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Find video protocols related to scientific articles indexed in Pubmed.
Identification of the translational start site of codon-optimized mCherry in Mycobacterium tuberculosis.
BMC Res Notes
PUBLISHED: 06-06-2014
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Fluorescent proteins are used widely as reporter genes in many organisms. We previously codon-optimized mCherry for Mycobacterium tuberculosis and generated expression constructs with high level expression in mycobacteria with multiple uses in vitro and in vivo. However, little is known about the expression of fluorescent proteins in mycobacteria and the translational start codon for mCherry has not been experimentally determined.
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Hypoglycaemia in a patient who is non-diabetic.
BMJ Case Rep
PUBLISHED: 03-12-2014
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A 59-year-old Caucasian man was seen in clinic following general practitioner's referral presenting with a 1-year history of hypoglycaemia. It was deemed important given his history that an insulinoma should be ruled out. The patient had an induced episode of hypoglycaemia while under medical supervision; C-peptide, insulin and insulin-like growth factor levels were measured. These results strongly suggested an insulinoma. He was subsequently referred to our local tertiary centre for surgical workup and excision of the insulinoma. This case reminds us that insulinoma, although rare, is an important differential to rule out in non-diabetic hypoglycaemia. Often the diagnosis is overlooked as their symptoms mimic many other conditions. Although there is no specific Driver and Vehicle Licensing Agency advice, we, as the healthcare provider, should be ensuring the patients inform them.
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Mastoid surgery under local anesthesia for medically unfit patients: techniques and outcome.
Ann. Otol. Rhinol. Laryngol.
PUBLISHED: 12-04-2013
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We present the surgical techniques and outcomes of mastoid surgery under local anesthesia in patients who were unfit for general anesthesia.
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Serum glucose and fructosamine in relation to risk of cancer.
PLoS ONE
PUBLISHED: 01-25-2013
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Impaired glucose metabolism has been linked with increased cancer risk, but the association between serum glucose and cancer risk remains unclear. We used repeated measurements of glucose and fructosamine to get more insight into the association between the glucose metabolism and risk of cancer.
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Methionine sulfoximine resistance in Mycobacterium tuberculosis is due to a single nucleotide deletion resulting in increased expression of the major glutamine synthetase, GlnA1.
Microb. Drug Resist.
PUBLISHED: 08-31-2011
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We investigated the effect of methionine sulfoximine (MetSox), a potent inhibitor of glutamine synthetase, on Mycobacterium tuberculosis. M. tuberculosis encodes four glutamine synthetases, of which MetSox targets the type I enzyme encoded by glnA1. Transcriptional profiling revealed that glutamate synthetase (gltB) and a type II glutamine synthetase (glnA3) were induced after exposure to MetSox. In addition, we observed a high rate (10(-5)) of spontaneous resistance to MetSox. All resistant strains had a single-nucleotide deletion in the 5 region of glnA1, and Western analysis revealed that GlnA1 expression was increased in resistant as compared with sensitive strains. These data show that M. tuberculosis can respond to the effect of MetSox inhibition either by up-regulation of GlnA3 or by GlnA1. The high frequency of resistance suggests that MetSox and other compounds specifically targeting GlnA1 are not likely to become successful anti-mycobacterial agents.
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Maintaining Breast Cancer Specimen Integrity and Individual or Simultaneous Extraction of Quality DNA, RNA, and Proteins from Allprotect-Stabilized and Nonstabilized Tissue Samples.
Biopreserv Biobank
PUBLISHED: 07-11-2011
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The Saint Jamess Hospital Biobank was established in 2008, to develop a high-quality breast tissue BioResource, as a part of the breast cancer clinical care pathway. The aims of this work were: (1) to ascertain the quality of RNA, DNA, and protein in biobanked carcinomas and normal breast tissues, (2) to assess the efficacy of AllPrep(®) (Qiagen) in isolating RNA, DNA, and protein simultaneously, (3) to compare AllPrep with RNEasy(®) and QIAamp(®) (both Qiagen), and (4) to examine the effectiveness of Allprotect(®) (Qiagen), a new tissue stabilization medium in preserving DNA, RNA, and proteins. One hundred eleven frozen samples of carcinoma and normal breast tissue were analyzed. Tumor and normal tissue morphology were confirmed by frozen sections. Tissue type, tissue treatment (Allprotect vs. no Allprotect), extraction kit, and nucleic acid quantification were analyzed by utilizing a 4 factorial design (SPSS PASW 18 Statistics Software(®)). QIAamp (DNA isolation), AllPrep (DNA, RNA, and Protein isolation), and RNeasy (RNA isolation) kits were assessed and compared. Mean DNA yield and A(260/280) values using QIAamp were 33.2?ng/?L and 1.86, respectively, and using AllPrep were 23.2?ng/?L and 1.94. Mean RNA yield and RNA Integrity Number (RIN) values with RNeasy were 73.4?ng/?L and 8.16, respectively, and with AllPrep were 74.8?ng/?L and 7.92. Allprotect-treated tissues produced higher RIN values of borderline significance (P=0.055). No discernible loss of RNA stability was detected after 6?h incubation of stabilized or nonstabilized tissues at room temperature or 4°C or in 9 freeze-thaw cycles. Allprotect requires further detailed evaluation, but we consider AllPrep to be an excellent option for the simultaneous extraction of RNA, DNA, and protein from tumor and normal breast tissues. The essential presampling procedures that maintain the diagnostic integrity of pathology specimens do not appear to compromise the quality of molecular isolates.
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Identifying vulnerable pathways in Mycobacterium tuberculosis by using a knockdown approach.
Appl. Environ. Microbiol.
PUBLISHED: 06-03-2011
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We constructed recombinant strains of Mycobacterium tuberculosis in which expression of specific genes was downregulated to identify vulnerable drug targets. Growth phenotypes in macrophages and culture were used to rank targets: the dprE1, clpP1, and fadD32 operons were the best targets and glnA1, glnE, pknL, regX3, and senX3 were poor targets.
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Surgical management of an Irish cohort of BRCA-mutation carriers.
Breast
PUBLISHED: 04-03-2011
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Little information is available regarding the management of BRCA-related breast cancer in Ireland. A cancer genetics programme was initiated in 1992 at our institution to provide counselling and expert management for those with cancers resulting from inherited predisposition. We examined a cohort of BRCA mutation-carriers treated at a single institution over 16 years. A total of 107 women from 57 families were found to be carriers of mutations in BRCA1/2. Bilateral salpingo-oophorectomy was the most common prophylactic surgery performed. Overall survival between BRCA-related and sporadic breast cancer was equivalent. This is the first publication on surgical management of BRCA-mutation carriers in Ireland. It is imperative that those considered likely to harbour a mutation are referred early to a dedicated clinic so that appropriate counselling, testing and subsequent management to reduce the risk of dying from cancer can be undertaken.
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Influence of the metabolic syndrome on leptin and leptin receptor in breast cancer.
Mol. Carcinog.
PUBLISHED: 01-19-2011
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Obesity and its associated metabolic syndrome (MetS) are recognized risk factors for breast cancer. The molecular basis for this association remains largely unknown. Adipokines, in particular leptin and adiponectin, are thought to form part of the mechanism linking obesity with cancer through their altered expression/production either systemically (endocrine pathway) or locally (paracrine/autocrine pathway). Using quantitative PCR, mRNA expression of adiponectin (AdipoQ) and leptin (Ob) in mammary adipose tissue (MAT), intratumoral leptin and associated ligand receptors (ObR, AdipoR1, and AdipoR2) was examined in 77 patients with complete anthropomorphic and serological data. Expression of Ob in MAT, and ObR in matched tumor tissue was significantly higher in patients with MetS compared to obese only or normal weight cancer patients (P?
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Cardiac valve disease and low-dose dopamine agonist therapy: an artefact of reporting bias?
Clin. Endocrinol. (Oxf)
PUBLISHED: 01-05-2011
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Chronic low-dose cabergoline treatment for microprolactinoma may cause cardiac valve pathology, but the evidence is contradictory. We investigated whether the expectation of the echocardiographer could influence the report.
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Health status of adults with congenital adrenal hyperplasia: a cohort study of 203 patients.
J. Clin. Endocrinol. Metab.
PUBLISHED: 08-18-2010
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No consensus exists for management of adults with congenital adrenal hyperplasia (CAH) due to a paucity of data from cohorts of meaningful size.
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Expression and characterization of Mycobacterium tuberculosis CYP144: common themes and lessons learned in the M. tuberculosis P450 enzyme family.
Biochim. Biophys. Acta
PUBLISHED: 04-02-2010
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CYP144 from Mycobacterium tuberculosis was expressed and purified. CYP144 demonstrates heme thiolate coordination in its ferric form, but the cysteinate is protonated to thiol in both the carbon monoxide-bound and ligand-free ferrous forms (forming P420 in the former). Tight binding of various azole drugs was shown, with affinity for miconazole (K(d)=0.98 ?M), clotrimazole (0.37 ?M) and econazole (0.78 ?M) being highest. These azoles are also the trio with the highest affinity for the essential CYP121 and for the cholesterol oxidase CYP125 (essential for host infection), and have high potency as anti-mycobacterial drugs. Construction of a Mtb gene knockout strain demonstrated that CYP144 is not essential for growth in vitro. However the deletion strain was more sensitive to azole inhibition in culture suggesting an important role for CYP144 in cell physiology and/or in mediating azole resistance. The biophysical and genetic features of CYP144 are compared to those of other characterized Mtb P450s, identifying both commonality in properties (including thiolate protonation in ferrous P450s) and intriguing differences in thermodynamic and spectroscopic features. Our developing knowledge of the Mtb P450s has revealed unusual biochemistry and gene essentiality, highlighting their potential as drug targets in this human pathogen.
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Co-morbidities, management and clinical outcome of auto-immune Addisons disease.
Endocrine
PUBLISHED: 02-24-2010
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There are no consensus guidelines on the optimum long-term care of patients with primary adrenal failure. Published data suggest increased morbidity and mortality in patients treated with current therapy. Investigations of bone mineral density (BMD) in adults with adrenal failure have reported conflicting results. The objectives of this study were to determine the prevalence of auto-immune and other co-morbidities, describe the treatment regimens and to assess the BMD of adults with auto-immune Addisons disease (AAD). A retrospective, cohort study of adults with primary adrenal failure was used. Electronic and paper records were used to collect demographic, biochemical, BMD data and details of other co-morbidities. 48 patients (35% male; 65% female; 50 ± 16, years, mean ± SD) with primary adrenal failure were identified. There was high prevalence of other auto-immune co-morbidities (hypothyroidism 58%, vitamin B(12) deficiency 29%, type 1 diabetes 10%). The presence of cardiovascular risk factors including dyslipidaemia (65% had total cholesterol >5 mmol/l) and excess weight (65% had a BMI >25 kg/m(2)) were high. Using WHO criteria, 17.9 and 53.5% of patients had spinal osteoporosis and osteopenia, respectively, at the spine. This did not relate to the duration or dose of glucocorticoid replacement. Our data shows a high prevalence of both auto-immune and non-autoimmune co-morbidities in patients with AAD. In addition to common auto-immune diseases, patients should be screened for other cardiovascular risk factors. Further studies are needed to assess the cause of the observed increased prevalence of reduced BMD at the lumbar spine. There is a need for internationally agreed long-term management guidelines.
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Sensitive detection of gene expression in mycobacteria under replicating and non-replicating conditions using optimized far-red reporters.
PLoS ONE
PUBLISHED: 01-26-2010
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Fluorescent reporter proteins have proven useful for imaging techniques in many organisms. We constructed optimized expression systems for several fluorescent proteins from the far-red region of the spectrum and analyzed their utility in several mycobacterial species. Plasmids expressing variants of the Discosoma Red fluorescent protein (DsRed) from the Mycobacterium bovis hsp60 promoter were unstable; in contrast expression from the Mycobacterium smegmatis rpsA promoter was stable. In Mycobacterium tuberculosis expression of several of the far-red reporters was readily visualised by eye and three reporters (mCherry, tdTomato, and Turbo-635) fluoresced at a high intensity. Strains expressing mCherry showed no fitness defects in vitro or in macrophages. Treatment of cells with antibiotics demonstrated that mCherry could also be used as a reporter for cell death, since fluorescence decreased in the presence of a bactericidal compound, but remained stable in the presence of a bacteriostatic compound. mCherry was functional under hypoxic conditions; using mCherry we demonstrated that the P(mtbB) is expressed early in hypoxia and progressively down-regulated. mCherry and other far-red fluorescent proteins will have multiple uses in investigating the biology of mycobacteria, particularly under non-replicating, or low cell density conditions, as well as providing a novel means of detecting cell death rapidly.
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Assaying promoter activity using LacZ and GFP as reporters.
Methods Mol. Biol.
PUBLISHED: 01-01-2009
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The ability of bacteria to survive in a variety of different niches is due, in part, to their ability to respond and adapt to the environment. Extracellular signals are recognized by bacilli, and their responses are generally conducted at the transcript level. RNA polymerases recognize specific promoter regions on the genome and initiate transcription. Therefore, the analysis of gene expression is paramount to understanding the biology of an organism. In the case of pathogens, gene expression can alter during the course of the infection, and, therefore, specific targets can be identified for drug development. Promoter activity can be determined by cloning a promoter sequence upstream of a reporter gene and assaying the reporter activity, either from whole cells or from cell lysates. This chapter describes two reporter systems (GFP and LacZ) used for determining promoter activity that have been widely used in mycobacteria.
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Deletion of SenX3-RegX3, a key two-component regulatory system of Mycobacterium smegmatis, results in growth defects under phosphate-limiting conditions.
Microbiology (Reading, Engl.)
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Two component regulatory systems are key elements in the control of bacterial gene expression in response to environmental perturbations. The SenX3-RegX3 system is implicated in the control of phosphate uptake in Mycobacterium smegmatis and Mycobacterium tuberculosis. regX3 is reported to be essential in M. smegmatis, but not in M. tuberculosis. We attempted to construct complete senX3-regX3 operon deletion strains of M. smegmatis; initially we found that the operon could only be deleted when another functional copy was provided. Using a strain in which the only functional copy of the operon was present on an integrating plasmid, we attempted to replace the functional copy with an empty vector. Surprisingly, we obtained strains in which the functional copy had been deleted from the chromosome at a low frequency. We deleted the senX3 gene in a similar fashion, but it was not possible to delete regX3 alone. To identify possible compensatory mutations we sequenced the whole genome of two deletion strains and the wild-type. A synonymous single nucleotide polymorphism (SNP) in a lipoprotein was found in all deletion strains, but not the parental strains, and a frameshift mutation in nhaA was identified in three of the four deletion strains. Operon deletion strains were more sensitive to phosphate limitation, showing a reduced ability to grow at lower phosphate concentrations. The M. tuberculosis operon was able to functionally complement the growth phenotype in M. smegmatis under phosphate-replete conditions, but not under low phosphate conditions, reinforcing the difference between the two species. Our data show that, in contrast with previous reports, it is possible to delete the operon in M. smegmatis, possibly due to the accumulation of compensatory mutations, and that the deletion does affect growth in phosphate.
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A new in vivo model to test anti-tuberculosis drugs using fluorescence imaging.
J. Antimicrob. Chemother.
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The current method for testing new drugs against tuberculosis in vivo is the enumeration of bacteria in organs by cfu assay. Owing to the slow growth rate of Mycobacterium tuberculosis (Mtb), these assays can take months to complete. Our aim was to develop a more efficient, fluorescence-based imaging assay to test new antibiotics in a mouse model using Mtb reporter strains.
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The Development of the Metabolic Syndrome and Insulin Resistance After Adjuvant Treatment for Breast Cancer.
Cancer Nurs
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Adjuvant breast cancer treatment is associated with a number of adverse physical changes, including weight gain, and therefore may represent a critical period for the development of metabolic disturbance.
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What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

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In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.