TDP-43 is found in cytoplasmic inclusions in 95% of amyotrophic lateral sclerosis (ALS) and 60% of frontotemporal lobar degeneration (FTLD). Approximately 4% of familial ALS is caused by mutations in TDP-43. The majority of these mutations are found in the glycine-rich domain, including the variant M337V, which is one of the most common mutations in TDP-43. In order to investigate the use of allele-specific RNA interference (RNAi) as a potential therapeutic tool, we designed and screened a set of siRNAs that specifically target TDP-43M337V mutation. Two siRNA specifically silenced the M337V mutation in HEK293T cells transfected with GFP-TDP-43wt or GFP-TDP-43M337V or TDP-43 C-terminal fragments counterparts. C-terminal TDP-43 transfected cells show an increase of cytosolic inclusions, which are decreased after allele-specific siRNA in M337V cells. We then investigated the effects of one of these allele-specific siRNAs in induced pluripotent stem cells (iPSCs) derived from an ALS patient carrying the M337V mutation. These lines showed a two-fold increase in cytosolic TDP-43 compared to the control. Following transfection with the allele-specific siRNA, cytosolic TDP-43 was reduced by 30% compared to cells transfected with a scrambled siRNA. We conclude that RNA interference can be used to selectively target the TDP-43M337V allele in mammalian and patient cells, thus demonstrating the potential for using RNA interference as a therapeutic tool for ALS.
Egawa et al. recently showed the value of patient-specific induced
pluripotent stem cells (iPSCs) for modeling amyotrophic lateral sclerosis in
vitro. Their study and our work highlight the need for complementary assays to
detect small, but potentially important, phenotypic differences between control
iPSC lines and those carrying disease mutations.
Imaging single fluorescent proteins in living mammalian cells is challenging due to out-of-focus fluorescence excitation by common microscopy schemes. We report the development of a novel fluorescence microscopy method, reflected light sheet microscopy (RLSM), which allows selective plane illumination throughout the nucleus of living mammalian cells, for reducing out-of-focus fluorescence signal. Generation of a thin light sheet parallel to the imaging plane and close to the sample surface is achieved by reflecting an elliptical laser beam incident from the top by 45° with a small mirror. The thin light sheet allows for an increased signal-to-background ratio superior to previous illumination schemes and enables imaging of single fluorescent proteins with up to 100 Hz time resolution. We demonstrate the sensitivity of RLSM by measuring the DNA-bound fraction of glucocorticoid receptor (GR) and determine the residence times on DNA of various oligomerization states and mutants of GR and estrogen receptor (ER), enabling us to resolve different modes of DNA binding of GR. Finally, we demonstrate two-color single molecule imaging by observing the spatio-temporal co-localization of two different protein pairs. The combination of our single molecule measurements and statistical analysis reveals dynamic properties of transcription factors in live mammalian cells.
The present study describes a case of a painful supraclavicular soft-tissue metastasis of a skin melanoma invading the brachial plexus in a 38-year-old male. The patient was treated twice with radiofrequency ablation (RFA) under computed tomography (CT) guidance, which caused tumoral necrosis. The patient was originally referred with a 7-cm metastasis in the right supraclavicular fossa, which caused intractable pain and a degree of numbness. These symptoms were unresponsive to chemotherapy and radiotherapy and the pain was not controlled using narcotic analgesics. The lesion was treated with CT-guided RFA causing necrosis, relieving the pain and sparing the patient from using analgesics. The pain recurred 19 months thereafter and a CT scan revealed an 8-cm mass in the right supraclavicular space. The patient underwent repeat CT-guided RFA, which reduced the pain to a level that was controlled with minor oral analgesics. In conclusion, in this case of a painful supraclavicular soft-tissue metastasis invading the brachial plexus, which was intractable to chemotherapy and radiotherapy, RFA was feasible and offered substantial palliation of the symptoms, freedom from the use of narcotic analgesics and improvements to the quality of life.
soft tissue metastasis; supraclavicular; radiofrequency ablation; palliation; computed tomography-guided
Gene expression profiling was performed via DNA microarrays in leukocytes from critically ill trauma patients nonseptic upon admission to the ICU, who subsequently developed either sepsis (n = 2) or severe sepsis and acute respiratory distress syndrome (n = 3). By comparing our results with published expression profiling studies in animal models of sepsis and lung injury, we found aquaporin-1 to be differentially expressed across all studies. Our aim was to determine how the water channel aquaporin-1 is involved in regulating the immune response in critically ill patients during infection acquired in the ICU.
Following the results of the initial genetic screening study, we prospectively followed aquaporin-1 leukocyte expression patterns in patients with ICU-acquired sepsis who subsequently developed septic shock (n = 16) versus critically ill patients who were discharged without developing sepsis (n = 13). We additionally determined aquaporin-1 expression upon lipopolysaccharide (LPS) exposure and explored functional effects of aquaporin-1 induction in polymorphonuclear granulocytes (PMNs).
Leukocyte aquaporin-1 expression was induced at the onset of sepsis (median 1.71-fold increase; interquartile range: 0.99 to 2.42, P = 0.012 from baseline) and was further increased upon septic shock (median 3.00-fold increase; interquartile range: 1.20 to 5.40, P = 0.023 from sepsis, Wilcoxon signed-rank test); no difference was observed between baseline and discharge in patients who did not develop sepsis. Stimulation of PMNs by LPS led to increased expression of aquaporin-1 in vitro, which could be abrogated by the NF-κB inhibitor EF-24. PMN hypotonic challenge resulted in a transient increase of the relative cell volume, which returned to baseline after 600 seconds, while incubation in the presence of LPS resulted in persistently increased cell volume. The latter could be abolished by blocking aquaporin-1 with mercury and restored by incubation in β-mercaptoethanol, which abrogated the action of mercury inhibition.
Aquaporin-1 is induced in leukocytes of patients with ICU-acquired sepsis and exhibits higher expression in septic shock. This phenomenon may be due to LPS-triggered NF-κB activation that can also lead to alterations in plasma membrane permeability.
This paper provides the first critical analysis of the financing and current capacity for REDD+ readiness in the Congo Basin, with a particular focus on the REDD+ component of national forest monitoring and measurement, reporting and verification (M&MRV). We focus on three areas of analysis: (i) general financing for REDD+ readiness especially M&MRV; (ii) capacity and information for REDD+ implementation and M&MRV; (iii) prospects and challenges for REDD+ and M&MRV readiness in terms of financing and capacity. For the first area of analysis, a REDD+ and M&MRV readiness financing database was created based on the information from the REDD+ voluntary database and Internet searches. For the second area of analysis, a qualitative approach to data collection was adopted (semi-structured interviews with key stakeholders, surveys and observations). All 10 countries were visited between 2010 and 2012. We find that: (i) a significant amount of REDD+ financing flows into the Congo Basin (±US$550 million or almost half of the REDD+ financing for the African continent); (ii) across countries, there is an important disequilibrium in terms of REDD+ and M&MRV readiness financing, political engagement, comprehension and capacity, which also appears to be a key barrier to countries receiving equal resources; (iii) most financing appears to go to smaller scale (subnational) REDD+ projects; (iv) four distinct country groups in terms of REDD+ readiness and M&MRV status are identified; and (v) the Congo Basin has a distinct opportunity to have a specific REDD+ financing window for large-scale and more targeted national REDD+ programmes through a specific fund for the region.
REDD+ readiness; national forest monitoring systems; measurement, reporting and verification; Central African Commission; financing
The mammalian Protocadherin (Pcdh) alpha, beta, and gamma gene clusters encode a large family of cadherin-like transmembrane proteins that are differentially expressed in individual neurons. The 22 isoforms of the Pcdhg gene cluster are diversified into A-, B- and C-types, and the C-type isoforms differ from all other clustered Pcdhs in sequence and expression. Here we show that mice lacking the 3 C-type isoforms are phenotypically indistinguishable from the Pcdhg null mutants, displaying virtually identical cellular and synaptic alterations resulting from neuronal apoptosis. By contrast, mice lacking 3 A-type isoforms exhibit no detectable phenotypes. Remarkably, however, genetically blocking apoptosis rescues the neonatal lethality of the C-type isoform knockouts, but not that of the Pcdhg null mutants. We conclude that the role of the Pcdhg gene cluster in neuronal survival is primarily, if not specifically mediated by its C-type isoforms, whereas a separate role essential for postnatal development, likely in neuronal wiring, requires isoform diversity.
Human pluripotent stem cells are a promising source of differentiated cells for developmental studies, cell transplantation, disease modeling, and drug testing. However, their widespread use even for intensely studied cell types like spinal motor neurons is hindered by the long duration and low yields of existing protocols for in vitro differentiation and by the molecular heterogeneity of the populations generated. We report a combination of small molecules that within 3 weeks induce motor neurons at up to 50% abundance and with defined subtype identities of relevance to neurodegenerative disease. Despite their accelerated differentiation, motor neurons expressed combinations of HB9, ISL1 and column-specific markers that mirror those observed in vivo in human fetal spinal cord. They also exhibited spontaneous and induced activity, and projected axons towards muscles when grafted into developing chick spinal cord. Strikingly, this novel protocol preferentially generates motor neurons expressing markers of limb-innervating lateral motor column motor neurons (FOXP1+/LHX3−). Access to high-yield cultures of human limb-innervating motor neuron subtypes will facilitate in-depth study of motor neuron subtype-specific properties, disease modeling, and development of large-scale cell-based screening assays.
ALS; astrocytes; stem cell; reactive gliosis; glia; TDP-43; SOD1; disease modeling
Although mangroves dominated by Avicennia germinans and Rhizophora mangle are extending over 6000 ha in the Tanbi Wetland National Park (TWNP) (The Gambia), their importance for local populations (both peri-urban and urban) is not well documented. For the first time, this study evaluates the different mangrove resources in and around Banjul (i.e., timber, non-timber, edible, and ethnomedicinal products) and their utilization patterns, including the possibility of ecotourism development. The questionnaire-based results have indicated that more than 80% of peri-urban population rely on mangroves for timber and non-timber products and consider them as very important for their livelihoods. However, at the same time, urban households demonstrate limited knowledge on mangrove species and their ecological/economic benefits. Among others, fishing (including the oyster—Crassostrea cf. gasar collection) and tourism are the major income-generating activities found in the TWNP. The age-old practices of agriculture in some parts of the TWNP are due to scarcity of land available for agriculture, increased family size, and alternative sources of income. The recent focus on ecotourism (i.e., boardwalk construction inside the mangroves near Banjul city) received a positive response from the local stakeholders (i.e., users, government, and non-government organizations), with their appropriate roles in sharing the revenue, rights, and responsibilities of this project. Though the guidelines for conservation and management of the TWNP seem to be compatible, the harmony between local people and sustainable resource utilization should be ascertained.
Electronic supplementary material
The online version of this article (doi:10.1007/s13280-012-0248-7) contains supplementary material, which is available to authorized users.
Mangroves; Socio-ecology; Tanbi Wetland National Park; Resource utilization; Participatory methods; The Gambia
The genome-wide identification of pairs of interacting proteins is an important step in the elucidation of cell regulatory mechanisms1,2. Much of our current knowledge derives from high-throughput techniques such as yeast two hybrid and affinity purification3, as well as from manual curation of experiments on individual systems4. A variety of computational approaches based, for example, on sequence homology, gene co-expression, and phylogenetic profiles have also been developed for the genome-wide inference of protein-protein interactions (PPIs)5,6. Yet, comparative studies suggest that the development of accurate and complete repertoires of PPIs is still in its early stages7–9. Here we show that three-dimensional structural information can be used to predict PPIs with an accuracy and coverage that are superior to predictions based on non-structural evidence. Moreover, an algorithm, PrePPI, that combines structural information with other functional clues is comparable in accuracy to high-throughput experiments, yielding over 30,000 high confidence interactions for yeast and over 300,000 for human. Experimental tests of a number of predictions demonstrate the ability of the PrePPI algorithm to identify unexpected PPIs of significant biological interest. The surprising effectiveness of three-dimensional structural information can be attributed to the use of homology models combined with the exploitation of both close and remote geometric relationships between proteins.
Pulmonary endothelium is a major metabolic organ affecting pulmonary and systemic vascular homeostasis. Brain death (BD)-induced physiologic and metabolic derangements in donors’ lungs, in the absence of overt lung pathology, may cause pulmonary dysfunction and compromise post-transplant graft function. To explore the impact of BD on pulmonary endothelium, we estimated pulmonary capillary endothelium-bound (PCEB)-angiotensin converting enzyme (ACE) activity, a direct and quantifiable index of pulmonary endothelial function, in eight brain-dead patients and ten brain-injured mechanically ventilated controls. No subject suffered from acute lung injury or any other overt lung pathology. Applying indicator-dilution type techniques, we measured single-pass transpulmonary percent metabolism (%M) and hydrolysis (v) of the synthetic, biologically inactive, and highly specific for ACE substrate 3H-benzoyl-Phe-Ala-Pro, under first order reaction conditions, and calculated lung functional capillary surface area (FCSA). Substrate %M (35 ± 6.8%) and v (0.49 ± 0.13) in BD patients were decreased as compared to controls (55.9 ± 4.9, P = 0.033 and 0.9 ± 0.15, P = 0.033, respectively), denoting decreased pulmonary endothelial enzyme activity at the capillary level; FCSA, a reflection of endothelial enzyme activity per vascular bed, was also decreased (BD patients: 1,563 ± 562 mL/min vs 4,235 ± 559 in controls; P = 0.003). We conclude that BD is associated with subtle pulmonary endothelial injury, expressed by decreased PCEB-ACE activity. The applied indicator-dilution type technique provides direct and quantifiable indices of pulmonary endothelial function at the bedside that may reveal the existence of preclinical lung pathology in potential lung donors.
angiotensin converting enzyme; brain death; pulmonary endothelium
Dendritic arbors of many neurons are patterned by a process called self-avoidance, in which branches arising from a single neuron repel each other1-7. By minimizing gaps and overlaps within the arbor, self-avoidance facilitates complete coverage of a neuron’s territory by its neurites1-3. Remarkably, some neurons that display self-avoidance interact freely with other neurons of the same subtype, implying that they discriminate self from non-self. Here, we demonstrate roles for the clustered protocadherins (Pcdhs) in dendritic self-avoidance and self/non-self discrimination. The Pcdh locus encodes ~60 related cadherin-like transmembrane proteins, at least some of which exhibit isoform-specific homophilic adhesion in heterologous cells and are expressed stochastically and combinatorially in single neurons7-11. Deletion of all 22 Pcdhs in the mouse gamma subcluster (Pcdhgs) disrupts self-avoidance of dendrites in retinal starburst amacrine cells (SACs) and cerebellar Purkinje cells. Further genetic analysis of SACs showed that Pcdhgs act cell-autonomously during development, and that replacement of the 22 Pcdhgs with a single isoform restores self-avoidance. Moreover, expression of the same single isoform in all SACs decreases interactions among dendrites of neighboring SACs (heteroneuronal interactions). These results suggest that homophilic Pcdhg interactions between sibling neurites (isoneuronal interactions) generate a repulsive signal that leads to self-avoidance. In this model, heteroneuronal interactions are normally permitted because dendrites seldom encounter a matched set of Pcdhgs unless they emanate from the same soma. In many respects, our results mirror those reported for Dscam1 in Drosophila: this complex gene encodes thousands of recognition molecules that exhibit stochastic expression and isoform-specific interactions, and mediate both self-avoidance and self/non-self discrimination4-7,12-15. Thus, although insect Dscams and vertebrate Pcdhs share no sequence homology, they appear to underlie similar strategies for endowing neurons with distinct molecular identities and patterning their arbors.
We have found that the γ2 subunit of the GABAA receptor (γ2-GABAAR) specifically interacts with protocadherin γ-C5 (Pcdh-γC5) in the rat brain. The interaction occurs between the large intracellular loop of the γ2-GABAAR and the cytoplasmic domain of Pcdh-γC5. In brain extracts, Pcdh-γC5 co-immunoprecipitates with GABAARs. In co-transfected HEK293 cells, Pcdh-γC5 promotes the transfer of γ2-GABAAR to the cell surface. We have previously shown that in cultured hippocampal neurons, endogenous Pcdh-γC5 forms clusters, some of which associate with GABAergic synapses. Overexpression of Pcdh-γC5 in hippocampal neurons increases the density ofγ2-GABAAR clusters but has no significant effect on the number of GABAergic contacts that these neurons receive, indicating that Pcdh-γC5 is not synaptogenic. Deletion of the cytoplasmic domain of Pcdh-γC5 enhanced its surface expression but decreased the association with both γ2-GABAAR clusters and presynaptic GABAergic contacts. Cultured hippocampal neurons from the Pcdh-γ triple C-type isoform knockout (TCKO) mouse (Pcdhgtcko/tcko) showed plenty of GABAergic synaptic contacts, although their density was reduced compared with sister cultures from wild type and heterozygous mice. Knocking down Pcdh-γC5 expression with shRNA decreased γ2-GABAAR cluster density and GABAergic innervation. The results indicate that although Pcdh-γC5 is not essential for GABAergic synapse formation or GABAAR clustering, i) Pcdh-γC5 regulates the surface expression of GABAARs via cis-cytoplasmic interaction with γ2-GABAAR; and ii) Pcdh-γC5 plays a role in the stabilization and maintenance of some GABAergic synapses.
GABAA receptors; synapses; GABAergic synapse; protocadherin; PCDHGC5; interaction
Mutations in the RNA binding protein FUS cause ALS, a fatal adult motor neuron disease. Decreased expression of SMN causes the fatal childhood motor neuron disorder SMA. The SMN complex localizes in both the cytoplasm and nuclear Gems, and loss of Gems is a cellular hallmark of SMA patient fibroblasts. Here, we report that FUS associates with the SMN complex, an interaction mediated by U1 snRNP and by direct interactions between FUS and SMN. Functionally, we show that FUS is required for Gem formation in HeLa cells, and expression of FUS containing a severe ALS-causing mutation (R495X) also results in Gem loss. Strikingly, a reduction in Gems is observed in ALS patient fibroblasts expressing either mutant FUS or TDP-43, another ALS-causing protein that interacts with FUS. The physical and functional interactions between SMN, FUS, TDP-43, and Gems indicate that ALS and SMA share a biochemical pathway, adding strong new support to the view that these motor neuron diseases are related.
Post-traumatic pulmonary pseudocyst is an uncommon cavitary lesion of the lung and develops after blunt chest trauma and even more rarely following penetrating injuries. It is generally seen in young adults presenting with cough, chest pain, hemoptysis, and dyspnea. Post-traumatic pulmonary pseudocyst should be included in the differential diagnosis of cavitary pulmonary lesions. We describe the case of a 60-year-old Caucasian Greek woman who sustained traumatic pulmonary pseudocyst with hemopneumothorax due to a blunt chest trauma after a traffic accident.
After a traffic accident, a 60-year-old Caucasian Greek woman sustained a hemopneumothorax due to a blunt chest trauma. There was evidence of an extensive contusion in the posterior and lateral segments of the right lower lobe, a finding that was attributed to an early sign of a cavitation, and the presence of a thin-walled air cavity was detected on the anterior segment of the right lower lobe in the control computed tomography taken 24 hours after admission. Our patient was treated by catheter aspiration, and the findings of computed tomography evaluation about one month later showed complete resolution of one of the two air-filled cavitary lesions. The second pseudocyst also disappeared completely, as shown by the control computed tomography scan performed six months later.
Traumatic pulmonary pseudocyst is a rare complication of blunt chest trauma, and computed tomography is a more valuable imaging technique than chest radiograph for early diagnosis.
Traumatic pulmonary pseudocyst; lung cyst; blunt chest trauma; pulmonary contusion
Caveolin-1 is a key regulator of pulmonary endothelial barrier function. Here, we tested the hypothesis that caveolin-1 expression is required for ventilator-induced lung injury (VILI). Caveolin-1 gene-disrupted (Cav-1-/-) and age-, sex-, and strain-matched wild-type (WT) control mice were ventilated using two protocols: volume-controlled with protective (8 mL/kg) versus injurious (21 mL/Kg) tidal volume for up to 6 hours; and pressure-controlled with protective (airway pressure = 12 cm H2O) versus injurious (30 cm H2O) ventilation to induce lung injury. Lung microvascular permeability (whole-lung 125I-albumin accumulation, lung capillary filtration coefficient [Kf, c]) and inflammatory markers (bronchoalveolar lavage [BAL] cytokine levels and neutrophil counts) were measured. We also evaluated histologic sections from lungs, and the time course of Src kinase activation and caveolin-1 phosphorylation. VILI induced a 1.7-fold increase in lung 125I-albumin accumulation, fourfold increase in Kf, c, significantly increased levels of cytokines CXCL1 and interleukin-6, and promoted BAL neutrophilia in WT mice. Lung injury by these criteria was significantly reduced in Cav-1-/- mice but fully restored by i.v. injection of liposome/Cav-1 cDNA complexes that rescued expression of Cav-1 in lung microvessels. As thrombin is known to play a significant role in mediating stretch-induced vascular injury, we observed in cultured mouse lung microvascular endothelial cells (MLECs) thrombin-induced albumin hyperpermeability and phosphorylation of p44/42 MAP kinase in WT but not in Cav-1-/- MLECs. Thus, caveolin-1 expression is required for mechanical stretch-induced lung inflammation and endothelial hyperpermeability in vitro and in vivo.
high tidal volume mechanical ventilation; lung inflammation; thrombin; caveolae; albumin permeability
Anthocyanin content is a trait of major interest in Vitis vinifera L. These compounds affect grape and wine quality, and have beneficial effects on human health. A candidate-gene approach was used to identify genetic variants associated with anthocyanin content in grape berries. A total of 445 polymorphisms were identified in 5 genes encoding transcription factors and 10 genes involved in either the biosynthetic pathway or transport of anthocyanins. A total of 124 SNPs were selected to examine association with a wide range of phenotypes based on RP-HPLC analysis and visual characterization. The phenotypes were total skin anthocyanin (TSA) concentration but also specific types of anthocyanins and relative abundance. The visual assessment was based on OIV (Organisation Internationale de la Vigne et du Vin) descriptors for berry and skin colour. The genes encoding the transcription factors MYB11, MYBCC and MYCB were significantly associated with TSA concentration. UFGT and MRP were associated with several different types of anthocyanins. Skin and pulp colour were associated with nine genes (MYB11, MYBCC, MYCB, UFGT, MRP, DFR, LDOX, CHI and GST). Pulp colour was associated with a similar group of 11 genes (MYB11, MYBCC, MYCB, MYCA, UFGT, MRP, GST, DFR, LDOX, CHI and CHSA). Statistical interactions were observed between SNPs within the transcription factors MYB11, MYBCC and MYCB. SNPs within LDOX interacted with MYB11 and MYCB, while SNPs within CHI interacted with MYB11 only. Together, these findings suggest the involvement of these genes in anthocyanin content and on the regulation of anthocyanin biosynthesis. This work forms a benchmark for replication and functional studies.
The primary function of the mammalian lung is to facilitate diffusion of oxygen to venous blood and to ventilate carbon dioxide produced by catabolic reactions within cells. However, it is also responsible for a variety of other important functions, including host defense and production of vasoactive agents to regulate not only systemic blood pressure, but also water, electrolyte and acid-base balance. Caveolin-1 is highly expressed in the majority of cell types in the lung, including epithelial, endothelial, smooth muscle, connective tissue cells, and alveolar macrophages. Deletion of caveolin-1 in these cells results in major functional aberrations, suggesting that caveolin-1 may be crucial to lung homeostasis and development. Furthermore, generation of mutant mice that under-express caveolin-1 results in severe functional distortion with phenotypes covering practically the entire spectrum of known lung diseases, including pulmonary hypertension, fibrosis, increased endothelial permeability, and immune defects. In this Chapter, we outline the current state of knowledge regarding caveolin-1-dependent regulation of pulmonary cell functions and discuss recent research findings on the role of caveolin-1 in various pulmonary disease states, including obstructive and fibrotic pulmonary vascular and inflammatory diseases.
Diabetic patients may develop acute lung injury less often than non-diabetics; a fact that could be partially ascribed to the usage of antidiabetic drugs, including metformin. Metformin exhibits pleiotropic properties which make it potentially beneficial against lung injury. We hypothesized that pretreatment with metformin preserves alveolar capillary permeability and, thus, prevents ventilator-induced lung injury.
Twenty-four rabbits were randomly assigned to pretreatment with metformin (250 mg/Kg body weight/day per os) or no medication for two days. Explanted lungs were perfused at constant flow rate (300 mL/min) and ventilated with injurious (peak airway pressure 23 cmH2O, tidal volume ≈17 mL/Kg) or protective (peak airway pressure 11 cmH2O, tidal volume ≈7 mL/Kg) settings for 1 hour. Alveolar capillary permeability was assessed by ultrafiltration coefficient, total protein concentration in bronchoalveolar lavage fluid (BALF) and angiotensin-converting enzyme (ACE) activity in BALF.
High-pressure ventilation of the ex-vivo lung preparation resulted in increased microvascular permeability, edema formation and microhemorrhage compared to protective ventilation. Compared to no medication, pretreatment with metformin was associated with a 2.9-fold reduction in ultrafiltration coefficient, a 2.5-fold reduction in pulmonary edema formation, lower protein concentration in BALF, lower ACE activity in BALF, and fewer histological lesions upon challenge of the lung preparation with injurious ventilation. In contrast, no differences regarding pulmonary artery pressure and BALF total cell number were noted. Administration of metformin did not impact on outcomes of lungs subjected to protective ventilation.
Pretreatment with metformin preserves alveolar capillary permeability and, thus, decreases the severity of ventilator-induced lung injury in this model.
BRCA1 promotes DNA repair through interactions with multiple proteins, including CtIP and FANCJ (also known as BRIP1/BACH1). While CtIP facilitates DNA end resection when de-acetylated, the function of FANCJ in repair processing is less well defined. Here, we report that FANCJ is also acetylated. Preventing FANCJ acetylation at lysine 1249 does not interfere with the ability of cells to survive DNA interstrand crosslinks (ICLs). However, resistance is achieved with reduced reliance on recombination. Mechanistically, FANCJ acetylation facilitates DNA end processing required for repair and checkpoint signaling. This conclusion was based on the finding that FANCJ and its acetylation were required for robust RPA foci formation, RPA phosphorylation, and Rad51 foci formation in response to camptothecin (CPT). Furthermore, both preventing and mimicking FANCJ acetylation at lysine 1249 disrupts FANCJ function in checkpoint maintenance. Thus, we propose that the dynamic regulation of FANCJ acetylation is critical for robust DNA damage response, recombination-based processing, and ultimately checkpoint maintenance.
The BRCA1–Fanconi anemia (FA) pathway is required for both tumor suppression and cell survival, particularly following treatment with DNA damaging agents that induce DNA interstrand crosslinks (ICLs). ICL processing by the BRCA–FA pathway includes promotion of homologous recombination (HR) and DNA damage tolerance through translesion synthesis. However, little is known about how the BRCA–FA pathway or these ICL processing mechanisms are regulated. Here, we identify acetylation as a DNA damage–dependent regulator of the BRCA–FA protein, FANCJ. FANCJ acetylation at lysine 1249 is enhanced by expression of the histone acetyltransferase CBP and reduced by expression of histone deacetylases HDAC3 or SIRT1. Furthermore, acetylation on endogenous FANCJ is induced upon treatment of cells with agents that generate DNA lesions. Consistent with this post-translation event regulating FANCJ function during cellular DNA repair, preventing FANCJ acetylation skews ICL processing. Cells have reduced reliance on HR factor Rad54 and greater reliance on translesion synthesis polymerase polη. Our data indicate that FANCJ acetylation contributes to DNA end processing that is required for HR. Furthermore, resection-dependent checkpoint maintenance relies on the dynamic regulation of FANCJ acetylation. The implication of these findings is that FANCJ acetylation contributes to DNA repair choice within the BRCA–FA pathway.