University Hospitals Geneva Medical Center

Toxic epidermal necrolysis (TEN, Lyell's syndrome) is a severe adverse drug reaction in which keratinocytes die and large sections of epidermis separate from the dermis. Keratinocytes normally express the death receptor Fas (CD95); those from TEN patients were found to express lytically active Fas ligand (FasL). Antibodies present in pooled human intravenous immunoglobulins (IVIG) blocked Fas-mediated keratinocyte death in vitro. In a pilot study, 10 consecutive individuals with clinically and histologically confirmed TEN were treated with IVIG; disease progression was rapidly reversed and the outcome was favorable in all cases. Thus, Fas-FasL interactions are directly involved in the epidermal necrolysis of TEN, and IVIG may be an effective treatment.

The cell surface receptor Fas (FasR, Apo-1, CD95) and its ligand (FasL) are mediators of apoptosis that have been shown to be implicated in the peripheral deletion of autoimmune cells, activation-induced T cell death, and one of the two major cytolytic pathways mediated by CD8+ cytolytic T cells. To gain further understanding of the Fas system., we have analyzed Fas and FasL expression during mouse development and in adult tissues. In developing mouse embryos, from 16.5 d onwards, Fas mRNA is detectable in distinct cell types of the developing sinus, thymus, lung, and liver, whereas FasL expression is restricted to submaxillary gland epithelial cells and the developing nervous system. Significant Fas and FasL expression were observed in several nonlymphoid cell types during embryogenesis, and generally Fas and FasL expression were not localized to characteristic sites of programmed cell death. In the adult mouse, RNase protection analysis revealed very wide expression of both Fas and FasL. Several tissues, including the thymus, lung, spleen, small intestine, large intestine, seminal vesicle, prostate, and uterus, clearly coexpress the two genes. Most tissues constitutively coexpressing Fas and FasL in the adult mouse are characterized by apoptotic cell turnover, and many of those expressing FasL are known to be immune privileged. It may be, therefore, that the Fas system is implicated in both the regulation of physiological cell turnover and the protection of particular tissues against potential lymphocyte-mediated damage.

BACKGROUND: The European Academy of Allergy and Clinical Immunology (EAACI) is developing Guidelines for Allergen Immunotherapy (AIT) for IgE-mediated Food Allergy. To inform the development of clinical recommendations, we sought to critically assess evidence on the effectiveness, safety and cost-effectiveness of AIT in the management of food allergy. METHODS: We undertook a systematic review and meta-analysis that involved searching nine international electronic databases for randomized controlled trials (RCTs) and nonrandomized studies (NRS). Eligible studies were independently assessed by two reviewers against predefined eligibility criteria. The quality of studies was assessed using the Cochrane Risk of Bias tool for RCTs and the Cochrane ACROBAT-NRS tool for quasi-RCTs. Random-effects meta-analyses were undertaken, with planned subgroup and sensitivity analyses. RESULTS: We identified 1814 potentially relevant papers from which we selected 31 eligible studies, comprising of 25 RCTs and six NRS, studying a total of 1259 patients. Twenty-five trials evaluated oral immunotherapy (OIT), five studies investigated sublingual immunotherapy, and one study evaluated epicutaneous immunotherapy. The majority of these studies were in children. Twenty-seven studies assessed desensitization, and eight studies investigated sustained unresponsiveness postdiscontinuation of AIT. Meta-analyses demonstrated a substantial benefit in terms of desensitization (risk ratio (RR) = 0.16, 95% CI 0.10, 0.26) and suggested, but did not confirm sustained unresponsiveness (RR = 0.29, 95% CI 0.08, 1.13). Only one study reported on disease-specific quality of life (QoL), which reported no comparative results between OIT and control group. Meta-analyses revealed that the risk of experiencing a systemic adverse reaction was higher in those receiving AIT, with a more marked increase in the risk of local adverse reactions. Sensitivity analysis excluding those studies judged to be at high risk of bias demonstrated the robustness of summary estimates of effectiveness and safety of AIT for food allergy. None of the studies reported data on health economic analyses. CONCLUSIONS: AIT may be effective in raising the threshold of reactivity to a range of foods in children with IgE-mediated food allergy whilst receiving (i.e. desensitization) and post-discontinuation of AIT. It is, however, associated with a modest increased risk in serious systemic adverse reactions and a substantial increase in minor local adverse reactions. More data are needed in relation to adults, long term effects, the impact on QoL and the cost-effectiveness of AIT.

Even though radiomics can hold great potential for supporting clinical decision-making, its current use is mostly limited to academic research, without applications in routine clinical practice. The workflow of radiomics is complex due to several methodological steps and nuances, which often leads to inadequate reporting and evaluation, and poor reproducibility. Available reporting guidelines and checklists for artificial intelligence and predictive modeling include relevant good practices, but they are not tailored to radiomic research. There is a clear need for a complete radiomics checklist for study planning, manuscript writing, and evaluation during the review process to facilitate the repeatability and reproducibility of studies. We here present a documentation standard for radiomic research that can guide authors and reviewers. Our motivation is to improve the quality and reliability and, in turn, the reproducibility of radiomic research. We name the checklist CLEAR (CheckList for EvaluAtion of Radiomics research), to convey the idea of being more transparent. With its 58 items, the CLEAR checklist should be considered a standardization tool providing the minimum requirements for presenting clinical radiomics research. In addition to a dynamic online version of the checklist, a public repository has also been set up to allow the radiomics community to comment on the checklist items and adapt the checklist for future versions. Prepared and revised by an international group of experts using a modified Delphi method, we hope the CLEAR checklist will serve well as a single and complete scientific documentation tool for authors and reviewers to improve the radiomics literature.

BACKGROUND: The prognostic significance of germline mutations in BRCA1 and BRCA2 in women with breast cancer remains unclear. A combined analysis was performed to address this uncertainty. METHODS: Two retrospective cohorts of Ashkenazi Jewish women undergoing breast-conserving treatment for invasive cancer between 1980 and 1995 (n = 584) were established. Archived tissue blocks were used as the source of DNA for Ashkenazi Jewish BRCA1/BRCA2 founder mutation analysis. Paraffin-embedded tissue and follow-up information was available for 505 women. RESULTS: Genotyping was successful in 496 women, of whom 56 (11.3%) were found to carry a BRCA1/BRCA2 founder mutation. After a median follow-up period of 116 months, breast cancer specific survival was worse in women with BRCA1 mutations than in those without (62% at 10 years versus 86%; P < 0.0001), but not in women with the BRCA2 mutation (84% versus 86% at 10 years; P = 0.76). Germline BRCA1 mutations were an independent predictor of breast cancer mortality in multivariate analysis (hazard ratio 2.4, 95% confidence interval 1.2-4.8; P = 0.01). BRCA1 status predicted breast cancer mortality only among women who did not receive chemotherapy (hazard ratio 4.8, 95% confidence interval 2.0-11.7; P = 0.001). The risk for metachronous ipsilateral cancer was not greater in women with germline BRCA1/BRCA2 founder mutations than in those without mutations (P = 0.68). CONCLUSION: BRCA1 mutations, but not BRCA2 mutations, are associated with reduced survival in Ashkenazi women undergoing breast-conserving treatment for invasive breast cancer, but the poor prognosis associated with germline BRCA1 mutations is mitigated by adjuvant chemotherapy. The risk for metachronous ipsilateral disease does not appear to be increased for either BRCA1 or BRCA2 mutation carriers, at least up to 10 years of follow up.

Voltage-gated proton (H+) channels are found in many human and animal tissues and play an important role in cellular defense against acidic stress. However, a molecular identification of these unique ion conductances has so far not been achieved. A 191-amino acid protein is described that, upon heterologous expression, has properties indistinguishable from those of native H+ channels. This protein is generated through alternative splicing of messenger RNA derived from the gene NOH-1 (NADPH oxidase homolog 1, where NADPH is the reduced form of nicotinamide adenine dinucleotide phosphate).

BACKGROUND: Electromagnetic navigation bronchoscopy (ENB) is an emerging endoscopic technique for the diagnosis of peripheral lung lesions. A thorough analysis of ENB's yield and safety is required for comparison to other sampling modalities. OBJECTIVES: To describe ENB's yield and safety profile. METHODS: The MEDLINE and EMBASE databases were systematically searched for studies reporting ENB's yield for peripheral lung lesions. Two independent investigators extracted data and rated each study on a scale of methodological quality. Clearly defined performance outcomes were reconstructed and meta-analyzed. Subgroup analysis and meta-regression were used to identify possible sources of study heterogeneity. RESULTS: A total of 15 trials were included (1,033 lung nodules). A positive and definitive diagnosis was obtained after 64.9% of all ENB procedures (95% CI 59.2-70.3). Overall diagnostic accuracy was 73.9% (95% CI 68.0-79.2). Sensitivity to detect cancer was 71.1% (95% CI 64.6-76.8), with a negative predictive value of 52.1% (95% CI 43.5-60.6). Pneumothorax occurred in 3.1% of patients, requiring chest tube drainage in 1.6% of these cases. Original trials identified 6 variables associated with higher ENB yields: nodule location in the upper or middle lobes, nodule size, lower registration error, presence of a bronchus sign on CT imaging, combined use of an ultrasonic radial probe, and catheter suctioning as a sampling technique. Heterogeneity exploration revealed that studies using general anesthesia or rapid on-site cytological evaluation reported better yields. CONCLUSIONS: ENB is effective and particularly safe. Prospective studies are needed to clarify the role of several variables conditioning the yield of this technique.

SIGNIFICANCE: There is increasing evidence that the generation of reactive oxygen species (ROS) in the central nervous system (CNS) involves the NOX family of nicotinamide adenine dinucleotide phosphate oxidases. Controlled ROS generation appears necessary for optimal functioning of the CNS through fine-tuning of redox-sensitive signaling pathways, while overshooting ROS generation will lead to oxidative stress and CNS disease. RECENT ADVANCES: NOX enzymes are not only restricted to microglia (i.e. brain phagocytes) but also expressed in neurons, astrocytes, and the neurovascular system. NOX enzymes are involved in CNS development, neural stem cell biology, and the function of mature neurons. While NOX2 appears to be a major source of pathological oxidative stress in the CNS, other NOX isoforms might also be of importance, for example, NOX4 in stroke. Globally speaking, there is now convincing evidence for a role of NOX enzymes in various neurodegenerative diseases, cerebrovascular diseases, and psychosis-related disorders. CRITICAL ISSUES: The relative importance of specific ROS sources (e.g., NOX enzymes vs. mitochondria; NOX2 vs. NOX4) in different pathological processes needs further investigation. The absence of specific inhibitors limits the possibility to investigate specific therapeutic strategies. The uncritical use of non-specific inhibitors (e.g., apocynin, diphenylene iodonium) and poorly validated antibodies may lead to misleading conclusions. FUTURE DIRECTIONS: Physiological and pathophysiological studies with cell-type-specific knock-out mice will be necessary to delineate the precise functions of NOX enzymes and their implications in pathomechanisms. The development of CNS-permeant, specific NOX inhibitors will be necessary to advance toward therapeutic applications.

The origin of soluble CD14 (sCD14) in the circulation is uncertain. To examine whether CD14 could be an acute-phase protein (APP), the levels of sCD14, IL-6, and C-reactive protein were determined by ELISA in serum and synovial fluid (SF) of patients with various arthropathies, and the regulation of CD14 synthesis was examined in liver cells. In patients with crystal-mediated or immunologically mediated arthritis (rheumatoid arthritis), serum levels of sCD14 were higher than or similar to those found in infection-mediated arthritis (reactive arthritis), precluding a relation with bacteria exposure. Levels of sCD14 were similar in SF and serum, and did not correlate with the number of SF leukocytes, excluding an important source from leukocyte membrane-bound CD14, by protease-mediated shedding. In contrast, serum levels of sCD14 in patients correlated with those of C-reactive protein, a classical APP, and IL-6, a cytokine known to regulate the synthesis of APP in the liver. Serum levels of sCD14 also correlated with disease activity in rheumatoid arthritis and reactive arthritis patients. IL-6 stimulated the production of CD14 by HepG2 hepatoma cells. By real-time PCR, the inducibility of CD14 by IL-6 was also observed at the mRNA level both in HepG2 cells and human primary hepatocytes. These in vitro results were confirmed by in vivo studies in IL-6(-/-) mice injected with turpentine, an experimental model of acute-phase response. Liver levels of CD14 mRNA increased in IL-6(+/+), but not in IL-6(-/-) mice. These results indicate that sCD14 can be considered as a type 2 APP.

PURPOSE: To propose a new quality scoring tool, METhodological RadiomICs Score (METRICS), to assess and improve research quality of radiomics studies. METHODS: We conducted an online modified Delphi study with a group of international experts. It was performed in three consecutive stages: Stage#1, item preparation; Stage#2, panel discussion among EuSoMII Auditing Group members to identify the items to be voted; and Stage#3, four rounds of the modified Delphi exercise by panelists to determine the items eligible for the METRICS and their weights. The consensus threshold was 75%. Based on the median ranks derived from expert panel opinion and their rank-sum based conversion to importance scores, the category and item weights were calculated. RESULT: In total, 59 panelists from 19 countries participated in selection and ranking of the items and categories. Final METRICS tool included 30 items within 9 categories. According to their weights, the categories were in descending order of importance: study design, imaging data, image processing and feature extraction, metrics and comparison, testing, feature processing, preparation for modeling, segmentation, and open science. A web application and a repository were developed to streamline the calculation of the METRICS score and to collect feedback from the radiomics community. CONCLUSION: In this work, we developed a scoring tool for assessing the methodological quality of the radiomics research, with a large international panel and a modified Delphi protocol. With its conditional format to cover methodological variations, it provides a well-constructed framework for the key methodological concepts to assess the quality of radiomic research papers. CRITICAL RELEVANCE STATEMENT: A quality assessment tool, METhodological RadiomICs Score (METRICS), is made available by a large group of international domain experts, with transparent methodology, aiming at evaluating and improving research quality in radiomics and machine learning. KEY POINTS: • A methodological scoring tool, METRICS, was developed for assessing the quality of radiomics research, with a large international expert panel and a modified Delphi protocol. • The proposed scoring tool presents expert opinion-based importance weights of categories and items with a transparent methodology for the first time. • METRICS accounts for varying use cases, from handcrafted radiomics to entirely deep learning-based pipelines. • A web application has been developed to help with the calculation of the METRICS score ( https://metricsscore.github.io/metrics/METRICS.html ) and a repository created to collect feedback from the radiomics community ( https://github.com/metricsscore/metrics ).

AIM: The goal of this European Society of Coloproctology (ESCP) guideline project is to give an overview of the existing evidence on the management of diverticular disease, primarily as a guidance to surgeons. METHODS: The guideline was developed during several working phases including three voting rounds and one consensus meeting. The two project leads (JKS and EA) appointed by the ESCP guideline committee together with one member of the guideline committee (WB) agreed on the methodology, decided on six themes for working groups (WGs) and drafted a list of research questions. Senior WG members, mostly colorectal surgeons within the ESCP, were invited based on publication records and geographical aspects. Other specialties were included in the WGs where relevant. In addition, one trainee or PhD fellow was invited in each WG. All six WGs revised the research questions if necessary, did a literature search, created evidence tables where feasible, and drafted supporting text to each research question and statement. The text and statement proposals from each WG were arranged as one document by the first and last authors before online voting by all authors in two rounds. For the second voting ESCP national representatives were also invited. More than 90% agreement was considered a consensus. The final phrasing of the statements with < 90% agreement was discussed in a consensus meeting at the ESCP annual meeting in Vienna in September 2019. Thereafter, the first and the last author drafted the final text of the guideline and circulated it for final approval and for a third and final online voting of rephrased statements. RESULTS: This guideline contains 38 evidence based consensus statements on the management of diverticular disease. CONCLUSION: This international, multidisciplinary guideline provides an up to date summary of the current knowledge of the management of diverticular disease as a guidance for clinicians and patients.

Insulin-, glucagon-, and somatostatin-contianing cells, identified by immunofluorescent staining, were quantitated morphometrically in sections of pancreas obtained from diabetic and nondiabetic humans and rats. Both the volume density and number of somatostatin- and glucagon-containing cells were significantly increased in the islets of juvenile-type human diabetics and of streptozotocin diabetic rats.

The classic organization of a gene structure has followed the Jacob and Monod bacterial gene model proposed more than 50 years ago. Since then, empirical determinations of the complexity of the transcriptomes found in yeast to human has blurred the definition and physical boundaries of genes. Using multiple analysis approaches we have characterized individual gene boundaries mapping on human chromosomes 21 and 22. Analyses of the locations of the 5' and 3' transcriptional termini of 492 protein coding genes revealed that for 85% of these genes the boundaries extend beyond the current annotated termini, most often connecting with exons of transcripts from other well annotated genes. The biological and evolutionary importance of these chimeric transcripts is underscored by (1) the non-random interconnections of genes involved, (2) the greater phylogenetic depth of the genes involved in many chimeric interactions, (3) the coordination of the expression of connected genes and (4) the close in vivo and three dimensional proximity of the genomic regions being transcribed and contributing to parts of the chimeric RNAs. The non-random nature of the connection of the genes involved suggest that chimeric transcripts should not be studied in isolation, but together, as an RNA network.

Producing and perceiving music engage a wide range of sensorimotor, cognitive, and emotional processes. Emotions are a central feature of the enjoyment of music, with a large variety of affective states consistently reported by people while listening to music. However, besides joy or sadness, music often elicits feelings of wonder, nostalgia, or tenderness, which do not correspond to emotion categories typically studied in neuroscience and whose neural substrates remain largely unknown. Here we review the similarities and differences in the neural substrates underlying these "complex" music-evoked emotions relative to other more "basic" emotional experiences. We suggest that these emotions emerge through a combination of activation in emotional and motivational brain systems (e.g., including reward pathways) that confer its valence to music, with activation in several other areas outside emotional systems, including motor, attention, or memory-related regions. We then discuss the neural substrates underlying the entrainment of cognitive and motor processes by music and their relation to affective experience. These effects have important implications for the potential therapeutic use of music in neurological or psychiatric diseases, particularly those associated with motor, attention, or affective disturbances.

The TNF ligand family member BAFF (B cell activating factor belonging to the TNF family, also called Blys, TALL-1, zTNF-4, or THANK) is an important survival factor for B cells [corrected]. In this study, we show that BAFF is able to regulate T cell activation. rBAFF induced responses (thymidine incorporation and cytokine secretion) of T cells, suboptimally stimulated through their TCR. BAFF activity was observed on naive, as well as on effector/memory T cells (both CD4+ and CD8+ subsets), indicating that BAFF has a wide function on T cell responses. Analysis of the signal transduced by BAFF into T cells shows that BAFF has no obvious effect on T cell survival upon activation, but is able to deliver a complete costimulation signal into T cells. Indeed, BAFF is sufficient to induce IL-2 secretion and T cell division, when added to an anti-TCR stimulation. This highlights some differences in the BAFF signaling pathway in T and B cells. In conclusion, our results indicate that BAFF may play a role in the development of T cell responses, in addition to its role in B cell homeostasis.

There are five members of the RFX family of transcription factors in mammals. While RFX5 plays a well-defined role in the immune system, the functions of RFX1 to RFX4 remain largely unknown. We have generated mice with a deletion of the Rfx3 gene. RFX3-deficient mice exhibit frequent left-right (LR) asymmetry defects leading to a high rate of embryonic lethality and situs inversus in surviving adults. In vertebrates, specification of the LR body axis is controlled by monocilia in the embryonic node, and defects in nodal cilia consequently result in abnormal LR patterning. Consistent with this, Rfx3 is expressed in ciliated cells of the node and RFX3-deficient mice exhibit a pronounced defect in nodal cilia. In contrast to the case for wild-type embryos, for which we document for the first time a twofold increase in the length of nodal cilia during development, the cilia are present but remain markedly stunted in mutant embryos. Finally, we show that RFX3 regulates the expression of D2lic, the mouse orthologue of a Caenorhabditis elegans gene that is implicated in intraflagellar transport, a process required for the assembly and maintenance of cilia. In conclusion, RFX3 is essential for the differentiation of nodal monocilia and hence for LR body axis determination.

Mitochondria modulate Ca2+ signals by taking up, buffering, and releasing Ca2+ at key locations near Ca2+ release or influx channels. The role of such local interactions between channels and organelles is difficult to establish in living cells because mitochondria form an interconnected network constantly remodeled by coordinated fusion and fission reactions. To study the effect of a controlled disruption of the mitochondrial network on Ca2+ homeostasis, we took advantage of hFis1, a protein that promotes mitochondrial fission by recruiting the dynamin-related protein, Drp1. hFis1 expression in HeLa cells induced a rapid and complete fragmentation of mitochondria, which redistributed away from the plasma membrane and clustered around the nucleus. Despite the dramatic morphological alteration, hFis1-fragmented mitochondria maintained a normal transmembrane potential and pH and took up normally the Ca2+ released from intracellular stores upon agonist stimulation, as measured with a targeted ratiometric pericam probe. In contrast, hFis1-fragmented mitochondria took up more slowly the Ca2+ entering across plasma membrane channels, because the Ca2+ ions reaching mitochondria propagated faster and in a more coordinated manner in interconnected than in fragmented mitochondria. In parallel cytosolic fura-2 measurements, the capacitative Ca2+ entry (CCE) elicited by store depletion was only marginally reduced by hFis1 expression. Regardless of mitochondria shape and location, disruption of mitochondrial potential with uncouplers or oligomycin/rotenone reduced CCE by ∼35%. These observations indicate that close contact to Ca2+ influx channels is not required for CCE modulation and that the formation of a mitochondrial network facilitates Ca2+ propagation within interconnected mitochondria. Mitochondria modulate Ca2+ signals by taking up, buffering, and releasing Ca2+ at key locations near Ca2+ release or influx channels. The role of such local interactions between channels and organelles is difficult to establish in living cells because mitochondria form an interconnected network constantly remodeled by coordinated fusion and fission reactions. To study the effect of a controlled disruption of the mitochondrial network on Ca2+ homeostasis, we took advantage of hFis1, a protein that promotes mitochondrial fission by recruiting the dynamin-related protein, Drp1. hFis1 expression in HeLa cells induced a rapid and complete fragmentation of mitochondria, which redistributed away from the plasma membrane and clustered around the nucleus. Despite the dramatic morphological alteration, hFis1-fragmented mitochondria maintained a normal transmembrane potential and pH and took up normally the Ca2+ released from intracellular stores upon agonist stimulation, as measured with a targeted ratiometric pericam probe. In contrast, hFis1-fragmented mitochondria took up more slowly the Ca2+ entering across plasma membrane channels, because the Ca2+ ions reaching mitochondria propagated faster and in a more coordinated manner in interconnected than in fragmented mitochondria. In parallel cytosolic fura-2 measurements, the capacitative Ca2+ entry (CCE) elicited by store depletion was only marginally reduced by hFis1 expression. Regardless of mitochondria shape and location, disruption of mitochondrial potential with uncouplers or oligomycin/rotenone reduced CCE by ∼35%. These observations indicate that close contact to Ca2+ influx channels is not required for CCE modulation and that the formation of a mitochondrial network facilitates Ca2+ propagation within interconnected mitochondria. Mitochondria actively participate to the cellular Ca2+ homeostasis and modulate the pattern of agonist-induced Ca2+ signals by their ability to sequester and release Ca2+ (1Duchen M.R. J. Physiol. 1999; 516: 1-17Crossref PubMed Scopus (527) Google Scholar). Because of the low Ca2+ affinity of the uniporter that constitutes the main mechanism of Ca2+ entry into mitochondria, it was proposed that the ability of these organelles to accumulate Ca2+ relies on their close location to Ca2+ release channels on the endoplasmic reticulum (ER) 1The abbreviations and trivial names used are: ER, endoplasmic reticulum; CCE, capacitative Ca2+ entry; CCCP, carbonylcyanide m-chlorophenylhydrazone; [Ca2+]mit, mitochondrial [Ca2+]; [Ca2+]cyt, cytosolic [Ca2+]; RP3.1mit, ratiometric pericam targeted to the mitochondrial matrix; SERCA, sarco/endoplasmic reticulum Ca2+ ATPase; ICRAC, Ca2+ release-activated Ca2+ current; Δψm, mitochondrial membrane potential; TMRM, tetramethylrhodamine methyl ester; GFP, green fluorescent protein. 1The abbreviations and trivial names used are: ER, endoplasmic reticulum; CCE, capacitative Ca2+ entry; CCCP, carbonylcyanide m-chlorophenylhydrazone; [Ca2+]mit, mitochondrial [Ca2+]; [Ca2+]cyt, cytosolic [Ca2+]; RP3.1mit, ratiometric pericam targeted to the mitochondrial matrix; SERCA, sarco/endoplasmic reticulum Ca2+ ATPase; ICRAC, Ca2+ release-activated Ca2+ current; Δψm, mitochondrial membrane potential; TMRM, tetramethylrhodamine methyl ester; GFP, green fluorescent protein. (2Rizzuto R. Brini M. Murgia M. Pozzan T. Science. 1993; 262: 744-747Crossref PubMed Scopus (985) Google Scholar, 3Rizzuto R. Pinton P. Carrington W. Fay F.S. Fogarty K.E. Lifshitz L.M. Tuft R.A. Pozzan T. Science. 1998; 280: 1763-1766Crossref PubMed Scopus (1748) Google Scholar). Mitochondria also interact with plasma membrane channels and thereby modulate the so-called capacitative Ca2+ entry (CCE) pathway, the ubiquitous Ca2+ entry mechanism triggered by emptying of the ER Ca2+ store (4Putney J.J. Cell Calcium. 1986; 7: 1-12Crossref PubMed Scopus (2077) Google Scholar, 5Putney J.J. Cell Calcium. 1990; 11: 611-624Crossref PubMed Scopus (1255) Google Scholar). Although the molecular identity of the channel(s) responsible for CCE as well as its mechanism of activation are still debated, recent evidence indicates that mitochondria represent a key organelle in CCE activity and/or activation. Indeed, CCE is inhibited by intracellular Ca2+ elevations, and mitochondria were shown to act as local buffers to prevent Ca2+-mediated inhibition of the CCE pathway (6Hoth M. Fanger C.M. Lewis R.S. J. Cell Biol. 1997; 137: 633-648Crossref PubMed Scopus (459) Google Scholar, 7Hoth M. Button D.C. Lewis R.S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10607-10612Crossref PubMed Scopus (229) Google Scholar, 8Parekh A.B. J. Biol. Chem. 1998; 273: 14925-14932Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 9Malli R. Frieden M. Osibow K. Graier W.F. J. Biol. Chem. 2003; 278: 10807-10815Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Local interactions between mitochondria and other subcellular structures are difficult to establish in living cells because mitochondria display a complex architecture that varies considerably between cell types. This ranges from a largely interconnected tubular network in COS-7, endothelial, or HeLa cells to round punctuated structures in hepatocytes (10Collins T.J. Berridge M.J. Lipp P. Bootman M.D. EMBO J. 2002; 21: 1616-1627Crossref PubMed Scopus (455) Google Scholar). Moreover, mitochondria are highly dynamic organelles that move in the cytosol and that constantly undergo fusion and fission. Both processes are under the control of certain GTPases and their associated proteins (11Shaw J.M. Nunnari J. Trends Cell Biol. 2002; 12: 178-184Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar). hFis1, the human orthologue of the yeast Fis1p (12Mozdy A.D. McCaffery J.M. Shaw J.M. J. Cell Biol. 2000; 151: 367-380Crossref PubMed Scopus (523) Google Scholar), is a 17-kDa transmembrane protein located in the outer membrane of the mitochondria that is involved in the machinery of mitochondria fission, and overexpression of this protein enhances the fission process in HeLa cells (13James D.I. Parone P.A. Mattenberger Y. Martinou J.C. J. Biol. Chem. 2003; 278: 36373-36379Abstract Full Text Full Text PDF PubMed Scopus (512) Google Scholar). In this study, we overexpressed the protein hFis1 in HeLa cells to induce a controlled fragmentation of mitochondria and measured the impact of these structural changes on cytosolic and mitochondria Ca2+ signals with fura-2 and with a targeted ratiometric pericam probe, respectively. This approach allowed us to investigate the role of mitochondria interconnection on cytosolic and mitochondrial Ca2+ homeostasis and to distinguish the local and global effects of mitochondria on the Ca2+ entry process. Materials—Minimum essential medium, fetal calf serum, penicillin, and streptomycin were obtained from Invitrogen. Histamine, thapsigargin, oligomycin, and rotenone were obtained from Sigma. Acetoxymethyl ester form of fura-2 (fura-2/AM) and Mitotracker Red were obtained from Molecular Probes Europe (Leiden, Netherlands). Carbonylcyanide m-chlorophenylhydrazone (CCCP) was obtained from Fluka (Buchs, Switzerland). Transfast transfection reagent was purchased from Promega. Cell Culture and Transfection—HeLa cells were grown in minimum essential medium containing 10% heat-inactivated fetal calf serum, 2 mml-glutamine, 50 units/ml penicillin, 50 μg/ml streptomycin and were maintained at 37 °C under 5% CO2. For experiments, cells were plated on 25-mm diameter glass coverslips 2-3 days before use. After reaching 40-60% of confluence, cells were transiently transfected with the the Transfast reagent to the by the For of cytosolic Ca2+ [Ca2+]cyt, hFis1 was with a targeted to the to cells were transfection with To mitochondrial [Ca2+]mit, cells were transfected with the ratiometric pericam targeted to the mitochondrial a from and with and was on an with an a Switzerland). were on a by the shown in and were with the The the Switzerland). To the of the cytosol by mitochondria, HeLa cells a cytosolic pericam were with Mitotracker Red for to mitochondria. The cytosolic and mitochondrial were and respectively. of in were the the were This for cells and to located at the of the this a of was to before mitochondria and membrane an was to the contrast, by an of the to the was to and a was used to the The of mitochondria and the membrane were from the For the mitochondrial than a of were not into The were in an and the impact between the membrane and the mitochondria were The impact to a of between mitochondrial and membrane or to the of the pH and is not by the fragmentation of the mitochondrial HeLa cells were transiently transfected with the RP3.1mit, and the pH and Ca2+ of the are in and was to the mitochondrial The in pH is by a of the at The the cell was with that induced a mitochondrial Ca2+ which is by a of the the only marginally HeLa cells were transfected with the and with the of CCCP, which the across the mitochondrial a of the at of the effect of are for the cells and for the of the mitochondrial Ca2+ with 50 in of the of Ca2+ induced by are for the cells and for the were in containing 2 pH with of for the experiments, was coverslips were in a with and for were on a a Switzerland). HeLa cells were for with 2 at in the and for to To [Ca2+]cyt, cells were at and with a a was a to the experiments, cells were with Mitotracker Red for and to with cells were by the of the and the of mitochondria was by the The of the was also at the of [Ca2+]cyt, the was For this we only cells with a and the of the cytosol used to not the nucleus. was a and were with the of was for Ca2+ to the Ca2+ channels, to the were at which to the of The of the at which the and the the was used as an for CCE changes in Δψm, cells were for with 50 tetramethylrhodamine methyl ester in and were in the were at and an in were as is the of the in the mitochondria by the cytosolic at a and is the of the mitochondrial cytosolic pH took advantage of the of RP3.1mit, is at and at T. A. A. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, M. Pozzan T. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar, also The cells were at and and was at a in pH were as is the at a and is the of at the of the in mitochondrial Ca2+ are shown as because at with Ca2+ of hFis1 on Mitochondria and in HeLa cells form a largely interconnected network constantly remodeled by fusion and fission reactions. To this fission, we overexpressed hFis1, the human orthologue of the yeast protein Fis1p to participate in mitochondrial shown in expression of hFis1 fragmented the mitochondrial network into organelles that clustered around the nucleus. The fragmentation process upon hFis1 expression was complete within as by of cells with a the cells the hFis1 a mitochondria with a study (13James D.I. Parone P.A. Mattenberger Y. Martinou J.C. J. Biol. Chem. 2003; 278: 36373-36379Abstract Full Text Full Text PDF PubMed Scopus (512) Google Scholar). To the of mitochondrial we took of mitochondria with Mitotracker and of the cell cytosol with the cytosolic protein ratiometric the mitochondrial as a we the of the cell by mitochondria on the cytosolic was in the cell shown in mitochondria to the and a of the cytosol in control the of the cell mitochondria was in cells with control and The were used to the of contact between mitochondria and the cell as the of the cytosolic shown in of the cell membrane was to mitochondria in control cells the of of a that was reduced by upon hFis1 expression. hFis1 not only mitochondrial fragmentation also mitochondria away from the plasma of the cell of mitochondria and between mitochondria and the cell of hFis1 on and the effects of hFis1 expression on mitochondrial we the mitochondrial membrane potential was For this cells were with and with μg/ml prevent and rotenone the complex of the shown in the elicited changes in in and control In the of rotenone a effect on their to a The of the induced a rapid and complete of the membrane The of in the of indicates that the was in hFis1 cells and that the mitochondrial membrane potential was not maintained by the mitochondrial in and we measured the and Ca2+ the mitochondrial of HeLa For these measurements, we took advantage of the to Ca2+ and pH of a ratiometric pericam targeted to mitochondria, by A. is highly to pH at an of not at T. A. A. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, M. Pozzan T. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). with of Ca2+ at is largely to Ca2+ at this by HeLa cells to the mitochondrial or to the agonist a in at as the mitochondria to its pH with the pH of the cytosol In contrast, of a only at that changes in mitochondrial Ca2+ [Ca2+]mit, at this used this approach to the effect of hFis1 expression on mitochondrial Ca2+ and pH shown in and of elicited a in at that was of in control and in The in to a because the were in and cells in the pH These the and indicate that the pH of the mitochondrial was not by hFis1 expression. In Ca2+ at of 50 of the of hFis1 In the of the the of the was in control and cells The measured in the of Ca2+ were also not the of the in cells and in hFis1 these indicate that mitochondrial pH and Ca2+ homeostasis is not by hFis1 expression the complete fragmentation of the mitochondrial of hFis1 on ER of normal in HeLa cells with fragmented and clustered mitochondria is because the of mitochondria to the Ca2+ the Ca2+ release was shown to for a mitochondrial Ca2+ Mitochondria proposed to form interactions with ER Ca2+ release channels to for the of Ca2+ between the organelles M. Pozzan T. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). Because hFis1 induced dramatic in mitochondrial we the ER was also shown in the pattern of the was not upon hFis1 that the mitochondrial was not by changes in ER of hFis1 on from to mitochondria to normally the Ca2+ released from the ER, as by the normal elicited by Because the main effect of hFis1, from is to move mitochondria away from the plasma membrane we hFis1 the ability of mitochondria to up Ca2+ from the plasma For this Ca2+ was to cells with 50 in the of shown in the of the were in control and in cells the to this was by hFis1 expression. The Ca2+ entering across the plasma membrane on to a in hFis1-fragmented mitochondria. were obtained in cells with the of and that ER Ca2+ were not involved in the of Ca2+ from the plasma membrane to mitochondria. To the structural of this we the pattern of the Ca2+ to control and hFis1 shown in in of control tubular mitochondria. In contrast, in of hFis1-fragmented mitochondria. The of hFis1-fragmented mitochondria was not because of a in the of Ca2+ from the plasma membrane to mitochondria, because the at the or in mitochondria from cells Ca2+ faster and in a more coordinated manner within tubular mitochondria that the propagation of the was by the fragmentation of the mitochondrial The in hFis1 cells a reduced or influx of Ca2+ across the plasma To this we measured Ca2+ influx with shown in the cytosolic Ca2+ changes upon Ca2+ to cells with were of and in control and To this CCE activity was measured by the of influx The of were not in control and cells that CCE was largely by the fragmentation and subcellular of the mitochondrial of hFis1 on CCE by mitochondria are required to CCE, it is not mitochondria act as Ca2+ buffers that or by the of the ER or by releasing a Because mitochondria in hFis1 cells were located away from the plasma membrane than in a to the local and global effects of mitochondria on For this cells were with to CCE and mitochondria was inhibited by or by a of rotenone and μg/ml The effects of the mitochondria on CCE were by the Ca2+ or by the shown in Ca2+ entry was reduced by in the of or of of hFis1 expression. entry was reduced to a in the of CCCP, in control to and in cells to These indicate that mitochondria are required for activation of CCE in HeLa the modulation of CCE by mitochondria is than in other cell types. In this study we the effect of a controlled disruption of the mitochondrial network on the Ca2+ homeostasis of mitochondria. For this we the protein hFis1 in HeLa cells to induce a rapid fragmentation and of their mitochondria. these dramatic morphological impact on the organelle because mitochondria were still to a normal membrane potential and pH and to up and release This allowed us to study Ca2+ by mitochondria located close or from the plasma to the role of mitochondria interconnection in the propagation of Ca2+ and to the local and global effects of mitochondria on plasma membrane Ca2+ channels. hFis1 Mitochondria the of the are dynamic organelles that form an tubular network the of fusion and fission the proteins the fusion and fission hFis1 was shown to induce mitochondrial fission in cells by recruiting the dynamin-related GTPases from the cytosol to the outer mitochondrial membrane (13James D.I. Parone P.A. Mattenberger Y. Martinou J.C. J. Biol. Chem. 2003; 278: 36373-36379Abstract Full Text Full Text PDF PubMed Scopus (512) Google Scholar, Y. Biol. 2003; PubMed Scopus Google Scholar). that expression of hFis1 in HeLa cells a complete fragmentation of mitochondria within This effect was for mitochondria because expression of hFis1 not the ER the mitochondria clustered around the of the cytosol of these that of the cellular was mitochondria in cells hFis1 with in control cells and that the fragmented mitochondria were located away from the plasma not the location of mitochondria to the of the plasma membrane the of the the of in the that upon hFis1 expression mitochondria of the hFis1 overexpression not the mitochondrial membrane potential as measured in with low of the and or in effects on This the that fragmented mitochondria maintained a normal membrane potential by the of because the not with the of a normal the pH of the mitochondrial was not transfection of hFis1 not because expression of hFis1 for shown to induce release and (13James D.I. Parone P.A. Mattenberger Y. Martinou J.C. J. Biol. 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This is to for the rapid that cell an of this it was proposed that the contact between the ER and mitochondria are highly that structural interactions between the organelles M. Pozzan T. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). This was on the of highly mitochondria cells and the than in to which indicates that mitochondria that of Ca2+ are not by other mitochondria from the In measurements, we not in the ability of fragmented or tubular mitochondria to up Ca2+ stimulation, in the in the of the Although this is not to the of a close between certain of the ER and mitochondria, it is to with the of that the ER was not by the fragmentation of the mitochondrial that hFis1 the and location of organelle the other Although it is that mitochondria move and interactions with other ER Ca2+ channels at a location, it is difficult to that move cells the ER to for the rapid of Ca2+ into fragmented mitochondria, the is that close between the ER and mitochondria at a the of the organelles in Although fragmented mitochondria normally the Ca2+ released from the ER, Ca2+ with a with tubular mitochondria the Ca2+ was the The was the Ca2+ was not Because that fragmented mitochondria are located in the Ca2+ ions on a before reaching a fragmented than a tubular mitochondria. this is to for the of Ca2+ to fragmented mitochondria, because fura-2 Ca2+ within in the The from the plasma membrane into a reduced Ca2+ around mitochondria. In this Ca2+ at a the mitochondrial Ca2+ uniporter its to accumulate Regardless of the the ability of mitochondria to up at that close to plasma membrane channels were not required for Ca2+ by mitochondria capacitative Ca2+ Moreover, observations that the of Ca2+ from the to mitochondria not the ER, because mitochondria located in the cell were still to up Ca2+ ER were inhibited by These indicate that Ca2+ not the ER to mitochondria and that Ca2+ are not required for the of Ca2+ that observations also that the formation of a tubular network facilitates the propagation of Ca2+ mitochondria. shown in Ca2+ was not only Ca2+ also more in fragmented than in tubular mitochondria. In fragmented mitochondria, Ca2+ in that Ca2+ in an In contrast, Ca2+ in an manner within of the tubular This indicates that the Ca2+ entering mitochondria within the and not mitochondria. between mitochondria in HeLa cells the (10Collins T.J. Berridge M.J. Lipp P. 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PubMed Scopus Google Scholar), of mitochondria the in local Ca2+ the Ca2+ inhibition on Ca2+ entry channels the for Ca2+ a local role of mitochondria on Ca2+ entry channels is not the mechanism by which mitochondria modulate CCE in HeLa Regardless of their location, mitochondria were required for CCE, because mitochondria with or oligomycin/rotenone reduced CCE in control and that in other cellular such as M.D. A.B. EMBO J. 2002; 21: PubMed Scopus Google Scholar), M. Button D.C. Lewis R.S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10607-10612Crossref PubMed Scopus (229) Google Scholar), or cells R. Frieden M. Osibow K. Graier W.F. J. Biol. 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Lipoproteins of Treponema pallidum and Borrelia burgdorferi possess potent proinflammatory properties and, thus, have been implicated as major proinflammatory agonists in syphilis and Lyme disease. Here we used purified B. burgdorferi outer surface protein A (OspA) and synthetic lipopeptides corresponding to the N-termini of OspA and the 47-kDa major lipoprotein immunogen of T. pallidum to clarify the contribution of CD14 to monocytic cell activation by spirochetal lipoproteins and lipopeptides. As with LPS, mouse anti-human CD14 Abs blocked the activation of 1,25-dihydroxyvitamin D3-matured human myelomonocytic THP-1 cells by OspA and the two lipopeptides. The existence of a CD14-dependent pathway was corroborated by using undifferentiated THP-1 cells transfected with CD14 and peritoneal macrophages from CD14-deficient BALB/c mice. Unlike LPS, cell activation by lipoproteins and lipopeptides was serum independent and was not augmented by exogenous LPS-binding protein. Two observations constituted evidence that LPS and lipoprotein/lipopeptide signaling proceed via distinct transducing elements downstream of CD14: 1) CHO cells transfected with CD14 were exquisitely sensitive to LPS but were lipoprotein/lipopeptide nonresponsive; and 2) substoichiometric amounts of deacylated LPS that block LPS signaling at a site distal to CD14 failed to antagonize activation by lipoproteins and lipopeptides. The combined results demonstrate that spirochetal lipoproteins and lipopeptides use a CD14-dependent pathway that differs in at least two fundamental respects from the well-characterized LPS recognition pathway.

Analysis of the human and mouse genomes identified an abundance of conserved non-genic sequences (CNGs). The significance and evolutionary depth of their conservation remain unanswered. We have quantified levels and patterns of conservation of 191 CNGs of human chromosome 21 in 14 mammalian species. We found that CNGs are significantly more conserved than protein-coding genes and noncoding RNAS (ncRNAs) within the mammalian class from primates to monotremes to marsupials. The pattern of substitutions in CNGs differed from that seen in protein-coding and ncRNA genes and resembled that of protein-binding regions. About 0.3% to 1% of the human genome corresponds to a previously unknown class of extremely constrained CNGs shared among mammals.

Increasing quantities of man-made organic chemicals are released each year into the biosphere. Some of these compounds are both toxic and relatively resistant to physical, chemical, or biological degradation, and they thus constitute an environmental burden of considerable magnitude. Genetic manipulation of microbial catabolic pathways offers a powerful means by which to accelerate evolution of biodegradative routes through which such compounds might be eliminated from the environment. In the experiments described here, a catabolic pathway for alkylbenzoates specified by the TOL plasmid of Pseudomonas was restructured to produce a pathway capable of processing a new substrate, 4-ethylbenzoate. Analysis of critical steps in the TOL pathway that prevent metabolism of 4-ethylbenzoate revealed that this compound fails to induce synthesis of the catabolic enzymes and that one of its metabolic intermediates inactivates catechol 2,3-dioxygenase (C23O), the enzyme that cleaves the aromatic ring. Consequently, the pathway was sequentially modified by recruitment of genes from mutant bacteria selected for their production of either an altered pathway operon regulator that is activated by 4-ethylbenzoate or an altered C23O that is less sensitive to metabolite inactivation. The redesigned pathway was stably expressed and enabled host bacteria to degrade 4-ethylbenzoate in addition to the normal substrates of the TOL pathway.