Chemical synthesis of the mouse mitochondrial genome

We describe a one-step, isothermal assembly method for synthesizing DNA molecules from overlapping oligonucleotides. The method cycles between in vitro recombination and amplification until the desired length is reached. As a demonstration of its simplicity and robustness, we synthesized the entire 16.3-kilobase mouse mitochondrial genome from 600 overlapping 60-mers.     

Weevil borne microbes contribute as much to the reduction of photosynthesis in water hyacinth as does herbivory

Arthropods released for weed biocontrol can have effects other than simply removing biomass and frequently decrease photosynthetic rate more than can be attributed to the mere loss of photosynthetic surface area. Some of this effect may result because biological control agents facilitate the transfer and ingress of deleterious microbes into plant tissues on which they feed. We evaluated this facilitation effect using water hyacinth (Eichhornia crassipes) and a weevil (Neochetina eichhorniae) and compared the reductions in photosynthetic rates between leaves subject to herbivory by adult weevils sterilized with 3.5% chlorine bleach, to those that were unsterilized. The results showed that weevils carried both fungi and bacteria, transferred these to leaves on which they fed, and that microbes and biomass removal contributed almost equally to the 37% decrease in photosynthetic productivity. Hence, maximising the effectiveness of using arthropods that damage leaf surfaces for biocontrol requires the presence of microorganisms that are deleterious to plants.     

Gamma-secretase-regulated proteolysis of the Notch receptor by mitochondrial intermediate peptidase

Notch is a transmembrane receptor that controls a diverse array of cellular processes including cell proliferation, differentiation, survival, and migration. The cellular outcome of Notch signaling is dependent on extracellular and intracellular signals, but the complexities of its regulation are not well understood. Canonical Notch signaling involves ligand association that triggers sequential and regulated proteolysis of Notch at several sites. Ligand-dependent proteolysis at the S2 site removes the bulk of the extracellular domain of Notch. Subsequent γ-secretase-mediated intramembrane proteolysis of the remaining membrane-tethered Notch fragment at the S3 site produces a nuclear-destined Notch intracellular domain (NICD). Here we show that following γ-secretase cleavage, Notch is proteolyzed at a novel S5 site. We have identified this S5 site to be eight amino acids downstream of the S3 site. Biochemical fractionation and purification resulted in the identification of the S5 site protease as the mitochondrial intermediate peptidase (MIPEP). Expression of the MIPEP-cleaved NICD (ΔNICD) results in a decrease in cell viability and mitochondria membrane potential. The sequential and regulated proteolysis by γ-secretase and MIPEP suggests a new means by which Notch function can be modulated.     

Neuropeptide Y inhibits cholangiocarcinoma cell growth and invasion

No information exists on the role of neuropeptide Y (NPY) in cholangiocarcinoma growth. Therefore, we evaluated the expression and secretion of NPY and its subsequent effects on cholangiocarcinoma growth and invasion. Cholangiocarcinoma cell lines and nonmalignant cholangiocytes were used to assess NPY mRNA expression and protein secretion. NPY expression was assessed by immunohistochemistry in human liver biopsies. Cell proliferation and migration were evaluated in vitro by MTS assays and matrigel invasion chambers, respectively, after treatment with NPY or a neutralizing NPY antibody. The effect of NPY or NPY depletion on tumor growth was assessed in vivo after treatment with NPY or the neutralizing NPY antibody in a xenograft model of cholangiocarcinoma. NPY secretion was upregulated in cholangiocarcinoma compared with normal cholangiocytes. Administration of exogenous NPY decreased proliferation and cell invasion in all cholangiocarcinoma cell lines studied and reduced tumor cell growth in vivo. In vitro, the effects of NPY on proliferation were blocked by specific inhibitors for NPY receptor Y2, but not Y1 or Y5, and were associated with an increase in intracellular d-myo-inositol 1,4,5-trisphosphate and PKCα activation. Blocking of NPY activity using a neutralizing antibody promoted cholangiocarcinoma growth in vitro and in vivo and increased the invasiveness of cholangiocarcinoma in vitro. Increased NPY immunoreactivity in human tumor tissue occurred predominantly in the center of the tumor, with less expression toward the invasion front of the tumor. We demonstrated that NPY expression is upregulated in cholangiocarcinoma, which exerts local control on tumor cell proliferation and invasion. Modulation of NPY secretion may be important for the management of cholangiocarcinoma.     

Knockout of the neurokinin-1 receptor reduces cholangiocyte proliferation in bile duct-ligated mice

In bile duct-ligated (BDL) rats, cholangiocyte proliferation is regulated by neuroendocrine factors such as α-calcitonin gene-related peptide (α-CGRP). There is no evidence that the sensory neuropeptide substance P (SP) regulates cholangiocyte hyperplasia. Wild-type (WT, (+/+)) and NK-1 receptor (NK-1R) knockout (NK-1R(-/-)) mice underwent sham or BDL for 1 wk. Then we evaluated 1) NK-1R expression, transaminases, and bilirubin serum levels; 2) necrosis, hepatocyte apoptosis and steatosis, and the number of cholangiocytes positive by CK-19 and terminal deoxynucleotidyl transferase biotin-dUTP nick-end labeling in liver sections; 3) mRNA expression for collagen 1α and α-smooth muscle (α-SMA) actin in total liver samples; and 4) PCNA expression and PKA phosphorylation in cholangiocytes. In cholangiocyte lines, we determined the effects of SP on cAMP and D-myo-inositol 1,4,5-trisphosphate levels, proliferation, and PKA phosphorylation. Cholangiocytes express NK-1R with expression being upregulated following BDL. In normal NK-1R(-/-) mice, there was higher hepatocyte apoptosis and scattered hepatocyte steatosis compared with controls. In NK-1R (-)/(-) BDL mice, there was a decrease in serum transaminases and bilirubin levels and the number of CK-19-positive cholangiocytes and enhanced biliary apoptosis compared with controls. In total liver samples, the expression of collagen 1α and α-SMA increased in BDL compared with normal mice and decreased in BDL NK-1R(-/-) compared with BDL mice. In cholangiocytes from BDL NK-1R (-)/(-) mice there was decreased PCNA expression and PKA phosphorylation. In vitro, SP increased cAMP levels, proliferation, and PKA phosphorylation of cholangiocytes. Targeting of NK-1R may be important in the inhibition of biliary hyperplasia in cholangiopathies.     

Castration inhibits biliary proliferation induced by bile duct obstruction: novel role for the autocrine trophic effect of testosterone

Increased cholangiocyte growth is critical for the maintenance of biliary mass during liver injury by bile duct ligation (BDL). Circulating levels of testosterone decline following castration and during cholestasis. Cholangiocytes secrete sex hormones sustaining cholangiocyte growth by autocrine mechanisms. We tested the hypothesis that testosterone is an autocrine trophic factor stimulating biliary growth. The expression of androgen receptor (AR) was determined in liver sections, male cholangiocytes, and cholangiocyte cultures [normal rat intrahepatic cholangiocyte cultures (NRICC)]. Normal or BDL (immediately after surgery) rats were treated with testosterone or antitestosterone antibody or underwent surgical castration (followed by administration of testosterone) for 1 wk. We evaluated testosterone serum levels; intrahepatic bile duct mass (IBDM) in liver sections of female and male rats following the administration of testosterone; and secretin-stimulated cAMP levels and bile secretion. We evaluated the expression of 17β-hydroxysteroid dehydrogenase 3 (17β-HSD3, the enzyme regulating testosterone synthesis) in cholangiocytes. We evaluated the effect of testosterone on the proliferation of NRICC in the absence/presence of flutamide (AR antagonist) and antitestosterone antibody and the expression of 17β-HSD3. Proliferation of NRICC was evaluated following stable knock down of 17β-HSD3. We found that cholangiocytes and NRICC expressed AR. Testosterone serum levels decreased in castrated rats (prevented by the administration of testosterone) and rats receiving antitestosterone antibody. Castration decreased IBDM and secretin-stimulated cAMP levels and ductal secretion of BDL rats. Testosterone increased 17β-HSD3 expression and proliferation in NRICC that was blocked by flutamide and antitestosterone antibody. Knock down of 17β-HSD3 blocks the proliferation of NRICC. Drug targeting of 17β-HSD3 may be important for managing cholangiopathies.     

Investigating the potential of conserved inner core oligosaccharide regions of Moraxella catarrhalis lipopolysaccharide as vaccine antigens: accessibility and functional activity of monoclonal antibodies and glycoconjugate derived sera

We investigated the conservation and antibody accessibility of inner core epitopes of Moraxella catarrhalis lipopolysaccharide (LPS) in order to assess their potential as vaccine candidates. Two LPS mutants, a single mutant designated lgt2 and a double mutant termed lgt2/lgt4, elaborating truncated inner core structures were generated in order to preclude expression of host-like outer core structures and to create an inner core structure that was shared by all three serotypes A, B and C of M. catarrhalis. Murine monoclonal antibodies (mAbs), designated MC2-1 and MC2-10 were obtained by immunising mice with the lgt2 mutant of M. catarrhalis serotype A strain. We showed that mAb MC2-1 can bind to the core LPS of wild-type (wt) serotype A, B and C organisms and concluded that mAb MC2-1 defines an immunogenic inner core epitope of M. catarrhalis LPS. We were unsuccessful in obtaining mAbs to the lgt2/lgt4 mutant. MAb MC2-10 only recognised the lgt2 mutant and the wt serotype A strain, and exhibited a strong requirement for the terminal N-acetyl-glucosamine residue of the lgt2 mutant core oligosaccharide, suggesting that this residue was immunodominant. Subsequently, we showed that both mAbs MC2-1 and MC2-10 could facilitate bactericidal killing of the lgt2 mutant, however neither mAb could facilitate bactericidal killing of the wt serotype A strain. We then confirmed and extended the candidacy of the inner core LPS by demonstrating that it is possible to elicit functional antibodies against M. catarrhalis wt strains following immunisation of rabbits with glycoconjugates elaborating the conserved inner core LPS antigen. The present study describes three conjugation strategies that either uses amidases produced by Dictyostelium discoideum, targeting the amino functionality created by the amidase activity as the attachment point on the LPS molecule, or a strong base treatment to remove all fatty acids from the LPS, thus creating amino functionalities in the lipid A region to conjugate via maleimide-thiol linker strategies targeting the carboxyl residues of the carrier protein and the free amino functionalities of the derived lipid A region of the carbohydrate resulted in a high loading of carbohydrates per carrier protein from these carbohydrate preparations. Immunisation derived antisera from rabbits recognised fully extended M. catarrhalis LPS and whole cells. Moreover, bactericidal activity was demonstrated to both the immunising carbohydrate antigen and importantly to wt cells, thus further supporting the consideration of inner core LPS as a potential vaccine antigen to combat disease caused by M. catarrhalis.     

Ubiquilin-1 Overexpression Increases the Lifespan and Delays Accumulation of Huntingtin Aggregates in the R6/2 Mouse Model of Huntington's Disease

Huntington''s Disease (HD) is a neurodegenerative disorder that is caused by abnormal expansion of a polyglutamine tract in huntingtin (htt) protein. The expansion leads to increased htt aggregation and toxicity. Factors that aid in the clearance of mutant huntingtin proteins should relieve the toxicity. We previously demonstrated that overexpression of ubiqulin-1, which facilitates protein clearance through the proteasome and autophagy pathways, reduces huntingtin aggregates and toxicity in mammalian cell and invertebrate models of HD. Here we tested whether overexpression of ubiquilin-1 delays or prevents neurodegeneration in R6/2 mice, a well-established model of HD. We generated transgenic mice overexpressing human ubiquilin-1 driven by the neuron-specific Thy1.2 promoter. Immunoblotting and immunohistochemistry revealed robust and widespread overexpression of ubiquilin-1 in the brains of the transgenic mice. Similar analysis of R6/2 animals revealed that ubiquilin is localized in huntingtin aggregates and that ubiquilin levels decrease progressively to 30% during the end-stage of disease. We crossed our ubiquilin-1 transgenic line with R6/2 mice to assess whether restoration of ubiquilin levels would delay HD symptoms and pathology. In the double transgenic progeny, ubiquilin levels were fully restored, and this correlated with a 20% increase in lifespan and a reduction in htt inclusions in the hippocampus and cortex. Furthermore, immunoblots indicated that endoplasmic reticulum stress response that is elevated in the hippocampus of R6/2 animals was attenuated by ubiquilin-1 overexpression. However, ubiquilin-1 overexpression neither altered the load of htt aggregates in the striatum nor improved motor impairments in the mice.     

Mycobactericidal Activity of Sutezolid (PNU-100480) in Sputum (EBA) and Blood (WBA) of Patients with Pulmonary Tuberculosis

Rationale

Sutezolid (PNU-100480) is a linezolid analog with superior bactericidal activity against Mycobacterium tuberculosis in the hollow fiber, whole blood and mouse models. Like linezolid, it is unaffected by mutations conferring resistance to standard TB drugs. This study of sutezolid is its first in tuberculosis patients.

Methods

Sputum smear positive tuberculosis patients were randomly assigned to sutezolid 600 mg BID (N = 25) or 1200 mg QD (N = 25), or standard 4-drug therapy (N = 9) for the first 14 days of treatment. Effects on mycobacterial burden in sputum (early bactericidal activity or EBA) were monitored as colony counts on agar and time to positivity in automated liquid culture. Bactericidal activity was also measured in ex vivo whole blood cultures (whole blood bactericidal activity or WBA) inoculated with M. tuberculosis H37Rv.

Results

All patients completed assigned treatments and began subsequent standard TB treatment according to protocol. The 90% confidence intervals (CI) for bactericidal activity in sputum over the 14 day interval excluded zero for all treatments and both monitoring methods, as did those for cumulative WBA. There were no treatment-related serious adverse events, premature discontinuations, or dose reductions due to laboratory abnormalities. There was no effect on the QT interval. Seven sutezolid-treated patients (14%) had transient, asymptomatic ALT elevations to 173±34 U/L on day 14 that subsequently normalized promptly; none met Hy''s criteria for serious liver injury.

Conclusions

The mycobactericidal activity of sutezolid 600 mg BID or 1200 mg QD was readily detected in sputum and blood. Both schedules were generally safe and well tolerated. Further studies of sutezolid in tuberculosis treatment are warranted.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01225640","term_id":"NCT01225640"}}NCT01225640     

Comparison of Xpert MTB/RIF with other nucleic acid technologies for diagnosing pulmonary tuberculosis in a high HIV prevalence setting: a prospective study

Background

The Xpert MTB/RIF (Cepheid) non-laboratory-based molecular assay has potential to improve the diagnosis of tuberculosis (TB), especially in HIV-infected populations, through increased sensitivity, reduced turnaround time (2 h), and immediate identification of rifampicin (RIF) resistance. In a prospective clinical validation study we compared the performance of Xpert MTB/RIF, MTBDRplus (Hain Lifescience), LightCycler Mycobacterium Detection (LCTB) (Roche), with acid fast bacilli (AFB) smear microscopy and liquid culture on a single sputum specimen.

Methods and Findings

Consecutive adults with suspected TB attending a primary health care clinic in Johannesburg, South Africa, were prospectively enrolled and evaluated for TB according to the guidelines of the National TB Control Programme, including assessment for smear-negative TB by chest X-ray, clinical evaluation, and HIV testing. A single sputum sample underwent routine decontamination, AFB smear microscopy, liquid culture, and phenotypic drug susceptibility testing. Residual sample was batched for molecular testing. For the 311 participants, the HIV prevalence was 70% (n = 215), with 120 (38.5%) culture-positive TB cases. Compared to liquid culture, the sensitivities of all the test methodologies, determined with a limited and potentially underpowered sample size (n = 177), were 59% (47%–71%) for smear microscopy, 76% (64%–85%) for MTBDRplus, 76% (64%–85%) for LCTB, and 86% (76%–93%) for Xpert MTB/RIF, with specificities all >97%. Among HIV+ individuals, the sensitivity of the Xpert MTB/RIF test was 84% (69%–93%), while the other molecular tests had sensitivities reduced by 6%. TB detection among smear-negative, culture-positive samples was 28% (5/18) for MTBDRplus, 22% (4/18) for LCTB, and 61% (11/18) for Xpert MTB/RIF. A few (n = 5) RIF-resistant cases were detected using the phenotypic drug susceptibility testing methodology. Xpert MTB/RIF detected four of these five cases (fifth case not tested) and two additional phenotypically sensitive cases.

Conclusions

The Xpert MTB/RIF test has superior performance for rapid diagnosis of Mycobacterium tuberculosis over existing AFB smear microscopy and other molecular methodologies in an HIV- and TB-endemic region. Its place in the clinical diagnostic algorithm in national health programs needs exploration. Please see later in the article for the Editors'' Summary