–$90,000 annually . Since fThe expenditure of adding a designated feeding team was not calculated because no additional full-time equivalent (FTE) positions were added for the project. Existing FTEs were simply reallocated for the intervention, optimizing efficiency. The addition of FTEs may be a financial consideration for other healthcare organizations.z-scores, almost a full standard deviation with decreased hospital lengths of stay. Hospital length of stay following first-stage surgical palliation decreased over 30% and up to 40% following second-stage surgical palliation representing considerable healthcare expenditure savings following the implementation of this quality improvement project. The interprofessional cardiac feeding team reduced provider variability in feeding practices and increased compliance with the adopted feeding protocol. Goal caloric consumption can be achieved through an evidence-based feeding protocol and intervention of an interprofessional feeding team. The process of parental engagement through home weight monitoring and feeding education contributes to improved outcomes and strengthens the relationship of the family and healthcare providers [The implementation of a standardized feeding approach which includes a feeding protocol and daily rounding by an interprofessional feeding team can demonstrate improved caloric intake and growth for infants with single ventricle physiology during the interstage. Due to the small sample size, the results should be viewed with caution; however, standardization in feeding practices yielded a positive change in weight-to-age roviders . Daily aroviders . Other vTranslation science provides an opportunity to bring teams together and enhance professional collegiality while improving patient care outcomes. Stakeholders, including parents, serve as effective partners in process improvement. Parental engagement through home weight monitoring supports a standardized feeding approach to infants with single ventricle physiology during the interstage. Vulnerable patients benefit from a deliberate approach to clinical care through thoughtful collaboration in interprofessional teams as opposed to the inconsistency associated with random provider variability. Standardization in feeding practices for infants with single ventricle physiology during the interstage improves growth and nutrition while lowering hospital lengths of stay.