Propionibacterium acnes is the only microorganism that has been isolated from sarcoid lesions. Many P. acnes have been detected in sarcoid lymph nodes using quantitative PCR and in sarcoid granulomas by in situ hybridization. P. acnes trigger factor protein causes a cellular immune response only in sarcoid patients and induces pulmonary granulomas in mice sensitized with the protein and adjuvant, but only those with latent P. acnes infection in their lungs. Eradication of P. acnes by antibiotics prevents the development of granulomas in this experimental model. Although P. acnes is the most common commensal bacterium in the lungs and lymph nodes, P. acnes-specific antibody detected the bacterium within sarcoid granulomas of these organs. P. acnes can cause latent infection in the lung and lymph node and persist in a cell-wall-deficient form. The dormant form is activated endogenously under certain conditions and proliferates at the site of latent infection. In patients with P. acnes hypersensitivity, granulomatous inflammation is triggered by intracellular proliferation of the bacterium. Proliferating bacteria may escape granulomatous isolation, spreading to other organs. Latent P. acnes infection in systemic organs can be reactivated by another triggering event, leading to systemic sarcoidosis.Sarcoidosis is a systemic granulomatous disease of unknown etiology.  Sarcoidosis is one of the best-known systemic granulomatous diseases. Despite intensive investigation, however, the etiology of sarcoidosis has remained unresolved for more than 100 years . SeveralMycobacterial and propionibacterial organisms are the most commonly implicated etiologic agents based on studies using polymerase chain reaction (PCR) of microbial DNA from these organisms in tissues from sarcoid patients around the world –7. DiffeMycobacterium tuberculosis and a recombinant trigger factor protein from Propionibacterium acnes, have been examined in sarcoid patients and control subjects [Immune responses against microbial antigens from these organisms, such as ESAT-6 and KatG peptides from subjects –12. ImmuGranuloma formation results from the persistence of a nondegradable product or a hypersensitivity response . The twoP. acnes is so far the only microorganism isolated from sarcoid lesions by bacterial culture [P. acnes is an anaerobic, nonspore-forming, gram-positive rod bacterium indigenous to the skin and mucosal surfaces. A series of Japanese studies has provided accumulating evidence for a role of P. acnes in sarcoidosis. In this paper, we propose mechanisms of granuloma formation in response to this indigenous bacterium in subjects with sarcoidosis based on our results obtained using histopathological and experimental approaches and introduce a new concept of endogenous infection caused by hypersensitivity to indigenous bacteria.  culture , 15. P. The lung and its draining lymph nodes are the organs most commonly affected by sarcoidosis. As the lung constantly encounters airborne substances, including pathogens, many researchers have considered infection to trigger sarcoidosis and have thus tried to identify possible causative transmissible agents and their contribution to the mechanism of sarcoid granuloma formation , 17. P. acnes, and no other microorganism, was isolated from the large number of samples [P. acnes was isolated in culture from biopsy samples of 31 (78%) of 40 lymph nodes from 40 patients with sarcoidosis [P. acnes during biopsy, and the results of both studies were the same.In the late 1970s, a large Japanese research project conducted by many clinicians and microbiologists with support by a grant from the Japanese Ministry of Health was organized to seek the pathogens responsible for sarcoidosis. Extensive trials were performed to isolate microorganisms, including bacteria, viruses, and fungi, from tissue samples  affected by sarcoidosis. Only  samples . P. acnecoidosis , whereasP. acnes was isolated from 24 of 43 lungs and 8 of 11 mediastinal lymph nodes, mostly in pure culture. P. acnes was isolated from 10 of 20 gastric and 3 of 12 intestinal lymph nodes; intestinal bacteria were also numerous. The number of P. acnes cells isolated was usually no more than 500 colony forming units (CFUs)/g in the lungs and lymph nodes. Of 43 lungs from patients without sarcoidosis, only 4 (9%) had exceptionally high numbers of P. acnes cells. Random amplified polymorphic DNA analysis was used to compare the DNA of 45 isolates of P. acnes from these patients, 39 isolates from sarcoid lymph nodes, and 67 isolates from normal skin, conjunctiva, and intestine. The P. acnes strains in the lung and mediastinal lymph nodes differed genetically from those in the skin. Therefore, contamination from the skin during operative or culture procedures seems unlikely. These findings suggest that P. acnes normally resides in peripheral lung tissue and mediastinal lymph nodes.Ishige et al. cultured peripheral lung tissue and various lymph nodes obtained from patients with diseases other than sarcoidosis . P. acneP. acnes are essential for linking this indigenous bacterium to the cause of sarcoidosis because infectious granulomas are commonly caused by intracellular pathogens. Furukawa et al. examined the cell invasiveness and serotype of P. acnes isolates from lymph nodes affected by sarcoidosis, together with isolates from nonsarcoid tissue obtained from the lymph nodes, lungs, prostate, skin, conjunctiva, and intestine [P. acnes isolates into HEK293T cells was examined by cell invasion assay according to the method described by Cue and Cleary [Studies of cell invading ntestine . The invd Cleary  and intrSome investigators in Europe using PCR assays detected mycobacterial DNA in samples of affected tissue from patients with sarcoidosis –23, but P. acnes, P. granulosum, and M. tuberculosis in histologic sections of lymph nodes from patients with sarcoidosis, tuberculosis, or gastric cancer [M. tuberculosis were found in samples from all 15 patients with tuberculosis, 3 patients with sarcoidosis, and 1 control sample. Genomes of P. acnes were found in 12 of 15 patients with sarcoidosis, 2 tuberculosis patients, and 3 controls. The difference in the estimated number of P. acnes genomes between individuals with and without sarcoidosis was similar to that in the number of M. tuberculosis between people with and without tuberculosis. Biopsy samples from the three patients with sarcoidosis but without P. acnes all contained many P. granulosum DNA. These findings suggest that propionibacteria resided in or proliferated ectopically in the sarcoid lesions, whether or not there was a connection with the disease. Propionibacteria are more likely than mycobacteria to cause sarcoidosis.Ishige et al. used quantitative PCR to search for bacterial genomes of c cancer . They exP. acnes, P. granulosum, M. tuberculosis, M. avium subsp. paratuberculosis, and Escherichia coli (as the control) were estimated by quantitative real-time PCR. Either P. acnes or P. granulosum was found in all but two of the sarcoid samples. M. avium subsp. paratuberculosis was not found in any sarcoid sample. M. tuberculosis was found in only 0% to 9% of the sarcoid samples, but in 65% to 100% of the tuberculosis samples. In sarcoid lymph nodes, the total numbers of genomes of P. acnes or P. granulosum far exceeded those of M. tuberculosis. P. acnes or P. granulosum was found in 0% to 60% of the tuberculosis and control samples, but the total numbers of genomes of P. acnes or P. granulosum in these samples were lower than those found in sarcoid samples. Propionibacteriium spp. are more likely than Mycobacteriium spp. to be involved in the etiology of sarcoidosis, not only in Japanese but also in the European patients with sarcoidosis.The international collaborative study evaluated the possible etiologic link between sarcoidosis and the suspected bacterial species . FormaliIn situ localization of P. acnes genomes in sarcoid lymph nodes may help to elucidate an etiologic link between sarcoidosis and this indigenous bacterium. Formalin-fixed and paraffin-embedded biopsy samples of lymph nodes from nine patients with sarcoidosis, nine patients with tuberculosis, and nine patients with nonspecific lymphadenitis as controls were examined by quantitative real-time PCR (QPCR) for P. acnes and by in situ hybridization (ISH) using catalyzed reporter deposition (CARD) for signal amplification with digoxigenin-labeled oligonucleotide probes that complemented 16S rRNA of P. acnes [μm2 of tissue sections from inside and outside sarcoidosis and tuberculosis granulomas and from control lymph nodes were counted. The number of genomes determined by QPCR was examined for correlation with the mean signal count by ISH with CARD. In sarcoid samples, one or several signals were detected in the cytoplasm of some epithelioid cells in granulomas . The accumulation of P. acnes genomes in and around sarcoid granulomas suggests that this indigenous bacterium is related to the cause of granulomatous inflammation in sarcoidosis.P. acnes . The siganulomas . The meaP. acnes, Negi et al. screened for this indigenous bacterium in sarcoid and nonsarcoid tissues using immunohistochemical methods with novel P. acnes-specific monoclonal antibodies that react with cell-membrane-bound lipoteichoic acid (PAB antibody) and ribosome-bound trigger factor protein (TIG antibody). They examined formalin-fixed and paraffin-embedded samples of lungs and lymph nodes from 196 patients with sarcoidosis and corresponding control samples from 275 patients with nonsarcoidosis diseases. The samples were mostly from Japanese patients, with 64 lymph node samples from German patients [Granulomatous reactions are basically a defense mechanism that the body uses to fight off poorly degradable antigens. granulomas begin as a small collection of lymphocytes and macrophages surrounding poorly degradable antigens. The aggregating macrophages, called an early focus of granuloma, then change to epithelioid cells and become organized into a cluster of cells, called an immature granuloma. Further progression results in ball-like clusters of cells and fusion of macrophages into giant cells, called a mature granuloma. The questions that must be asked in searching for the cause of sarcoidosis, therefore, are: “What is the antigen that the granulomas are fighting?” and “How is the antigen localized within the sarcoid lesion?” To evaluate the pathogenic role of patients .Immunohistochemistry with the PAB antibody revealed small round bodies within sarcoid granulomas in 20/27 (74%) video-assisted thoracic surgery lung samples, 24/50 48%) transbronchial lung biopsy samples, 71/81 (88%) Japanese lymph node samples, and 34/38 (89%) German lymph node samples. The PAB antibody did not react with nonsarcoid granulomas in any of the 45 tuberculosis samples or the 34 samples with sarcoid reaction. The appearance of the small round bodies detected by the PAB antibody within sarcoid granulomas did not differ between lungs and lymph nodes. In sarcoid granulomas with many small round bodies, the cytoplasm of some granuloma cells was filled with small round bodies, consistent with the intracellular proliferation of the bacterium . In many% transbrIn nongranulomatous areas small round bodies detected by the PAB antibody were found in alveolar macrophages of lungs and paracortical macrophages of lymph nodes from many sarcoid and some nonsarcoid patients. In the lymph nodes, paracortical macrophages with many small round bodies detected by the PAB antibody  were obsP. acnes, but rather intact forms of intracellular bacteria because the original distribution pattern  of these bacterial components was preserved in terms of the morphologic structure of the bacterium. Furthermore, conventional electron microscopy revealed that these bodies lack a cell-wall structure and occasionally exhibit protrusions from the body that appear to be yeast-like proliferating features , characteristic of cell-wall-deficient (L-form) bacteria (P. acnes.Hamazaki-Wesenberg (HW) bodies frequently appear in sarcoid lymph nodes although these bodies are not specific to sarcoidosis –32. The bacteria . HW bodiP. acnes causes latent infection and persists in macrophages. HW bodies are dormant and cell-wall-deficient P. acnes. This dormant form of P. acnes can be activated endogenously under certain environmental conditions and proliferate in cells at the sites of latent infection. Small round bodies proliferating in macrophages are infective forms of P. acnes. When these bodies spread out of macrophages, they infect other cells or organs via the lymph and blood streams. Sarcoid granulomas are formed as a host defense mechanism at the sites of activated bacteria proliferating intracellularly in patients with hypersensitive immune responses to P. acnes to prevent the spread of the infectious agent.Histopathological analysis with the PAB antibody led us to formulate a hypothesis for the mechanism of granuloma formation in sarcoidosis . P. acneP. acnes because an epitope of lipoteichoic acid detected by this antibody is more exposed in cell-wall-deficient forms than in conventional forms of the bacterium. The high frequency and specificity of P. acnes detected by the PAB antibody within sarcoid granulomas suggest an etiologic link between sarcoidosis and this indigenous bacterium. The PAB antibody may be useful for diagnosing sarcoidosis caused by P. acnes, when the reactivity is detected in idiopathic granulomas  of the P. acnes trigger factor, which has 529 amino acid residues and a calculated molecular mass of 57,614 Da. The C-terminal sequence (Asp-463 to Lys-529) seems to be unique to P. acnes, with no similarity to sequences of other bacterial proteins deposited in the Swiss-Prot database. Conformational analysis of the Ser-491 to Lys-529 regions at the C terminus revealed it to be highly antigenic. RP35 caused sarcoidosis-specific proliferation of peripheral blood mononuclear cells (PBMCs) from 9 (18%) of 50 patients with sarcoidosis  or ESAT-6 recombinant protein were not significantly different. Sarcoidosis may arise from an imbalance of Th1/Th17 immune responses to viable P. acnes.Recently, Furusawa et al.  reportedP. acnes was obtained in studies of BAL cells. When stimulated with a crude extract of P. acnes with pyridine, BAL cells from patients with sarcoidosis proliferated more than BAL cells from healthy subjects or from patients with lung cancer [P. acnes antigen was greater in sarcoidosis patients than in healthy subjects or patients with other lung diseases [P. acnes DNA was detected in BAL cells from 21 (70%) of 30 sarcoid patients and 7 (23%) of 30 control patients with other lung diseases [In situ signals of P. acnes DNA were detected in the cytoplasm of a few alveolar macrophages among the BAL cells from sarcoid patients, but from no other kinds of BAL cells, including alveolar lymphocytes and neutrophils. Gallium-67 uptake by lung parenchyma was found in about half of the 30 sarcoid patients with P. acnes DNA, but in none of the other sarcoid patients [Additional evidence of the hypersensitivity of sarcoid patients to g cancer . Interlediseases . P. acnediseases . In situpatients . P. acnes activates NF-κB in both an NOD1- and NOD2-dependent manner [P = 0.006 for trend). Functional studies indicated that the NOD1 796A-allele is associated with reduced expression leading to diminished NF-κB activation in response to intracellular P. acnes , TLR4, and TLR9 mediate the effects of P. acnes infection [P. acnes. TLRs are likely to serve as first-line receptors for P. acnes, but NOD proteins might play a major role in a subsequent phase of intracellular infection. NOD1 was recently reported to be a critical regulator of beta-defensin 2 during Helicobacter pylori infection [κB enables P. acnes to survive and persist intracellularly, leading to the pathogenesis of sarcoidosis.nfection , 46. Thenfection . It is pP. acnes. A single intravenous injection of P. acnes into mice leads to the development of many granulomas in the liver [P. acnes into sensitized rats [P. acnes was used as a sensitizer, and a challenge by a single intravenous injection of the bacterium was essential for granulomas to form in the lungs.In experimental animals, granulomatous lesions can be induced by he liver –50, but zed rats  and rabbzed rats . In thesP. acnes trigger factor protein (RP35) or heat-killed P. acnes causes pulmonary granulomas in some (25%–57%) mice sensitized with the protein and complete Freund's adjuvant (CFA) [P. acnes as a challenge was not essential for granulomas to form in the lungs. Granulomas were scattered throughout the lungs, especially in subpleural areas (P. acnes. nt (CFA) . An intral areas . The graP. acnes trigger factor, such as has been experimentally induced, has been reported in some patients with sarcoidosis. Second, situations resembling intravenous challenge with P. acnes are rare in humans, and sarcoidosis can start in asymptomatic persons without evidence of septicemia.This experimental protocol may provide a satisfactory model of sarcoidosis. First, hypersensitivity to P. acnes trigger factor protein or heat-killed P. acnes in CFA induces granulomatous inflammation confined to the lungs. This finding suggests that such antigens from P. acnes exist in the lungs of mice even before the experiment. Experimental models of allergic diseases, such as encephalomyelitis , thyroidP. acnes was cultured from the lungs, liver, and lymph nodes from some of the untreated normal mice, and culture was most often successful with the lungs. There was an unexpected concordance in the rate (33%) of culture from normal lungs and the frequency of detection of pulmonary granulomas in mice sensitized with the trigger factor protein. The concordance suggests that mice without granulomas may have been free from P. acnes in the normal indigenous flora of their lungs before and during the experiment. Similar to the results obtained by bacterial culture of human samples from lung and lymph nodes, P. acnes trigger factor antigen developed more severe and diffuse pulmonary granulomatosis than did sensitized mice. In fact, the severe granulomatous inflammation that developed could be even identified macroscopically  before and during the experiments prevented granuloma formation in these experimental models . P. acnes was taken up by lung cells, and the P. acnes-bearing cells expressed F4/80 rather than CD11c or DEC205, consistent with the ability of macrophages to phagocytose and deliver antigens to dendritic cells in the lung. As far away as the end of the airway, airborne organisms are impacted and eliminated by mechanical defenses, including mucociliary clearance and coughing. Nevertheless, a small number of P. acnes might escape this system and reside on the alveolar surface. P. acnes genomes were detected in normal pulmonary lymph nodes as well as the lungs, and lymphocytes from lymph nodes showed P. acnes-specific proliferation, suggesting that these cells had already been exposed to P. acnes by lung-derived antigen-presenting-cells and had established a memory response. Additionally, these results indicate that P. acnes were continuously transported to pulmonary regional lymph nodes in the steady state. Because of this constant delivery of antigens to the pulmonary lymph nodes for a long period, the small number of indigenous P. acnes in the normal lung would be enough to produce a specific immune response, but not for the formation of a steady-state granuloma. Although mycobacterial, atypical mycobacterial, and other propionibacterial antigens are potential candidate endogenous microorganisms that trigger pulmonary granuloma formation, genomic analyses revealed an absence of these organisms in the lungs of specific-pathogen-free C57BL/6 mice. Nishiwaki et al. further examined the mechanism of pulmonary granulomatosis caused by the sensitization of heat-killed P. acnes , using aP. acnes . In the P. acnes-sensitized lymph node CD4+ T cells into naïve mice resulted in granulomatous changes in the lung, indicating that extrapulmonary lymph node CD4+ T cells primed with P. acnes can interact with pulmonary resident cells via the circulation and induce granuloma formation in the normal lung. It was therefore hypothesized that a continuous supply of P. acnes-sensitized T cells should lead to chronic pulmonary granuloma formation, and consequently, we performed continuous remote sensitization of normal mice with P. acnes. These mice exhibited distinct pulmonary granulomas, distributed in lymph-rich spaces such as the subpleural, peribronchial, and perivascular areas and had typical cellular components of granuloma and preferential Th1 cytokine expression. These features are similar to those of pulmonary sarcoidosis. In addition, the ratio of CD4 to CD8 BAL lymphocytes was elevated in the group immunized twice, and serum calcium levels were also increased. Thus, the characteristics of this P. acnes-immunization model, without any direct exposure of antigen to the lung, showed several similarities to those of sarcoid patients.The adoptive transfer of P. acnes cells in the lung affected pulmonary granuloma formation. As expected, preloading of P. acnes exacerbated pulmonary disorders, whereas reduction of the P. acnes population by antimicrobial treatment reduced the pulmonary lesions. These findings suggest a pivotal role of normally localized P. acnes in the formation of pulmonary granuloma by extrapulmonary P. acnes sensitization, as well as the potential clinical usefulness of antimicrobial eradication targeting lung-indigenous P. acnes for the treatment of pulmonary granulomatosis induced by similar pathogenesis.That study also examined whether changes in the number of preexisting Pneumocystis carinii pneumonia, are well known to be associated with immunodeficiency in AIDS patients. Combination type infections, such as Candida and Aspergillus, not only cause opportunistic infections, but may also cause hypersensitivity pneumonitis. The hypersensitivity type of endogenous infection does not cause any tissue damage until the hypersensitive immune response is triggered. P. acnes as a cause of sarcoidosis can therefore be classified within the group of endogenous diseases that results from hypersensitivity.In the past, once the germ theory of disease was accepted, microbes were considered to be pathogens if they met the stipulations of Koch's postulates. Although there are many microbes, however, most human infections are caused by only a few. Some microbes have been classified as pathogens although they do not cause disease in every host. In addition, some microbes have been classified as nonpathogenic although they cause disease in certain hosts. For these reasons, in a redefinition of the concepts of virulence and pathogenicity of microbes, Casadevall and Pirofski suggested a classification system for pathogens based on their ability to cause damage as a function of the host's immune response . Koch's P. acnes is the most [P. acnes is found in 20% of nonsarcoid lymph nodes by bacterial culture [P. acnes in nongranulomatous areas of the lungs and lymph nodes from nonsarcoid patients suggests that latent infection and endogenous reactivation of this indigenous bacterium occurs in these organs, even in patients without sarcoidosis. Sarcoidosis involves many organs, and the lungs and mediastinal lymph nodes are involved at the highest frequency [P. acnes in these organs may explain why they are frequently involved in sarcoidosis.the most  common cthe most . Some P. culture , 15% of  culture , 18% of  culture . Occasiorequency . CommensP. acnes, indigenous low-virulence bacterium, can cause latent infection in the lungs and lymph nodes and persist in a cell-wall-deficient form. This dormant form of P. acnes can be activated endogenously under certain environmental conditions and then proliferate in cells at the site of the latent infection. In patients hypersensitive to this endogenous bacterium, granulomatous inflammation is triggered by intracellular proliferation of the bacterium. Some proliferating bacteria may escape from isolation by the granuloma and spread to other organs via the lymph and blood streams. The spread of infective P. acnes might cause a new latent infection in systemic organs, such as eyes, skin, and heart. Latent infection established in certain systemic organs will be reactivated simultaneously by the next triggering event, resulting in the onset of systemic sarcoidosis  latent infection with cell-wall-deficient P. acnes, (2) endogenous activation of dormant P. acnes triggered by certain environmental factors, and (3) a hypersensitive Th1 immune response towards the intracellular proliferation of P. acnes. The formation of sarcoid granulomas might be induced by a Th1 immune response to one or more antigens of P. acnes proliferating in the affected organ or tissue in an individual with a hereditary or acquired abnormality of the immune system. Sarcoidosis is most likely the result of a complex interaction between infection, immunity, and allergic reaction. There are three conditions essential to the development of sarcoidosis caused by M. tuberculosis infection usually occurs in childhood and produces lesions termed the “primary complex,” which is a combination of lesions in the lung and lung hilar lymph nodes. Around 90% of subjects with primary infection by M. tuberculosis exhibit a so-called “unapparent infection;” that is, they are asymptomatic. Latent infection by this pathogen is characterized by healed lesions comprising consolidated scar tissue or necrotic lesions that often becomes calcified. The persistent mycobacteria in this dormant phase are thought to be cell-wall-deficient. Active tuberculosis occurs when the latent mycobacterial infection is endogenously activated under certain environmental conditions, especially in older people. The risk for activation is also significantly increased by immunosuppressive triggers, such as HIV infection and diabetes. Recent immunologic data provide evidence of latent tuberculosis in about one-third of the global population, which corresponds to more than 2 billion individuals [Tuberculosis shares many common features with sarcoidosis, not only their histopathological features, but also aspects of their pathogenesis. Many tuberculosis cases arise from the endogenous activation of latent tuberculosis infection. Primary ividuals .α drugs administered to patients with rheumatoid arthritis [α treatment is thought to reactivate latent tuberculosis infection [P. acnes might be reactivated by antitumor necrosis factor-α treatment, resulting in sarcoidosis in certain susceptible subjects among these patients.Endogenous reactivation of latent bacteria is well known to occur in tuberculosis, which shares many common features with sarcoidosis, not only the histopathological features, but also the pathogenic features. Many cases of adult tuberculosis are caused by endogenous activation of latent tuberculosis infection , 61. Tubrthritis , 63. Antnfection . In the P. acnes infection.Immunosuppressive, mainly corticosteroid, therapy has been used for sarcoidosis for more than 50 years, but the long-term effects of steroidal treatment in chronic sarcoidosis are still disputed . FurtherAntibiotics not only kill the bacteria proliferating in cells, but they also prevent the endogenous activation of latent bacteria. Long-term administration of antibiotics may therefore be effective for patients with progressive sarcoidosis by preventing inflammatory relapses caused by reactivation of the latent bacteria . If lateP. acnes antigens responsible for granuloma formation, such as the trigger factor protein. Recent advances in the modulation of epitope-specific immune responses by synthetic peptides might help to improve the treatment of sarcoidosis. Another approach for treating sarcoidosis is specific suppression of the hypersensitivity to P. acnes. In an observational study, Bachelez and colleagues [P. acnes is sensitive, for the treatment of chronic forms of cutaneous sarcoidosis. The authors treated 12 patients with biopsy-proven cutaneous sarcoidosis with 200 mg daily minocycline over a median 12-month period. With a median followup of 26 months, the authors noted complete and partial responses to treatment in 8 and 2 patients, respectively. The mean time to reach a maximal response was 3.2 months. Of the 8 patients who had a complete response, minocycline could be withdrawn in 7 patients, 3 of whom experienced recurrent lesions and received further treatment with 200 mg daily doxycycline. Complete remission was maintained for a mean of 15.3 months. Regression of pulmonary infiltrates and mediastinal lymphadenopathy was noted in the 2 patients with concurrent pulmonary involvement. Minocycline is the first-choice antibiotic for patients with acne vulgaris caused by lleagues  reportedP. acnes in the pathophysiology of sarcoidosis and the uncertainty of the long-term effects of corticosteroids. The lesions were worsening before the antibiotic therapy was initiated, so the improvements appeared to be due to the therapy. Park and colleagues [The results of a nationwide questionnaire survey, performed by a Japanese research group in 2005 (reported in Japanese), indicated that antibiotic therapy was effective in 43% of 87 patients with sarcoidosis treated with many kinds of antibiotics, including minocycline, doxycycline, and clarithromycin. Baba and colleagues  used minlleagues  reportedlleagues  reportedin vitro studies and corroborated by clinical trials. Tetracycline suppresses neutrophil migration and chemotaxis [in vitro [P. acnes proposed in this chapter suggest that these antibiotics have many roles in the process of granuloma formation, including the events triggering granuloma formation caused by intracellular proliferation of the bacterium.Based on the studies described in this section, the antimicrobial properties of tetracyclines are effective for treating sarcoidosis. Many researchers have questioned the antimicrobial role of tetracyclines because tetracyclines also have anti-inflammatory properties which were demonstrated by emotaxis , and minemotaxis . Both miin vitro . Althoughttp://marshallprotocol.com/ and also at http://autoimmunityresearch.org/.The Marshall protocol  is an erP. acnes is currently the only microorganism that has been isolated from sarcoid lesions by bacterial culture. In the late 1970s, Homma et al. reported frequent isolation of P. acnes from biopsy specimens of Japanese patients with sarcoidosis. In the 1980s, Abe et al. isolated the same microbe from 31 (78%) of 40 sarcoid lymph nodes. These Japanese researchers also cultured this organism from 20% of 141 nonsarcoid lymph nodes. In 1999, Ishige et al. reported many genomes of P. acnes in biopsy samples of lymph nodes from 12 of 15 Japanese patients with sarcoidosis. Biopsy samples of the three patients without P. acnes all showed many genomes of P. granulosum. Similar results were obtained in an international collaborative study of samples from European patients with sarcoidosis. P. acnes genomes in and around sarcoid granulomas, suggesting that this indigenous bacterium is related to granulomatous inflammation in sarcoidosis. In 2012, Negi et al. reported that a novel monoclonal antibody specific to P. acnes that recognizes lipoteichoic acid of the plasmalemma reacted with small round bodies within sarcoid granulomas in 88% of lymph node biopsy samples, 77% of video-assisted thoracic surgery lung samples, 57% of transbronchial lung biopsy samples from Japanese patients with sarcoidosis, and in 89% of lymph node biopsy samples from German patients with sarcoidosis. Reactivity to the antibody was not observed in nonsarcoid granulomas, including those from patients with tuberculosis and so-called sarcoid reaction. The high frequency and specificity of P. acnes detected within sarcoid granulomas indicate that this indigenous bacterium might be the cause of granuloma formation in many patients with sarcoidosis. In 2002, Yamada et al. found P. acnes is the most common commensal bacterium in the lungs and lymph nodes of subjects without sarcoidosis. P. acnes is found in 20% of nonsarcoid lymph nodes by bacterial culture and in 15% of nonsarcoid lymph nodes by polymerase chain reaction. In nonsarcoid samples examined by immunohistochemistry, P. acnes was found in 18% of lung samples and in 22% of lymph node samples. Occasional detection of P. acnes in nongranulomatous areas of the lungs and lymph nodes from nonsarcoid patients indicates that host factors may be more critical than agent factors in the sarcoidosis etiology. P. acnes causes a cellular immune response in some sarcoid patients, but not in subjects without sarcoidosis. The P. acnes trigger factor protein induces pulmonary granulomas in mice sensitized with the protein and adjuvant, but only in mice with latent P. acnes infection in their lungs. This pathogenesis was confirmed by further experiments using mice sensitized with P. acnes and adjuvant, in which the eradication of P. acnes by antibiotics prevented granulomas in this experimental model. The findings from this experimental model of sarcoidosis might explain the recently reported effectiveness of tetracyclines for treating sarcoidosis. A particular protein, referred to as a trigger factor, from P. acnes can be activated endogenously under certain environmental conditions and then proliferate in cells at the site of latent infection. In patients who are hypersensitive to this endogenous bacterium, granulomatous inflammation is triggered by intracellular proliferation of the bacterium. Some of the proliferating bacteria may escape from isolation by the granuloma and spread to other organs via the lymphatic and blood streams. The spread of infective P. acnes might cause a new latent infection in systemic organs such as the eyes, skin, and heart. Latent infection established in certain systemic organs will be reactivated simultaneously by the next triggering event, resulting in the onset of systemic sarcoidosis.This indigenous low-virulence bacterium can cause latent infection in the lungs and lymph nodes and persist in a cell-wall-deficient form. This dormant form of


TAR DNA-binding protein 43 (TDP-43) is a protein that is involved in the pathology of Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Lobar Degeneration (FTLD). In patients with these neurodegenerative diseases, TDP-43 does not remain in its normal nuclear location, but instead forms insoluble aggregates in both the nucleus and cytoplasm of affected neurons.We used high density peptide array analysis to identify regions in TDP-43 that are bound by TDP-43 itself and designed candidate peptides that might be able to reduce TDP-43 aggregation. We found that two of the synthetic peptides identified with this approach could effectively inhibit the formation of TDP-43 protein aggregates in a concentration-dependent manner in HeLa cells in which a mutated human TDP-43 gene was overexpressed. However, despite reducing aggregation, these peptides did not reduce or prevent cell death. Similar results were observed in HeLa cells treated with arsenite. Again we found reduced aggregation, in this case of wild type TDP-43, but no difference in cell death.Our results suggest that TDP-43 aggregation is associated with the cell death process rather than being a direct cause. Recent evidence links TDP-43 pathology to at least two forms of neurodegeneration that had heretofore been thought to be quite separate. FTLD is the second most common type of early-onset neurodegenerative dementia after Alzheimer’s disease, and ALS is the most common adult-onset progressive motor neuron disease (MND). The TAR DNA binding protein 43 (TDP-43) has been found to be the major protein constituent of the intracellular aggregated inclusions in both FTLD with ubiquitin-positive inclusions (FTLD-U) and ALS ,2.TDP-43 is a 414 amino acid protein encoded by the TARDBP gene on chromosome 1. It was originally identified as a transcriptional repressor of the human immunodeficiency virus type 1 (HIV-1) gene . TDP-43 The mechanism through which TDP-43 is involved in neuronal death and degeneration remains unclear. One of the best characterized pathological features of TDP-43 proteinopathies is the cytoplasmic inclusions of TDP-43 aggregates. As with other protein misfolding diseases, TDP-43 mediated toxicity may result from a toxic gain of function associated with its aggregation. Johnson and colleagues have established a yeast model involving overexpressed full-length human TDP-43 or various TDP-43 truncation products . They foTo further elucidate the links between TDP-43 aggregation and disease mechanisms, we used two different models. The first was an aggregation model pioneered by Nonaka and colleagues . They foThe protein array described in the Methods section was used to identify candidate binding regions at which TDP-43 might interact with itself. Given the nature of the overlapping sequences associated with each neighboring peptide (frame shift of 2 aa), only positive spots in a series were considered as possible binding sites. Five separate and robust binding regions were found on the TDP-43 peptide array after incubating the membrane with recombinant TDP-43 protein followed by visualization of an antibody against TDP-43 Figure . This reTo verify whether the five regions of TDP-43 identified through the array assay are actually involved in self binding of the protein, the five peptides indicated in Figure To examine whether the peptides blocking interaction of recombinant TDP-43 protein with the peptide array could also inhibit TDP-43 aggregation in cells, a construct expressing a mutant TDP-43 lacking residues 187–192 [To investigate whether the synthetic peptides could inhibit the aggregation of mutant TDP-43, N-terminally TAT conjugated peptides A, B, C, D, E and/or TC were added to the cell culture medium after transfection. The peptides were added either individually or combined together in various ways . The TAT peptide has been used to deliver large molecules and small particles across both the plasma membrane and the nuclear membrane . The numTo investigate whether our peptides could inhibit aggregation of wild-type TDP-43 under stress conditions, HeLa cells were treated with arsenite with or without addition of synthetic peptides. After exposure to 0.5 μM arsenite with or without 10 μM of the synthetic peptide mixture (ABCDE) for 4 hours, the cell lysates were sequentially extracted and separated into soluble and insoluble proteins. The relative ratio of insoluble to soluble TDP-43 was used as a measure of TDP-43 aggregation propensity. After the treatment with arsenite, soluble endogenous TDP-43 was transformed into predominantly insoluble protein and showed a significant increased aggregation propensity (243.23 ± 22.97%) compared with control cells without treatment (41.02 ± 24.02%)  compared to untreated cells. Treatment with Peptide B and C, which significantly reduced the aggregation, led to similar levels of cell viability  as was found in untreated and scrambled peptide treated cells   compared to the basal condition. Our synthetic peptides decreased insoluble TDP-43 but did not prevent cell death  Figure . These rTo further elucidate the relationship between the cell death and TDP-43 aggregation, the cells were triple-labeled with TDP-43, GFP and the cell death marker, TUNEL. The numbers of transfected cells, aggregation-positive cells, or TUNEL-positive cells were counted by a blinded independent investigator. We measured four fields and 1000–1200 cells per microscope slide. We found that almost all the cells with TDP-43 aggregation that were untreated, Peptide B and C treated, or scrambled peptide (TC) treated showed TUNEL positive signals. In addition, even though Peptides B and C combined reduced TDP-43 aggregation, a much higher percentage of dead cells were observed in Peptides B and C treated cells without aggregation (12.22% ± 0.75%) than in scrambled peptide treated cells (5.95% ± 0.95%). Finally, we also measured cell death in cells without mutant TDP-43 overexpression but treated with the peptides. Cell death percentage in the untransfected cells of wild-type TDP-43, mutant TDP-43, mutant TDP-43 plus peptides BC and mutant TDP-43 plus scrambled peptide groups were 3.14% ± 0.56%, 2.28% ± 0.36%, 3.22% ± 0.35%, and 3.57% ± 0.39%, respectively  can induce TDP-43 aggregation and cell death in HeLa cells 7-192 can. Accordi2O2 and NMDA. Despite this increased neuronal vulnerability, we did not observe either nuclear or cytoplasmic TDP-43 aggregation. These results suggesting that reduced levels of TDP-43 in the nucleus may be critical for cytoxicity are consistent with evidence reviewed by Z.S. Xu in a recent paper [Our results indicating that aggregation may not be the causative factor for cytotoxicity in cells expressing mutant TDP-43, appear consistent with other emerging evidence that aggregation is not necessarily the cause of cellular toxicity. Previous work from our lab  showed tnt paper . These rnt paper . Recent nt paper . Moreovent paper ,32. Overnt paper . Togethein vivo. Furthermore, the same approach as the one taken here may be used to study the role of aggregation of other proteins, e.g. Huntingtin, α-synuclein, β-amyloid, in other neurodegenerative diseases.Protein aggregation has been observed in a number of neurodegenerative diseases. While aggregation is generally to be associated with cytotoxicity, it can also be a phenomenon associated with excessive ER stress, which is caused by an unusually large amount of misfolded proteins in the cell -38. In fIn this study, we are the first group to identify the regions in TDP-43 protein that involve in its self-aggregation. Peptides derived from those regions can effectively reduce the aggregation in cells transfected with an aggregation prone mutant TDP-43 or treated with arsenite. We then showed that reducing TDP-43 aggregation did not protect cells from cell death caused by either the expression of mutant TDP-43 or arsentie treatment. Thus, our study is the first to separate aggregation of TDP-43 from other factors involved in TDP-43 proteinopathy and demonstrate that TPD-43 aggregation may not be the cause of cytotoxicity.The peptides were synthesized on derivatized cellulose-based membranes  by the UBC Peptide Synthesis facility using a previously described protocol . The pepCell-penetrating peptides consisting of the truncated TAT domain at the N-terminal were synthesized by GL biochem Ltd. (Shanghai) and purified by HPLC. TAT, the trans-acting activator of transcription of the human immunodeficiency virus (HIV-1), has been used as an efficient way of delivering proteins or peptides into cells . The resMembranes were incubated with the mixture of TDP-43 protein (5 μg/ml) and/or single synthetic peptides (100 μg/ml) overnight at 4°C. Then the membranes were incubated sequentially with primary antibody and secondary antibody and washed with TBST. The protein interaction was visualized using an enhanced chemiluminescence reaction assay (PerkinElmer Life Sciences). Optical densities of each peptide array were measured using NIH ImageJ software.2. Wild type TDP-43 plasmid and mutant TDP-43 plasmid lacking residues 187–192 were obtained from Dr. Nonaka and Dr. Arai. The plasmids were transfected into the HeLa cells using the Lipofectamine 2000 system  according to the manufacturer’s instructions. After incubating the cells with the transfection mixture for 5 hours, the medium was replaced with fresh DMEM  with 2.5–20 μM synthetic peptides. After 24 hours, the medium was replaced with fresh peptides. The cells were returned to the incubator for an additional 24 hours before study.HeLa cells were obtained from the American Type Culture Collection (ATCC) and grown in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum  and 1% antibiotics . Cultures were maintained at 37°C in a humidified incubator  with 5% COFor studies using arsenite, arsenite  was added to the cell culture medium to a final concentration of 0.5 μM with or without the presence of synthetic peptides. The cells were then returned to the incubator for 4 hours before harvesting and analysis.Cells were fixed with pre-warmed 4% Paraformaldehyde  containing 4% sucrose  in PBS for 10 minutes at room temperature and permeabilized with 0.1% Trition X-100  in PBS for 2–3 minutes at room temperature. Then the reaction was blocked with 10% Bovine serum albumin  in PBS for 1 hour at room temperature and incubated with a primary antibody against TDP-43  overnight at 4°C. Reactions were visualized with anti-rabbit antibody conjugated with Alexa 488 . Cell nuclei were labeled with DAPI  for 2–3 minutes at room temperature. Images were obtained with an Olympus Fluoview FV1000 Confocal scanning microscope.The viability of cultured cells was assessed using the MTT assay. MTT -2,5-diphenyltetrazolium bromide)  was added to each well . After 4 h incubation, cells were lysed overnight. The optical density of test and control samples was measured at 590 nm absorbance with a reference filter of 750 nm using a “uQuant” microplate spectrophotometer . The absorbance was translated into cell viability ratios for comparison: cell viability ratio = (test sample absorbance/control sample absorbance) × 100%.in situ cell death detection kit . Cells were seeded on poly-D-lysine coated cover slips at a density of 2.5 × 104 cells per well in 24 well plate. After treatment, cells were fixed and stained with anti-TDP-43 , anti-GFP , Alexa 647 anti-rabbit  and Alexa 488 anti-chicken . The enzyme solution  and label solution were mixed in a volume ratio of 1:9 to obtain the TUNEL reaction mixture. The cells were incubated with the 50 μl TUNEL reaction mixture at 37°C for 1 hour and then washed three times with PBS. Then the cover slips were mounted on glass slides with antifade reagent . Images were obtained with an Olympus Fluoview FV1000 Confocal scanning microscope.Deoxynucleotidyl-transferase-mediated dUTP nick end-labeling (TUNEL) staining was performed using an To examine the effect of arsenite on solubility of endogenous TDP-43, preparation of soluble/insoluble protein were performed. HeLa cells were washed twice with cold PBS, lysed in cold lysis buffer . Cell lysates were rotated for 15 min and then centrifuged at 55,000 rpm for 15 min at 4°C. The supernatants were collected as soluble proteins. To prevent contamination caused by carrying over, the pellets were re-centrifuged at 55,000 rpm for 15 min at 4°C and washed once with lysis buffer. Then the pellets were lysed in PBS with 1% SDS and sonicated. The supernatants were collected as insoluble protein. Soluble and insoluble proteins were analyzed by immunoblotting.Protein concentrations of cell lysate and medium were measured using a standard BCA assay (Bio-Rad) as per the manufacturer’s instructions. Proteins were separated in SDS–PAGE (10%) and transferred to nitrocellulose membranes (Bio-Rad) at 4°C. Membranes were blocked with 5% nonfat dry milk in TBST for 1 hour, then incubated in 5% nonfat dry milk/TBST overnight at 4°C with antibodies for TDP-43 or β-actin . After washing with TBST, membranes were incubated with anti-rabbit secondary antibody conjugated with horseradish peroxidase for 1 hour at room temperature. Blots were developed using an enhanced chemiluminescence reaction assay.All data were expressed as mean ± SD and analyzed by one-way ANOVA. A value of p < 0.05 was considered statistically significant.The authors declare that they have no competing interests.RL, WJ and MSC conceived and designed the experiments. RL performed the experiments and analyzed the data. GY provided assistance in application of immunofluorescence methods. TN and TA were involved in plasmid construction. RL, WJ and MSC drafted and edited the manuscript. All Authors read and approved the final manuscript.


The tbx5 mutation in human causes Holt-Oram syndrome, an autosomal dominant condition characterized by a familial history of congenital heart defects and preaxial radial upper-limb defects. We report aberrant apoptosis and dormant cell growth over head, heart, trunk, fin, and tail of zebrafish embryos with tbx5 deficiency correspond to the dysmorphogenesis of tbx5 morphants.Wild-type zebrafish embryos at the 1-cell stage were injected with 4.3 nl of 19.4 ng of tbx5 morpholino or mismatch-tbx5-MO respectively in tbx5 morphants and mismatched control group. Semi-quantitative RT-PCR was used to for expression analysis of apoptosis and cell cycle-related genes. TUNEL and immunohistochemical assay showed the apoptosis spots within the local tissues. Ultra-structure of cardiac myocardium was examined by transmission electron microscope.Apoptosis-related genes , and cell cycle-related genes  showed remarkable increases in transcriptional level by RT-PCR. Using a TUNEL and immnuohistochemical assay, apoptosis was observed in the organs including the head, heart, pectoral fins, trunk, and tail of tbx5 knockdown embryos. Under transmission electron microscopic examination, mitochondria in cardiomyocytes became swollen and the myocardium was largely disorganized with a disarrayed appearance, compatible with reduced enhancement of myosin in the cardiac wall. The ATP level was reduced, and the ADP/ATP ratio as an apoptotic index significantly increased in the tbx5 deficient embryos.Our study highlighted that tbx5 deficiency evoked apoptosis, distributed on multiple organs corresponding to dysmorphogenesis with the shortage of promising maturation, in tbx5 knockdown zebrafish embryos. We hypothesized that mesenchymal cell apoptosis associated with altered TBX5 level may subsequently interfered with organogenesis and contributed to dysmorphogenesis in tbx5 deficiency zebrafish embryos. Tbx5 belongs to the T-box family of transcription factors and is required for the embryonic development of the heart and forelimbs [Tbx5 mutations in humans cause Holt-Oram syndrome (HOS), an autosomal dominant condition characterized by a familial history of congenital heart defects and preaxial radial ray upper-limb defects [tbx5 deficiency in different vertebrates are quite similar. Tbx5 deficiency in zebrafish causes multiple organ defects during organogenesis, including a shortened trunk, failure of cardiac looping formation, and hypogenesis or agenesis of the pectoral fins [tbx5 genotype and phenotype.orelimbs ,2. Tbx5  defects ,4. The pral fins . Either tbx5 encodes a transcription factor, tbx5 deficiency is presumed to disrupt development by altering the transcriptional function of the TBX5 protein, thereby affecting downstream target gene expressions, particularly affiliation binding proteins or transcription factors [Since  factors -9. Thougtbx5 leading to cardiac cell cycle arrest have been proved to elicit defect in cardiac sacromere formation and decreased cardiac cell number [The syndrome caused by mutations in T-box transcription factor 5 (TBX5) was described as "premature stops" or "developmental delay" in embryogenesis, though the mechanism how to delay or stop the development of the embryo, especially the cardiogenesis, remained indistinct . Some rel number .Apoptosis is a key event in many biological processes including embryogenesis and occurs in a variety of circumstance in specific time window in embryos. Zebrafish undergoes different levels of developmental cell death throughout embryonic stages precisely, including brain, spinal cord, ear, eyes, germ cells, tail bud and excretory system . Apoptostbx5 deficiency; meanwhile, the excessive apoptosis is in coincidence with the dysmorphogenesis in individuals with tbx5 deficiency.In this study, we reported that aberrant apoptosis and cell cycle arrest are increasing in zebrafish embryos with The approval of this study was granted by the National Taiwan Ocean University Aquaculture Animal ethics review board with annual evaluation.Zebrafish were maintained in 45-liter aquaria heated to 28.5°C with 25 fish per tank. The water was filtered, and about half of the water was replaced at least once a week. Adult zebrafish were fed 1 or 2 times per day with a variety of food, and the tank was cleaned by siphoning off any excess food after the second daily feeding. The day-night cycle was controlled with an automatic timer (14 h light/10 h dark).Zebrafish reach sexual maturity in 10~12 weeks, but breeding fish should be between 7 and 18 months of age for maximum embryo production. The day before breeding, 1/3 of the water was replaced and the tank was cleaned after feeding (1~2 h before the end of the light period). Finally, a collection box was placed at the bottom of the tank and preparation was made to collect the embryos the next day.When the light came on, we removed the collection box and placed the collected embryos into an incubator maintained at a temperature of 28.5°C.Total RNA was isolated from 50 embryos using the guanidine isothiocyanate-based TRIzol solution. RNA samples were resuspended in DEPC-treated water and quantified spectrophotometrically at 260 nm. The RNA quality was then checked by 1.2% agarose gel electrophoresis, after staining with 1 μg/ml ethidium bromide. The RNA stock was stored at -80°C.bcl2 , bad , and bax , p27 , p57 , and pcna , cdk2 . PCR conditions were as follows: denaturation at 95°C for 3 min, followed by 50 cycles of amplification . Quantification of PCR product was performed on an electrophoresis agarose gel using Kodak image 3.5 software. All measurements were performed in triplicate (n = 3).Total RNA was prepared from 50 morpholino (MO) injected or normal embryos . Three microliters of 1st-strand cDNA was amplified. Amplification primers for each specific mRNA were deduced from published sequences and included tbx5-MO (5 GAAAGGTGTCTTCACTGTCCGCCAT-3), was designed against the tbx5 translational start site  and a mismatch tbx5-MO (5'-GTCTCTTGACTCTCCGCGATCTCGG-3') was designed for control . Wild-type embryos primarily at the 1-cell stage with the chorion intact were injected with 19.4 ng/4.3 nl of stock morpholino diluted in Danieau's solution. The injected embryos were raised at 28.5°C. Embryos used for analyzing the expression of various markers were fixed with 4% paraformaldehyde. Otherwise, embryos were scored after 2 days of development for late effects. In our previous study, three control groups, including 3' end of tbx5-MO(2) (5'-GCCTGTACGATGTCTACCGTGAGGC-3') and embryos with blank microinjection and wild-type ones without microinjection, were included to identify the specific blockage of tbx5 mRNA translation effect of tbx5-MO.6 In order to examine the knockdown effectiveness of the tbx5-MO, a tbx5-GFP construct was generated by fusing a 468-bp fragment of tbx5, which included the morpholino target side into a GFP producing vector. Tbx5-MO at 19.4 ng and tbx5-GFP DNA at 150 μg were co-injected into 1-cell stage embryos. In control group, only 150 μg of tbx5-GFP was microinjected into 1-cell stage embryos [The morpholino antisense oligonucleotide, Zebrafish embryos were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS). Deparaffinized sections (3 μm) of zebrafish embryo tissues were placed on slides and processed for immunohistochemistry. After blocking with a biotin blocking system  for 30 min, sections were incubated with target-purified rabbit primary antibodies, including BCL2 , BAD , CASPASE-3 , CASPASE-8 ,CDK2 , P27  and MF20 , washed in PBS, and then incubated with a rhodamine-conjugated secondary antibody: goat anti-rabbit immunoglobulin G (IgG). After washing in PBS, sections were incubated with mounting medium and kept at 4°C.4 in Sorenson's phosphate buffer followed by dehydration through a graded series of ethanol washes, and embedded in Spurr's EPON. Blocks were heated in an oven for 8 h at 70°C. Semithin (1-μm) sections were cut and stained with toluidine blue for adequate preview under a microscope. Ultrathin sections (900 Å) were cut with a diamond knife, stained with uranyl acetate and lead citrate, and examined with an electron microscope.Embryos were fixed at 48 h post-fertilization (hpf) with 2.5% glutaraldehyde in Sorenson's phosphate buffer, postfixed with 1% OsOBoth whole mount and sectioned TdT-UTP nick end labeling (TUNEL) assays were performed using an ApopTag kit . Briefly, zebrafish embryos were fixed with 4% paraformaldehyde in PBS. Proteinase K-treated whole embryos or deparaffinized sections (5 μm) of embryos were incubated with the TdT enzyme followed by anti-digoxigenin. Finally, embryos or slides were stained with DAB for 5 min.Embryos were homogenized on ice in lysis buffer . Then, cellular debris was pelleted by centrifugation at 12,000 rpm for 20 min, and the supernatant was collected and measured. Proteins were mixed with sample buffer before separating in 10% sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels. The SDS-PAGE was then transferred onto nitrocellulose membranes at 100 V for 1 h. Membranes were blocked with blocking buffer ) at room temperature for 1 h. The TBX5 primary antibody  was incubated overnight at 4°C using the appropriate dilution (1: 1000). The nitrocellulose membranes were washed with PBST followed by incubating with a horseradish peroxidase (HRP)-conjugated secondary antibody (1: 5000) for 1 h at room temperature before the images were digitized.n = 10 in each subgroup) were separately placed in 96-well round-bottomed, flat, white plates for detection. The RLU (relative luminescence unit) value was detected using the PARADIGM™ Detection Platform .ATP and ADP/ATP ratio assay were performed using an ApoSENSOR™ ADP/ATP Ratio Assay Kit . Ten embryos  directly into embryos at the 1-cell stage resulted in retardation of the looping process, and the most prominent cardiac finding in tbx5 morphants was a string-like heart  embryos at 48 hpf, meanwhile the ventricle overlapped with the atria during the diastolic phase Figure . Injectit Figure . At 96 ht Figure ; howevers Figure . The tais Figure  and truns Figure  of knocks Figure  and truns Figure , both oftbx5 knockdown embryos  construct [us study . The effg Figure  and tbx5onstruct .bad, bax, and bcl2, showed remarkable increases in tbx5 morphants in all studied periods but not in embryos injected with mismatch-tbx5-MO, which has no significant different with the wild-type and control mismatch   Figure .tbx5 knockdown embryos. In addition, myofibrils appeared to be disarrayed  was remarkably decreased  or pro-apoptosis related genes (bad and bax) were all transcriptionally activated, and so were the cell proliferation markers (cdk2 and pcna) and cyclin kinase inhibitors (p27 and p57). In protein level, remarkably plentiful production of apoptosis-related proteins including CASPASE-3, CASPASE-8, and BAD were proved by immunohistochemistry These data disclose that a deficiency of tbx5, a transcription factor, in zebrafish embryos may trigger multiple signal pathways including promoting or inhibiting molecules and eventually come to the end sequel of apoptosis and cease of cell growth.Our data revealed activation of apoptosis and inhibition of cell growth in embryonic tbx5 in the embryos might be associated with the dysmorphogenesis, especially engendering the string-like heart, short fins, and malformed trunk during zebrafish embryonic development.Besides, the distribution of aberrant apoptosis and deviant cell cycle caused by a deficiency of tbx5-deficient embryos at the late stage of organogenesis [There are two main apoptosis pathways including cell-intrinsic  pathway and cell-extrinsic (death receptor) pathway; the intrinsic pathway is largely regulated by mitochondria, whereas the extrinsic pathway interacts with death receptors . Mitochoogenesis ,26.tbx5 knockdown embryos, rather than small and dense in WT embryos. This phenomenon is compatible with the morphological changes of mitochondria during apoptosis, which culminates in permeabilization of the mitochondrial outer membrane and release of soluble proteins from the mitochondrial intermembrane space. Once the mitochondrial permeability transition pore is opened, proapoptotic proteins are released, and the membrane potential collapses, which results in swelling of mitochondria. The number of mitochondria in tbx5 deficiency embryos also increased in addition to their size. Although a similar phenomenon was described during apoptosis induced by anticancer drugs in colon cancer cells and apoptosis in mitochondrial myopathy, mitochondrial proliferation accompanied by mitochondrial swelling was first reported in apoptosis of zebrafish embryos with tbx5 deficiency [The mitochondrial apoptotic process causes the outer membrane of mitochondria to release proapoptotic proteins and eventually gives rise to swollen mitochondria ,28. Our ficiency . Until ntbx5 knockdown embryos appeared eminently disorganized. The myofibrils were quantitatively diminished at both the atrial and ventricular levels. We used MF20, an anti-myosin antibody, to prove a decrease in cardiac myosin. This result is in accordance with conclusion of our previous study that cardiomyogenesis-related genes were perturbed in tbx5 knockdown embryos [Under TEM, the ultrastructure of the myocardium in  embryos .tbx5 activated aberrant apoptosis and dormant cell growth in the head, heart, fins, and trunk. Although abnormal apoptosis-related genes were expressed throughout the entire process of organogenesis in tbx5 knockdown embryos, the degree and distribution of apoptosis varied in different locations at 48 hpf. Such aberrant apoptosis may contribute to consequent morphologic anomalies including a string-like heart, a curled trunk, and hypogenesis of the pectoral fins, but it did not interfere with the migration and differentiation of progenitor cells during early development. For example, no ectopic fin was found in embryos, although hypogenesis or agenesis of the pectoral fin was frequently observed in tbx5 morphants, as was the string-like heart. We hypothesized that the aberrant apoptosis may play a role in the interruption of cardiac looping, chamber formation, and growth of pectoral fins in zebrafish. Nevertheless, mutations in tbx5 gene underlie Holt-Oram syndrome, the majority of which is assumed as sequel "premature stops" or "developmental delay", may induce inappropriate apoptotic processes during embryogenesis and lead to the dysmorphogenesis including cardiac and limb malformation.The TUNEL assay showed that a shortage of From a genomic analysis, dozens of genes were related to the underlying causes of isolated congenital heart disease and genetic syndrome associated with cardiac anomalies. However, genotype-phenotype distinctions exist. The discrepancy was frequently related to multiple genetic pathways, the size of the deletion, different allele/loci, point mutations, or a haploinsufficiency. Our observation presumed that the genotype-phenotype distinction might be a consequence of variable degrees of apoptosis in progenitor cells.tbx5 insufficiency ultimately resulted in activation of apoptosis and inhibition of cell growth in whole individual of zebrafish embryo, though knockdown of TBX5 leads to overexpression of both anti-apoptotic and pro-apoptotic gene than wild type embryos and the controls. Those data implied that abnormal tbx5 level play a role in aberrant apoptosis and cell growth contributing to dysmorphogenesis.Some study declared that exogenous TBX5 could induce apoptosis and inhibit cell proliferation in vitro . Our restbx5 deficiency including string-like heart, curled trunk, curled tail, and pectoral fins might be a consequence of aberrant apoptosis and dormant cell growth in early embryonic development, which affected late differentiation and growth of organs as well as maturation.In summary, abnormal appearances in zebrafish embryos with The authors declare that they have no competing interests.JHL and JKL conceived of the study, participated in its design, coordination and drafted the manuscript. TT participated in its design and drafted the manuscript, SC, SY and AY carried out the molecular genetic studies. RT and HL participated in its design and coordination. All authors read and approved the final manuscrpt.


Bacillus cereus group.Group II introns are widespread genetic elements endowed with a dual functionality. They are catalytic RNAs (ribozymes) that are able of self-splicing and they are also mobile retroelements that can invade genomic DNA. The group II intron RNA secondary structure is typically made up of six domains. However, a number of unusual group II introns carrying a unique extension of 53-56 nucleotides at the 3' end have been identified previously in bacteria of the In the present study, we conducted combined sequence comparisons and phylogenetic analyses of introns, host gene, plasmid and chromosome of host strains in order to gain insights into mobility, dispersal, and evolution of the unusual introns and their extension. We also performed in vitro mutational and kinetic experiments to investigate possible functional features related to the extension.B. cereus group, Bacillus pseudofirmus OF4 and Bacillus sp. 2_A_57_CT2, an uncharacterized species phylogenetically close to B. firmus. Interestingly, the B. pseudofirmus intron has a longer extension of 70 bases. From sequence comparisons and phylogenetic analyses, several possible separate events of mobility involving the atypical introns could be identified, including both retrohoming and retrotransposition events. In addition, identical extensions were found in introns that otherwise exhibit little sequence conservation in the rest of their structures, with the exception of the conserved and catalytically critical domains V and VI, suggesting either separate acquisition of the extra segment by different group II introns or a strong selection pressure acting on the extension. Furthermore, we show by in vitro splicing experiments that the 3' extension affects the splicing properties differently in introns belonging to separate evolutionary branches.We report the identification of novel copies of group II introns carrying a 3' extension including the first two copies in bacteria not belonging to the Altogether this study provides additional insights into the structural and functional evolution of unusual introns harboring a 3' extension and lends further evidence that these introns are mobile with their extension. Group II introns are genetic elements that are widespread in bacteria and in the organelles of eukaryotes. They are self-splicing catalytic RNAs (ribozymes) that remove themselves from precursor mRNA transcripts and ligate their flanking sequences (exons). Group II introns are also mobile retroelements which can invade genomic DNA sites -5. SplicBacillus cereus group, including B. cereus, B. thuringiensis, B. mycoides, and B. pseudomycoides. These bacterial species are genetically closely related and are known to harbor a range of mobile elements such as plasmids and introns [B. cereus group. Using the complete and diverse set of introns with an extra segment we conducted a detailed sequence and phylogenetic analysis of the introns together with their host genes and strains in order to gain insight into mobility, dispersal, and evolution of these elements and their domain VII. Functional studies were also carried out to investigate possible features related to the extension.The secondary structure of the group II intron RNA typically consists of six domains (numbered I to VI) that are linked by a network of tertiary interactions, and introns are classified based on structural features and IEP phylogeny ,5,14-16. introns ,20-23. F introns ,18,24. P introns ,18. In tB. cereus group, two of the newly discovered elements are encoded by strains of unrelated species, namely Bacillus pseudofirmus OF4  and theB.th.I6 intron from B. thuringiensis kurstaki BGSC 4D1/HD1 (Table B.th.I6-like introns (B.th.I6 and B.c.I16) are all inserted in the same homing site within the pXO1-42 plasmid gene, and the two intron copies (B.th.I6a and b) found in B. thuringiensis kurstaki BGSC 4D1/HD1 were suggested to be the result of intron mobility [With respect to the phylogeny of the introns themselves, many of the introns with a 3' extension belong to the β subgroup within the bacterial B class ,18, and mobility ,18. HereB. cereus group. Indeed, large plasmids are the main vectors of group II intron spread in this bacterial group [B.th.I6-like introns are distributed in both groups. Identical copies are present in distantly related pXO1-42 sequences, as exemplified by B.th.I6b and c/d and by B.c.I16a and b/c. This strongly suggests mobility of the individual introns rather than transfer of the whole locus containing host gene and intron  belonging to the "pXO1-like" family . Phylogeal group . Secondln Figure . A diffee Figure . The lat Figures  and 2A. B.th.I7a and b copies indicated that the B.th.I7 intron as well must be mobile with its extension. These two intron copies are inserted in different genes in B. thuringiensis kurstaki BGSC 4D1/HD1 . While the nucleotide sequences of the two intron copies are identical, the insertion sites exhibit little similarity, with the exception of the IBS2 motif  is identical to that of B.th.I5 from B. thuringiensis BGSC 4D1/HD1 and CT-43  has adapted to function with the 3' extra domain, the extension was not essential for splicing since the intron could splice nearly as efficiently as wildtype (WT) when the entire extension was deleted [B.c.I4_dS1S2) appeared to produce somewhat more linear form of the intron, suggesting more hydrolytic splicing without the 3' extension [B.c.I4 WT and dS1S2 constructs. These analyses confirmed that the linear form is not the major product of the splicing reaction of WT B.c.I4, even in buffers containing KCl, which is known to promote the hydrolytic splicing pathway [B.c.I4_dS1S2 construct produced a linear and lariat fraction of ~40% and ~45%, respectively, after the same time period . Even though splicing of the B.th.I6a construct deleted of the full 54-nt 3' extension (B.th.I6a_dS1S2) also appeared to give an increased amount of free linear intron in KCl-containing buffer 2SO4 and KCl buffers showed a dramatic inhibition of the second splicing step. This was revealed by the accumulation of the first step intermediate "lariat + 3' exon" product compared to the B.th.I6a WT construct containing the extension  was searched as well. Strain HD1 corresponds to strain BGSC 4D1, whose genome has also been sequenced by our laboratory and the Norwegian High-Throughput Sequencing Centre, University of Oslo, Norway . Introns were named following the nomenclature used in the Group II Intron Database [The nucleotide sequences of the previously identified xtension -19 were xtension  using BLxtension  for addiDatabase .B. cereus group strains publicly available in Genbank and Microgen at the time of analysis were screened for the B.th.I6 intron's host gene, pXO1-42, using BLAST. The BLAST search was conducted using the pXO1-42 sequence of the pXO1 plasmid of B. anthracis Ames Ancestor strain (locus tag GBAA_pXO1_0064) as query, and was performed both at the amino acid and nucleotide levels .The 86 completely sequenced B. cereus group were selected for screening, including strains closely related to those harboring B.th.I6 and the B.th.I6-like B.c.I16 intron, based on the multiple locus sequence typing (MLST) supertree of chromosomal housekeeping genes available in the SuperCAT database at the University of Oslo's typing website ([http://mlstoslo.uio.no/).An additional 36 isolates were screened by PCR, and positive products were sequenced. PCR was performed as previously described using the B.th.I6a_exon_left/right and B.th.I6b_exon_left/right oligonucleotide primer pairs . Strainswebsite  were computationally predicted by constrained folding using the MFOLD 3.1 package [The secondary structures of the  package ,35 follo package ,36. ThatB.th.I6-like introns was available for the strains that were screened by PCR. Thus, sites with gaps were removed in a pairwise manner when computing distances. Statistical support for branches in the tree was assessed by 1000 bootstrap replicates [Homologous pXO1-42 nucleotide sequences were aligned using CLUSTALW 2 ,38, follplicates . Phylogeplicates .B. cereus group strains encoding the pXO1-42 gene was reconstructed using the MLST data available in the SuperCAT database ([http://mlstoslo.uio.no/). The data included the nucleotide sequences of 7 to 26 chromosomal housekeeping genes, depending on the strain. According to information in culture collections, B. thuringiensis kurstaki BGSC 4D1 and ATCC 33679 should be the same strain and both correspond to strain HD1. However, typing studies revealed that the former two strains exhibit genotypic differences [B. cereus group isolates was reconstructed using the matrix representation by parsimony (MRP) technique as done in SuperCAT  according to the manufacturer's instructions using two complementary oligonucleotides (of ~40 bases) containing the desired mutation(s) with either B.c.I4 or B.th.I6a ΔORF constructs as templates [B.th.I6a (B.th.I6a_dS1S2 construct) was performed in the same manner as done previously for B.c.I4  according to the manufacturer's instructions. Transcription and gel-purification of radiolabelled and unlabelled RNA were conducted as previously described .2, and either 500 mM (NH4)2SO4 or 500 mM KCl at 47°C. Reactions were initiated by adding pre-warmed splicing buffer to the transcript RNA giving a total reaction volume of 40 μl. At each time point of the time-course analysis, 2 μl were taken out, quenched with loading buffer (Ambion) and storing samples on dry ice. Samples were then heated to 95°C and cooled on ice, before being separated on a 7.5 M Urea 4% polyacrylamide gel. Gels were then vacuum dried, exposed, and analyzed using a Molecular Dynamics Storm 860 Phosphorimager.In vitro generated transcripts were denatured and refolded using a GenAmp 2700 PCR machine (Applied Biosystems), by incubating the transcripts in 10 mM MOPS, pH 7.5 at 90°C for 1 min, 75°C for 5 min, and then slow cooling to the splicing temperature of 47°C. Intron transcripts were spliced with 70000 cpm RNA or ~0.1 μg unlabelled transcripts in 40 mM MOPS, pH 7.5, 100 mM MgClFor subsequent RT-PCR and sequencing of these splicing products, either unlabeled spliced transcripts, purified with Nucleotide purification kit (Qiagen), or labeled spliced transcript species, excised from gels, were used as templates.For kinetic analysis, the intensities of the radioactive bands were quantified using the ImageQuant 5.0 software. The relative fractions of unspliced precursor and free lariat RNA were computed from the intensities of the radioactive bands of all intron-containing products.The authors declare that they have no competing interests.NJT and FBS jointly conceived the study and wrote the manuscript. NJT conducted the bioinformatic analyses. FBS performed the laboratory experiments. ABK supervised the study and contributed to the interpretation of the data. All authors have read and approved the current manuscript.Figure S1-S3 and Table S4. The file includes three supplementary figures (S1-S3) along with the corresponding legends and associated references. The figures show respectively a phylogenetic tree of Bacillus species , comparisons of sequence homology between large plasmids , and drawings of the secondary structure of unusual group II introns . The file also includes a supplementary table (S4) listing the oligonucleotide primers used for the in vitro splicing experiments.Click here for file


Most of our time is spent indoors where we are exposed to a wide array of different microorganisms living on surfaces and in the air of our homes. Despite their ubiquity and abundance, we have a limited understanding of the microbial diversity found within homes and how the composition and diversity of microbial communities change across different locations within the home. Here we examined the diversity of bacterial communities found in nine distinct locations within each of forty homes in the Raleigh-Durham area of North Carolina, USA, using high-throughput sequencing of the bacterial 16S rRNA gene. We found that each of the sampled locations harbored bacterial communities that were distinct from one another with surfaces that are regularly cleaned typically harboring lower levels of diversity than surfaces that are cleaned infrequently. These location-specific differences in bacterial communities could be directly related to usage patterns and differences in the likely sources of bacteria dispersed onto these locations. Finally, we examined whether the variability across homes in bacterial diversity could be attributed to outdoor environmental factors, indoor habitat structure, or the occupants of the home. We found that the presence of dogs had a significant effect on bacterial community composition in multiple locations within homes as the homes occupied by dogs harbored more diverse communities and higher relative abundances of dog-associated bacterial taxa. Furthermore, we found a significant correlation between the types of bacteria deposited on surfaces outside the home and those found inside the home, highlighting that microbes from outside the home can have a direct effect on the microbial communities living on surfaces within our homes. Together this work provides the first comprehensive analysis of the microbial communities found in the home and the factors that shape the structure of these communities both within and between homes. Globally, homes vary from simple stick structures similar in their climate to the outdoor environment to climate-invariant mansions. But even within regions, house conditions can vary in subtler but important ways, and it is likely that microbial communities also vary among such houses. Such variation could be stochastic–a function of the complex dynamics influencing the fate of the many species and their populations among which we live. Alternately, composition might be influenced deterministically by differences between homes in outdoor or indoor environmental conditions, surface materials, and the inhabitants (both human and otherwise) of the homes. Both indoor humidity and temperature appear to influence the diversity and composition of building-associated bacterial communities To understand the differences in bacterial communities found across specific house locations (habitats) within homes and the variation that exists within habitats between homes, we sampled bacteria in nine standardized locations within each of forty homes in the Raleigh-Durham area of North Carolina. The sampled homes spanned a range of designs, sizes, numbers and types of occupants , degrees of urbanization, and socioeconomic status. They were identified as part of a volunteer-based citizen science project in which participants sampled their own homes. We compared the diversity and composition of the bacterial communities in the 360 collected samples using high-throughput sequencing of the bacterial 16S rRNA gene.http://www.yourwildlife.org/projects/wild-life-of-our-homes/).Historically, most efforts to engage the public in scientific research have rallied participation around charismatic organisms or easily observable environmental phenomena Here we present the results from the first forty households we recruited to participate in our study in North Carolina in autumn 2011. Each participant was provided with a written Informed Consent form approved by the North Carolina State University’s Human Research Committee  as well as instructions for sampling their home and a home microbe sampling kit.Each home sampling kit contained nine dual-tipped sterile BBL™ CultureSwabs™. Participants were instructed to sample nine standardized locations within their home: kitchen cutting board, kitchen counter, a shelf inside a refrigerator, toilet seat, pillowcase, exterior handle of the main door into the house, television screen, the upper door trim on the outside surface of an exterior door, and the upper door trim on an interior door . These lWe used the direct PCR approach described in Flores et al. Singletons and phylotypes classified as chloroplasts were removed from further analyses. Phylotypes observed in extraction controls were removed as per Flores et al. http://dx.doi.org/10.6084/m9.figshare.674588).Data have now been deposited online at figshare  in R In order to identify the relative importance of different potential source environments to the bacterial communities found on the sampled surfaces we used a source-tracking approach similar to that described previously Across all samples, a total of 7726 unique phylotypes were identified with nearly all individual samples having at least 100 unique OTUs (out of 1000 reads per sample) . We obseP<0.05 for all pairwise comparisons). In general, samples clustered into the following general groups: depositional environments , kitchen-associated , and frequently touched surfaces   were Proteobacteria, Firmicutes, and Actinobacteria which represented 34%, 32%, and 20%, respectively, of all 16S rRNA gene sequences. The bacterial families with the highest relative abundances across all of the collected samples were the Streptococcaceae (8.9%), Corynebacteriaceae (5.6%), and Lactobacillaceae (5.1%). Bacterial community composition differed significantly between each sampling site across the 40 homes ( handle) . The rel handle) .As we have previously analyzed a large number of samples from possible bacterial source environments inside and outside the home, including soil P = 0.001). The between-home differences accounted for one-quarter to one-half as much variation as the location-specific differences within homes. This was particularly evident for the communities collected from the kitchen environments that were highly variable in composition across the homes (As evident from he homes .P = 0.001). In particular, the presence of dogs was associated with a different community composition on pillowcases and TV screens  and 57% (P = 0.004) more OTUs on pillowcases and TV screens, respectively, compared to homes without pets.Although we had limited data on the specific environmental conditions found within the homes, we could use collected data on house occupancy, location, and design to ascertain which of these factors contributed to the inter-home variability in community composition for the individual sampling locations. We found that this variation could be attributed, in part, to the presence of dogs in homes , suggesting that information on the types of bacteria found in the air outside the home can be used to predict, in part, the composition of the bacterial communities found within homes. We tested other factors, including number of human occupants, presence of cats, presence of children, use of pesticides, presence of carpet, and the presence of and types of allergies, but none of these factors had a significant influence on the bacterial communities at any of the sampling locations . Either these factors were not important in structuring house-associated bacterial communities across the sampled locations or our sampling effort was insufficient to quantify the importance of those factors. For example, we could not detect an effect of cats on house-associated microbial communities; however, with only three homes having cats and no dogs, we may have lacked sufficient replication to detect such an effect. Likewise, we could not test the influence of other factors that may influence the structure of house-associated bacterial communities, including house environmental conditions , cleaning frequency, or specific surface materials, as we did not collect sufficient information on these factors from the homes sampled.We also found a significant relationship between exterior and interior microbial community composition  to have higher diversity  common on other types of moist surfaces Compared to the samples collected from inside homes, the exterior door trim had far lower abundances of human-associated bacteria and appeared to be dominated by microbes commonly found in soil and on leaf surfaces . Both so2 so any potential differences in airborne microbial taxa may be minimal if they are regionally driven. Future work would benefit from a broader sampling of homes across larger geographic regions to determine the extent to which the outside environment may shape the composition of those bacterial communities found within our homes.While strong differences across locations within houses exist and were consistent from one house to the next, some of the locations harbored bacterial communities that were highly variable in composition across the homes. A number of factors could account for this inter-home variability, including factors that we did not include in our analyses because they were either unmeasured  a phenomenon that has also been observed in other studies e.g., . The preOur work highlights that our homes are composed of many distinct habitats that harbor distinct bacterial communities. The composition of these communities is likely driven by numerous factors, including the differential sources of bacteria dispersed onto the surfaces, and many of the community assembly patterns appear to be predictable. The microbial communities that live within homes clearly provide unique opportunities for testing ecological theories of community assembly, theories that have been derived almost exclusively from research on plant and animal communities. Practically speaking, however, the more significant result is our observation of strong differences in bacterial communities among houses, even for particular habitats. In our dataset, a single feature of the home environment (the presence of a dog) explained a large portion of the variability in bacterial community composition and diversity in some of the habitats sampled – but clearly there are other unmeasured or undetected factors that are likely important in structuring communities across homes. To the extent that the differences in household microbial communities are associated with differences in health and well-being, understanding these missing factors seems important. However, given the large number of potential explanatory variables, such work will require much larger sample sizes than were possible here.Figure S1Diagram of the nine locations sampled within each of the 40 homes. Insets emphasize the habitat characteristics of each sampling site.(TIFF)Click here for additional data file.Figure S2Principle coordinates plots showing effect of dogs on bacterial community composition on (A) pillowcases and (B) TV screens. Points closer together are more similar in terms of their bacterial composition.(TIF)Click here for additional data file.Figure S3Presence of dogs in home influences relative abundance of bacterial taxa. Differences in relative abundance of selected taxa between homes with dogs and those without pets on pillowcases and TV screens (A) and between dog fur and human skin Click here for additional data file.Table S1OTUs considered to be contaminants and removed prior to downstream analyses. OTU IDs and taxonomy labels are from the 97% similarity OTUs of the Greengenes February 2011 release.(DOCX)Click here for additional data file.Table S2Indicator taxa used for source tracking analysis.(DOCX)Click here for additional data file.


Abnormal distribution, modification and aggregation of transactivation response DNA-binding protein 43 (TDP-43) are the hallmarks of multiple neurodegenerative diseases, especially frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U) and amyotrophic lateral sclerosis (ALS).M337V). We found hTDP-43M337V was expressed primarily in the nuclei of neurons in the brain and spinal cord, intranuclear and cytoplasmic phosphorylated TDP-43 aggregates were frequently detected, and the levels of TDP-43 LMW products of ~25 kDa and ~35 kDa species were also increased. Overexpression of hTDP-43M337V dramatically down regulated the levels of mouse TDP-43 (mTDP-43) protein and RNA, indicating TDP-43 levels are tightly controlled in mammalian systems. TDP-43M337V mice displayed reactive gliosis, widespread ubiquitination, chromatolysis, gait abnormalities, and early lethality. Abnormal cytoplasmic mitochondrial aggregates and abnormal phosphorylated tau were also detected in the mice.To explore the pathogenic properties of mutant forms of TDP-43, we generated and characterized two mouse lines expressing human TDP-43 carrying the M337V mutation (hTDP-43M337V mouse models produces similar phenotypes, our results suggest that overexpression of the hTDP-43M337V can cause neuronal dysfunction due to its effect on a number of cell organelles and proteins, such as mitochondria and TDP-43, that are critical for neuronal activity.While overexpression of hTDP-43 in wild-type TDP-43 and TDP-43