Investigating the role of somatic sequencing platforms for phaeochromocytoma and paraganglioma in a large UK cohort.

OBJECTIVES: Phaeochromocytomas and paragangliomas (PPGL) are rare neuroendocrine tumours with malignant potential and a hereditary basis in almost 40% of patients. Germline genetic testing has transformed the management of PPGL enabling stratification of surveillance approaches, earlier diagnosis and predictive testing of at-risk family members. Recent studies have identified somatic mutations in a further subset of patients, indicating that molecular drivers at either a germline or tumour level can be identified in up to 80% of PPGL cases. The aim of this study was to investigate the clinical utility of somatic sequencing in a large cohort of patients with PPGL in the United Kingdom. DESIGN AND PATIENTS: Prospectively collected matched germline and tumour samples (development cohort) and retrospectively collected tumour samples (validation cohort) of patients with PPGL were investigated. MEASUREMENTS: Clinical characteristics of patients were assessed and tumour and germline DNA was analysed using a next-generation sequencing strategy. A screen for variants within 'mutation hotspots' in 68 human cancer genes was performed. RESULTS: Of 141 included patients, 45 (32%) had a germline mutation. In 37 (26%) patients one or more driver somatic variants were identified including 26 likely pathogenic or pathogenic variants and 19 variants of uncertain significance. Pathogenic somatic variants, observed in 25 (18%) patients, were most commonly identified in the VHL, NF1, HRAS and RET genes. Pathogenic somatic variants were almost exclusively identified in patients without a germline mutation (all but one), suggesting that somatic sequencing is likely to be most informative for those patients with negative germline genetic test results. CONCLUSIONS: Somatic sequencing may further stratify surveillance approaches for patients without a germline genetic driver and may also inform targeted therapeutic strategies for patients with metastatic disease.

SDHC phaeochromocytoma and paraganglioma: A UK-wide case series.

OBJECTIVE: Phaeochromocytomas and paragangliomas (PPGL) are rare, but strongly heritable tumours. Variants in succinate dehydrogenase (SDH) subunits are identified in approximately 25% of cases. However, clinical and genetic information of patients with SDHC variants are underreported. DESIGN: This retrospective case series collated data from 18 UK Genetics and Endocrinology departments. PATIENTS: Both asymptomatic and disease-affected patients with confirmed SDHC germline variants are included. MEASUREMENTS: Clinical data including tumour type and location, surveillance outcomes and interventions, SDHC genetic variant assessment, interpretation, and tumour risk calculation. RESULTS: We report 91 SDHC cases, 46 probands and 45 non-probands. Fifty-one cases were disease-affected. Median age at genetic diagnosis was 43 years (range: 11-79). Twenty-four SDHC germline variants were identified including six novel variants. Head and neck paraganglioma (HNPGL, n = 30, 65.2%), extra-adrenal paraganglioma (EAPGL, n = 13, 28.2%) and phaeochromocytomas (PCC) (n = 3, 6.5%) were present. One case had multiple PPGLs. Malignant disease was reported in 19.6% (9/46). Eight cases had non-PPGL SDHC-associated tumours, six gastrointestinal stromal tumours (GIST) and two renal cell cancers (RCC). Cumulative tumour risk (95% CI) at age 60 years was 0.94 (CI: 0.79-0.99) in probands, and 0.16 (CI: 0-0.31) in non-probands, respectively. CONCLUSIONS: This study describes the largest cohort of 91 SDHC patients worldwide. We confirm disease-affected SDHC variant cases develop isolated HNPGL disease in nearly 2/3 of patients, EAPGL and PCC in 1/3, with an increased risk of GIST and RCC. One fifth developed malignant disease, requiring comprehensive lifelong tumour screening and surveillance.

Differences between immunotherapy-induced and primary hypophysitis-a multicenter retrospective study.

OBJECTIVE: Immune checkpoint inhibitors can cause various immune-related adverse events including secondary hypophysitis. We compared clinical characteristics of immunotherapy-induced hypophysitis (IIH) and primary hypophysitis (PH) DESIGN: Retrospective multicenter cohort study including 56 patients with IIH and 60 patients with PH. METHODS: All patients underwent extensive endocrine testing. Data on age, gender, symptoms, endocrine dysfunction, MRI, immunotherapeutic agents and autoimmune diseases were collected. RESULTS: Median time of follow-up was 18 months in IIH and 69 months in PH. The median time from initiation of immunotherapy to IIH diagnosis was 3 months. IIH affected males more frequently than PH (p < 0.001) and led to more impaired pituitary axes in males (p < 0.001). The distribution of deficient adenohypophysial axes was comparable between both entities, however, central hypocortisolism was more frequent (p < 0.001) and diabetes insipidus considerably less frequent in IIH (p < 0.001). Symptoms were similar except that visual impairment occurred more rarely in IIH (p < 0.001). 20 % of IIH patients reported no symptoms at all. Regarding MRI, pituitary stalk thickening was less frequent in IIH (p = 0.009). Concomitant autoimmune diseases were more prevalent in PH patients before the diagnosis of hypophysitis (p = 0.003) and more frequent in IIH during follow-up (p = 0.002). CONCLUSIONS: Clinically, IIH and PH present with similar symptoms. Diabetes insipidus very rarely occurs in IIH. Central hypocortisolism, in contrast, is a typical feature of IIH. Preexisting autoimmunity seems not to be indicative of developing IIH.

Effectiveness of Positive Discipline Parenting Program on Parenting Style, and Child Adaptive Behavior.

In this study, a community sample of parents attending free 7-week Positive Discipline parenting workshops were recruited, as well as a non-randomized community control. Both samples consisted of primarily Hispanic parents with similar demographic information and attrition rates (initial N = 91), as well as children of similar age (mean age 6.89 and 6.95 years) and gender. Parenting stress, parenting style, and parent-reported child adaptive behavior were assessed at baseline and after three months. Longitudinal analysis was performed using mixed-effects regression modeling. Results indicate that attendance in Positive Discipline parenting workshops was related to a decrease in authoritarian parenting style, a decrease in permissive parenting style, and a decrease in parental stress. It was also related to an increase in child academic competence, and a decrease in externalizing-hyperactive behavior (both parent-report). These results suggest that positive discipline parenting workshops may alter parenting style and may positively impact children of parents who attend.

Comparison of Preoperative MRI With Intraoperative Findings for Peroneal Tendon Pathologies.

Magnetic resonance imaging (MRI) is commonly used to evaluate soft tissue pathology of the foot and ankle. Prior investigations have reported limitations of this modality, however, in evaluation of pathologies related to the peroneal tendons. This article investigates the correlation of pre-operative MRI studies with intraoperative findings. Five board-certified radiologists interpreted MRIs of 80 ankles that subsequently underwent surgical procedures performed by one board-certified foot and ankle surgeon, after which comparison was made between their findings. Statistically significant disagreement was found between radiologist and surgeon findings of a normal peroneus brevis (PB), PB and peroneus longus (PL) tendinosis, PB and PL hypertrophy, PB and PL partial linear tears, PB and PL flattening, PB longitudinal split tears, and the PB attritional spectrum (combined analysis of flattening, partial linear tearing, and longitudinal split tears). These results suggest that given the disconcordance between MRI and intraoperative findings, surgeons should remain cautious in their reliance upon this imaging modality when evaluating this anatomic region.

Pregnancy outcomes in women receiving growth hormone replacement therapy enrolled in the NordiNet® International Outcome Study (IOS) and the American Norditropin® Studies: Web-Enabled Research (ANSWER) Program.

PURPOSE: Data on the safety of growth hormone (GH) replacement therapy during pregnancy are limited. We report a combined analysis of data from pregnant women treated with GH while enrolled in two non-interventional, multicenter studies: NordiNet® International Outcome Study (IOS) and the American Norditropin® Studies: Web-Enabled Research (ANSWER) Program. METHODS: Pregnancy data were pooled from NordiNet® IOS and the ANSWER Program. Data were collected during routine clinic visits by participating physicians using a web-based system. Patients exposed to GH replacement therapy during pregnancy were included in the analysis. RESULTS: The study population included 40 female patients with typical causes of adult GH deficiency (GHD). Overall, there were 54 pregnancies. Of these, 47 were exposed to GH between conception and delivery. In 48.9% of pregnancies exposed to GH, the dose was > 0.6 mg/day. GH was continued past conception and then stopped during the first, second, and third trimester, in 27.7%, 17.0%, and 2.1% of pregnancies, respectively. In 29.8%, GH was continued throughout pregnancy, with an unchanged dose in most cases. Of the 47 GH-exposed pregnancies, 37 (78.7%) progressed to normal delivery. There were three adverse events reported in two pregnancies. CONCLUSION: These real-world data suggest that there were no new safety signals related to GH exposure in women with GHD during pregnancy. These results are consistent with findings from previous studies reporting data in pregnancies exposed to GH at conception or throughout pregnancy. This observational study in additional pregnancies provides further evidence that GH exposure does not adversely affect pregnancy outcome. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov NCT00960128 (date of registration: August 13, 2009) and NCT01009905 (date of registration: November 5, 2009).

Ankle Disarticulation: An Underutilized Approach to Staged Below Knee Amputation-Case Series and Surgical Technique.

The staged approach to below knee amputation has proven to be an effective method of achieving functionality after amputation and reducing risk for postamputation complications. Transtibial or "guillotine" amputations are often used as the first-stage amputation. Disarticulation at the ankle joint is an alternative method that can be used with favorable results and benefits. These include rapid infection decompression while minimizing the blood loss of traditional tibial osteotomy. Here we describe our operative technique, perioperative management, and indications for this surgical procedure.

A multicentre validation study for the EU-TIRADS using histological diagnosis as a gold standard.

OBJECTIVE: Many systems for risk stratification of thyroid nodule with ultrasound (US) have been proposed and the EU-TIRADS issued by the ETA in 2017 was the last to have been published. The present study was undertaken to evaluate whether the malignancy risk of each category corresponded to the given range of the guidelines and assess the diagnostic value of EU-TIRADS in a multi-institutional trial with histology as gold standard. DESIGN: Three institutions in Switzerland, France and United Kingdom shared this retrospective study. Enrolment period was 2013-2017. Included were patients who had undergone surgery with a detailed preoperative thyroid US. METHODS: Cancer risk was calculated for each EU-TIRADS score. Predictivity tests were estimated. Nonparametric statistical analysis was used. RESULTS: The final series included 1058 nodules of which 257 (24.3%) carcinomas. Nodules were classified as EU-TIRADS 2, 3, 4 and 5 in 6.7, 46.4, 26.2 and 20.7%, respectively. Cancer prevalence was 1.4, 3.5, 17 and 87.7% in classes 2-5, respectively (P < 0.0001). EU-TIRADS 5 had a significantly higher cancer rate than the other summed categories (7.7%; P < 0.0001) with OR 84.7. When EU-TIRADS 4 and 5 were combined, 93% sensitivity and 97% NPV were found and findings of the three institutions were quite similar. Using the recommended criteria for FNA negative predictive value was 90.9%. CONCLUSIONS: The cancer rate was within or close to the given range described in the EU-TIRADS guidelines. The diagnostic value was satisfactory. The results were similar in the three institutions participating in the study.

Imaging Features of Succinate Dehydrogenase-deficient Pheochromocytoma-Paraganglioma Syndromes.

Pheochromocytoma (PC) and paraganglioma (PGL) are rare neuroendocrine tumors that occur throughout the body from the base of the skull to the pelvis. Sympathetic catecholamine-secreting tumors may be associated with hyperadrenergic symptoms and long-term morbidity if they are untreated. Typically biochemically silent, head and neck PGLs may result in cranial nerve palsies and symptoms due to localized mass effect. Tumors can arise sporadically or as part of an inheritable PC-PGL syndrome. Up to 40% of tumors are recognized to be associated with germline mutations in an increasing array of susceptibility genes, including those that appear to arise sporadically. Most commonly, up to 25% of all PC-PGLs are associated with mutations in one of the succinate dehydrogenase (SDH) enzyme subunit genes. The resulting familial PC-PGL syndrome varies according to the affected enzyme subunit (most commonly SDHB and SDHD mutations) with respect to tumor prevalence, location, age of onset, and risk of malignancy. Patients with SDH enzyme mutations have increased lifetime risk of developing multifocal tumors and malignancy. Early recognition of individuals at high risk, genetic testing, screening of family members, and lifelong surveillance programs are recommended, but not without health, economic, and psychologic implications. Anatomic and functional imaging is key to diagnosis, staging, treatment planning, and lifelong surveillance of these individuals. Radiologists must be aware of the imaging appearance of these varied tumors.©RSNA, 2019.

Practical Aspects of Introduction of Multiplex Bead Technology into the Routine Diagnostic Immunology Laboratory.

BACKGROUND: Multiplex bead assays, also known as addressable laser bead immunoassays (ALBIA) or Luminex® technology, have provided an alternative to enzyme-linked immunoassay, which is still the most widely utilized routine immunoassay for detection of specific autoantibodies. Our laboratory adopted the ALBIA technology early into its routine service. METHODS: We report the performance and utility of measurement of three different autoantibody types tested using the FIDIS (BMD, Marne La Vallee, France) ALBIA system. The analytes discussed are thyroid antibodies (thyroglobulin [TG], thyroid peroxidase [TPO]), anti-neutrophil cytoplasmic antibodies (ANCA), and ribonucleo-protein (RNP) antibodies. RESULTS: In single antibody analysis, TPO antibody testing was superior to TG antibody in identifying patients with Graves' disease and Hashimoto's thyroiditis. However, testing only TPO antibody would result in missing 8.6% of Graves' and 11.9% of Hashimoto's thyroiditis patients, hence demonstrating an advantage for the multiplex TG plus TPO assay. With respect to ANCA, the FIDIS ALBIA produced an overall similar level of performance to our comparator method, the Phadia fluorescent enzyme linked immunoassay. Sensitivity of the ALBIA for RNP antibodies was low in comparison to countercurrent immunoelectrophoresis, but performance was improved by altering the cutoff value for the assay. CONCLUSIONS: ALBIA technology has many potential advantages in the routine laboratory, but as with any new assay, evaluation must be thorough and ongoing to ensure satisfactory clinical performance is obtained. Both false positive and false negative results have been reported in ALBIA studies. It may be necessary to re-evaluate assay performance and cutoff and consider further clinical correlation.

Management of aggressive variants of papillary thyroid cancer.

PURPOSE OF REVIEW: The aim of this study was to provide a timely and relevant review of the latest findings and explore appropriate management of aggressive variants of papillary thyroid cancer (AVPTC). RECENT FINDINGS: In general, AVPTCs tend to exhibit more invasive characteristics, a lack of responsiveness to radioiodine, increased occurrences of regional spreading, distant metastases and higher mortality rates. Meanwhile, each variant showcases unique clinical and molecular profiles. SUMMARY: Given the elevated risk of recurrence postsurgery, a more aggressive strategy may be necessary when suspected preoperatively, particularly for those presenting with invasive features. Decision on the extent of surgical treatment and adjuvant therapy is individualized and made by experienced clinicians and multidisciplinary teams based on the clinical presentation, presence of aggressive features and molecular profile. Future studies on development of personalized medicine and molecular target therapy may offer tailored treatment options.

Dissociating the impact of alexithymia and impaired self-awareness on emotional distress and aggression after traumatic brain injury.

OBJECTIVE: Alexithymia, a deficit in identifying and describing feelings, is prevalent in traumatic brain injury (TBI). Sometimes referred to as "emotional unawareness," we sought to investigate whether alexithymia after TBI was related to, or distinct from, impaired self-awareness (ISA) and whether the two predicted differentiable emotional and aggression profiles. Further, the mediating role of frontal system behaviors (disinhibition, dysexecutive function, apathy) was explored. METHOD: Participants with TBI (N = 40) from diverse backgrounds completed self-report measures of alexithymia, emotional distress, aggression, and frontal system behaviors. For the assessment of ISA, significant other ratings were obtained to identify discrepancies from self-ratings. Data were analyzed quantitatively using independent samples t tests, correlations, partial correlations, and simple mediation. RESULTS: There was a negative correlation between alexithymia and ISA. Alexithymia, but not ISA, was associated with higher expressions of emotional distress and aggression even after controlling for the effects of ISA via partial correlations. Exploratory analyses found that frontal system behaviors mediated the relationships between alexithymia and aggression and alexithymia and emotional distress. CONCLUSIONS: Alexithymia is more accurately conceptualized as an emotional processing deficit than an awareness deficit. Indeed, self-awareness may be a prerequisite for the ability to identify alexithymic tendencies. Negative psychological effects of alexithymia are compounded by poorer executive function and disinhibition and call for the development of TBI-specific alexithymia screening tools and interventions. Alexithymia interventions are best delivered in conjunction with rehabilitation of emotion regulation and executive function. (PsycInfo Database Record (c) 2024 APA, all rights reserved).

The effect of a pre- and post- operative exercise program versus standard care on physical fitness of patients with oesophageal and gastric cancer undergoing neoadjuvant treatment prior to surgery (The PERIOP-OG Trial): a randomized controlled trial.

INTRODUCTION: Although the benefits of post-operative rehabilitation in cancer surgery are well established, the role of prehabilitation is less defined. Oesophagogastric cancers present a unique opportunity to study the impact of prehabilitation during the neoadjuvant window, whether with chemotherapy or chemoradiotherapy (NCT) in patients who are frequently nutritionally depleted. This trial examines the impact of a community-based exercise program on patient fitness during and after the neoadjuvant window. METHODS: A pragmatic, randomized controlled multi-centre trial was undertaken in three centres. Inclusion criteria were patients aged ≥ 18 years planned for NCT and esophagectomy or gastrectomy. Participants were randomized 1:1 to an exercise prehabilitation group (EX) or to usual care (UC). The primary endpoint was cardiorespiratory fitness between baseline and pre-surgery timepoint using the 6-min walk test. Secondary endpoints included hand dynamometer, 10-sec sit to stand, activity behaviour, body mass index, semi-structured interviews, questionnaires assessing quality of life, surgical fear, general self-efficacy and mastery. RESULTS: Between March 2019 and December 2020, 71 participants were recruited: EX (n=36) or UC (n=35). From baseline to pre-surgery, the difference-in-difference for EX showed a significant improvement in 6MWT of 50.7m (P=0.05) compared to UC [mean (SD): 522.1m (+/-104.3) to 582.1m (+/-108) vs. 497.5m (+/-106.3) to 506.0 m (+/-140.4). There was no statistically significant DID for secondary outcome measures. CONCLUSIONS: This community exercise prehabilitation program significantly improves physical fitness for surgery, is feasible and provides a standardized framework for prescription of exercise in esophagogastric cancer patients undergoing NCT.

Mastoid surgery under local anesthesia for medically unfit patients: techniques and outcome.

OBJECTIVES: We present the surgical techniques and outcomes of mastoid surgery under local anesthesia in patients who were unfit for general anesthesia. METHODS: Five tertiary-referred patients with multiple comorbidities and failed conservative treatment for chronic otitis media were operated on under local anesthesia. No sedation was administered. The principles of cholesteatoma surgery were observed, but the technique was adapted to keep surgical time to a minimum. RESULTS: None of the patients had perioperative problems, and all have dry, waterproof ears with preservation of hearing after surgery. So far, none of the patients have had recurrent or residual disease. CONCLUSIONS: Cholesteatoma surgery can be successfully performed with a local anesthetic in patients who are medically unfit for general anesthesia. Surgery requires a good coordination of the operating team in order to shorten the operating time. Otologists should develop and maintain their skills by performing ear surgery with local anesthetic on a regular basis.

Thyroid lymphoma.

PURPOSE OF REVIEW: To highlight recent advances in our understanding of the epidemiology, incidence, evaluation, management and outcomes of primary thyroid lymphoma (PTL), and highlight the indications and limitations of surgery. RECENT FINDINGS: The differential diagnosis of a rapidly enlarging thyroid mass with or without obstructive symptoms should include PTL and anaplastic thyroid cancer. When PTL is suspected, initial investigations should include blood tests and ultrasound-guided biopsy preferably core need biopsy to allow tissue typing and immunohistochemistry analysis. Systemic imaging with FDG PET-CT is required for staging. Surgery is not recommended for treatment purposes and should be reserved for diagnosis and airway management. Treatment includes chemotherapy and radiotherapy and offer an excellent prognosis. SUMMARY: PTL is a rare malignancy making diagnosis and management challenging. Initial investigations of suspected PTL should include blood tests and ultrasound-guided biopsy, preferably core needle biopsy and systemic imaging is required for staging. Surgery is reserved for diagnosis and airway management. Chemotherapy and radiotherapy are the treatment of choice.

Thyroglossal duct cyst carcinoma case series-Management strategy and outcomes.

OBJECTIVE: To review the clinical presentation, diagnosis, pathology and management strategies in a modern cohort of patients with thyroglossal duct cyst carcinoma. STUDY DESIGN: Retrospective case series following PROCESS Guidelines. SETTING: Comprehensive cancer centre. METHODS: Data recorded included: gender, age at diagnosis, clinical presentation, thyroid function, diagnostic investigations, cytological results, final histology, staging and follow up status. The risk of malignancy in cytological analysis was stratified according to the Royal College of Pathologists classification in United Kingdom. RESULTS: Twelve patients were included. The majority of patients (66.7%) presented with an isolated thyroglossal duct cyst. Only 4 patients had preoperative cytological suspicion of carcinoma (sensitivity: 33.3%). At the time of presentation all patients were euthyroid. Following diagnosis of malignancy, a total thyroidectomy was performed in all patients, with the exception of 2, who had a thyroglossal duct cyst carcinoma of less than 10mm. Among the 10 patients who underwent total thyroidectomy, 7 (70%) patients had proven carcinoma in the thyroid gland, 3 with deposits of less than 10mm. The average size of the thyroid cancer deposits was 7.2mm (1-20mm). With a mean follow-up of is 44 months (5-120), all patients were alive and free of recurrence at the end of the study period. CONCLUSION: Thyroglossal duct cyst carcinoma is a rare condition and its management should be discussed in a multidisciplinary meeting. As with differentiated thyroid cancer originating in the thyroid gland, it bears extraordinary survival rates. Accordingly, the management of these cancers has shifted towards a more conservative approach although its peculiarities must be taken into account: ease of extracystic invasion and possible different lymph node invasion.

Rehabilitation needs of people with brain tumours in Ireland: Protocol for a prospective, mixed methods action research study ("Brain-RESTORE").

Background: Approximately 480 people annually in Ireland are diagnosed with a primary brain tumour. Brain tumours are a heterogeneous group of conditions, varying in histopathology, location, and progression. A consistent feature is neurological impairment, which can lead to profound effects on physical and cognitive function. There is evidence that people with brain tumours can benefit from rehabilitation, but pathways are poorly described, and no best practice is defined. This leads to significant unmet need. The aim of this study is to understand the rehabilitation needs of people diagnosed with a brain tumour in Ireland, and gain insight to inform policy and practice. Methods: A prospective, mixed methods study with embedded action research will be conducted. Patients (n=122) with a new diagnosis of primary brain tumour, and optionally, a nominated carer or family member, will be recruited through a national neuro-oncology service. Rehabilitation need (Mayo-Portland Adaptability Inventory), quality of life (European Organisation for Research and Treatment of Cancer Quality of Life Questionnaire Brain Cancer Module, EuroQol-5D-5L), healthcare utilisation and, optionally, carer needs (Carer Support Needs Assessment Tool) will be assessed at four, eight and 12 months post diagnosis. An embedded qualitative study will invite 30 patients and carers to a semi-structured interview to explore their lived experience of rehabilitation needs and services following brain tumour diagnosis. Finally, using an Action Research approach, healthcare professionals involved in caring for people with brain tumours will be invited to participate in co-operative inquiry groups, to reflect on emerging aggregate findings and identify actions that could be undertaken while the study is underway. Conclusions: By understanding rehabilitation need, the findings will help healthcare professionals and health service providers understand how to prioritise the supports required and encourage policy makers to adequately resource neurorehabilitation to meet the needs of people with a brain tumour diagnosis.

Expression and characterization of Mycobacterium tuberculosis CYP144: common themes and lessons learned in the M. tuberculosis P450 enzyme family.

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 µM), clotrimazole (0.37 µM) and econazole (0.78 µM) 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.

Deletion of SenX3-RegX3, a key two-component regulatory system of Mycobacterium smegmatis, results in growth defects under phosphate-limiting conditions.

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.

A new in vivo model to test anti-tuberculosis drugs using fluorescence imaging.

OBJECTIVES: 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. METHODS: A commercial IVIS Kinetic® system and a custom-built laser scanning system with fluorescence molecular tomography (FMT) capability were used to detect fluorescent Mtb in living mice and lungs ex vivo. The resulting images were analysed and the fluorescence was correlated with data from cfu assays. RESULTS: We have shown that fluorescent Mtb can be visualized in the lungs of living mice at a detection limit of ∼8 × 107 cfu/lung, whilst in lungs ex vivo a detection limit of ∼2 × 105 cfu/lung was found. These numbers were comparable between the two imaging systems. Ex vivo lung fluorescence correlated to numbers of bacteria in tissue, and the effect of treatment of mice with the antibiotic moxifloxacin could be visualized and quantified after only 9 days through fluorescence measurements, and was confirmed by cfu assays. CONCLUSIONS: We have developed a new and efficient method for anti-tuberculosis drug testing in vivo, based on fluorescent Mtb reporter strains. Using this method instead of, or together with, cfu assays will reduce the time required to assess the preclinical efficacy of new drugs in animal models and enhance the progress of these candidates into clinical trials against human tuberculosis.