The High Mutational Sensitivity of ccdA Antitoxin Is Linked to Codon Optimality.

Deep mutational scanning studies suggest that synonymous mutations are typically silent and that most exposed, nonactive-site residues are tolerant to mutations. Here, we show that the ccdA antitoxin component of the Escherichia coli ccdAB toxin-antitoxin system is unusually sensitive to mutations when studied in the operonic context. A large fraction (∼80%) of single-codon mutations, including many synonymous mutations in the ccdA gene shows inactive phenotype, but they retain native-like binding affinity towards cognate toxin, CcdB. Therefore, the observed phenotypic effects are largely not due to alterations in protein structure/stability, consistent with a large region of CcdA being intrinsically disordered. E. coli codon preference and strength of ribosome-binding associated with translation of downstream ccdB gene are found to be major contributors of the observed ccdA mutant phenotypes. In select cases, proteomics studies reveal altered ratios of CcdA:CcdB protein levels in vivo, suggesting that the ccdA mutations likely alter relative translation efficiencies of the two genes in the operon. We extend these results by studying single-site synonymous mutations that lead to loss of function phenotypes in the relBE operon upon introduction of rarer codons. Thus, in their operonic context, genes are likely to be more sensitive to both synonymous and nonsynonymous point mutations than inferred previously.

Protein model discrimination attempts using mutational sensitivity, predicted secondary structure, and model quality information.

Structure prediction methods often generate a large number of models for a target sequence. Even if the correct fold for the target sequence is sampled in this dataset, it is difficult to distinguish it from other decoy structures. An attempt to solve this problem using experimental mutational sensitivity data for the CcdB protein was described previously by exploiting the correlation of residue depth with mutational sensitivity (r ~ 0.6). We now show that such a correlation extends to four other proteins with localized active sites, and for which saturation mutagenesis datasets exist. We also examine whether incorporation of predicted secondary structure information and the DOPE model quality assessment score, in addition to mutational sensitivity, improves the accuracy of model discrimination using a decoy dataset of 163 targets from CASP. Although most CASP models would have been subjected to model quality assessment prior to submission, we find that the DOPE score makes a substantial contribution to the observed improvement. We therefore also applied the approach to CcdB and four other proteins for which reliable experimental mutational data exist and observe that inclusion of experimental mutational data results in a small qualitative improvement in model discrimination relative to that seen with just the DOPE score. This is largely because of our limited ability to quantitatively predict effects of point mutations on in vivo protein activity. Further improvements in the methodology are required to facilitate improved utilization of single mutant data.

Molecular Determinants of Mutant Phenotypes, Inferred from Saturation Mutagenesis Data.

Understanding how mutations affect protein activity and organismal fitness is a major challenge. We used saturation mutagenesis combined with deep sequencing to determine mutational sensitivity scores for 1,664 single-site mutants of the 101 residue Escherichia coli cytotoxin, CcdB at seven different expression levels. Active-site residues could be distinguished from buried ones, based on their differential tolerance to aliphatic and charged amino acid substitutions. At nonactive-site positions, the average mutational tolerance correlated better with depth from the protein surface than with accessibility. Remarkably, similar results were observed for two other small proteins, PDZ domain (PSD95pdz3) and IgG-binding domain of protein G (GB1). Mutational sensitivity data obtained with CcdB were used to derive a procedure for predicting functional effects of mutations. Results compared favorably with those of two widely used computational predictors. In vitro characterization of 80 single, nonactive-site mutants of CcdB showed that activity in vivo correlates moderately with thermal stability and solubility. The inability to refold reversibly, as well as a decreased folding rate in vitro, is associated with decreased activity in vivo. Upon probing the effect of modulating expression of various proteases and chaperones on mutant phenotypes, most deleterious mutants showed an increased in vivo activity and solubility only upon over-expression of either Trigger factor or SecB ATP-independent chaperones. Collectively, these data suggest that folding kinetics rather than protein stability is the primary determinant of activity in vivo This study enhances our understanding of how mutations affect phenotype, as well as the ability to predict fitness effects of point mutations.

HIGH-PRECISION CONFORMAL FRACTIONATED RADIOTHERAPY IS EFFECTIVE IN ACHIEVING REMISSION IN PATIENTS WITH ACROMEGALY AFTER FAILED TRANSSPHENOIDAL SURGERY.

OBJECTIVE: Variable efficacy of pituitary radiotherapy in acromegaly is reported. Here we sought to assess the efficacy of high-precision conformal fractionated radiotherapy (CRT) in patients with acromegaly after failed TSS. METHODS: A retrospective analysis was conducted a in tertiary care referral center between 1999 to 2013 on 36 acromegaly patients (M: 16, F: 20; median age: 36.0 years) with macroadenoma and mean growth hormone (GH) and insulin-like growth factor-1 (IGF1) upper limits of normal (ULN) of 15.9 ± 14.3 ng/mL and 1.74 ± 0.43, respectively. The cohort was divided into 2 groups: 30 patients (M: 13, F: 17) who were medical treatment naïve, and 6 patients (M: 3, F: 3) who received medical treatment after CRT. RESULTS: Normalization of GH (fasting GH <1 ng/mL), normalization of IGF1 (ULN <1), and remission (normalization of GH and IGF1) were achieved in 20 (55%), 23 (63%) and 20 (55%) patients, respectively. The mean time required to achieve remission was 63 ± 33.4 months. Follow-up duration was the only predictor of achieving remission. GH level declined exponentially by 65% and 89% at 2 and 5 years, respectively. New onset hypopituitarism was noted in 33% of patients. Tumor control was achieved in 100% of patients. In groups 1 and 2, 18 (60%) and 2 (33.3%) achieved remission post-CRT, and the mean times required to achieve remission were 58.6 ± 30.7 months and 102 ± 42.4 months, respectively. CONCLUSION: High-precision CRT is an effective modality to achieve remission in patients with acromegaly after failed TSS.

TSpred: a web server for the rational design of temperature-sensitive mutants.

Temperature sensitive (Ts) mutants of proteins provide experimentalists with a powerful and reversible way of conditionally expressing genes. The technique has been widely used in determining the role of gene and gene products in several cellular processes. Traditionally, Ts mutants are generated by random mutagenesis and then selected though laborious large-scale screening. Our web server, TSpred (http://mspc.bii.a-star.edu.sg/TSpred/), now enables users to rationally design Ts mutants for their proteins of interest. TSpred uses hydrophobicity and hydrophobic moment, deduced from primary sequence and residue depth, inferred from 3D structures to predict/identify buried hydrophobic residues. Mutating these residues leads to the creation of Ts mutants. Our method has been experimentally validated in 36 positions in six different proteins. It is an attractive proposition for Ts mutant engineering as it proposes a small number of mutations and with high precision. The accompanying web server is simple and intuitive to use and can handle proteins and protein complexes of different sizes.

Primary (autoimmune) hypophysitis: a single centre experience.

BACKGROUND: Autoimmune hypophysitis (AH) is a rare autoimmune inflammatory disorder of pituitary gland. OBJECTIVE: To analyse clinical, hormonal, radiological features and management outcomes of AH. DESIGN: Retrospective analysis of patients with primary hypophysitis (where secondary causes of hypophysitis were ruled out) was carried out from 2006 to 2012. AH emerged as the most plausible aetiology and the diagnosis of exclusion. RESULTS: Twenty-four patients with AH (21 females and 3 males) were evaluated. They presented with symptoms of expanding sellar mass (83.3%), symptoms of anterior pituitary hormone deficiencies (58.3%), and diabetes insipidus (16.7%). The anterior pituitary hormonal axes affected were cortisol (75%), thyroid (58.33%) and gonadotropin (50%). All had sellar mass on magnetic resonance imaging, which was symmetrical (91.7%) and homogenously enhancing (91.7%). Stalk thickening, suprasellar extension, loss of posterior pituitary hyperintensity and parasellar T2 dark sign were seen in 87.5, 87.5, 71.5, and 50% respectively. In addition to hormone replacement, five (20.83%) patients underwent trans-sphenoidal surgery, fifteen (62.5%) were watchfully monitored, while four cases (16.67%) received steroid pulse therapy. On follow up imaging, the sellar mass regressed in all, while, stalk thickening was persistent in 13/19 (68.4%) non-operated patients at median follow up of 1 year. Pituitary hormone axis recovery was seen in 10 (41.67%) and was seen in cortisol 10/18 (55.5%) followed by gonadotropin 5/12 (41.67%) axis. CONCLUSION: Characteristic radiology helps in diagnosis of AH even without tissue diagnosis. Non-operative treatment is the preferred treatment modality. Steroid pulse therapy potentially improves pituitary axis recovery.

Clinical, biochemical and imaging characteristics of Cushing's macroadenomas and their long-term treatment outcome.

OBJECTIVE: Cushing's macroadenoma as a cause of Cushing's disease is less common than microadenoma. The data on nature and behaviour of Cushing's macroadenoma are limited to a few case series. We studied clinical, biochemical and imaging characteristics of macroadenoma and their long-term treatment outcomes. METHOD: Retrospective analysis of 40 patients with macroadenoma managed at our centre from 1997 to 2013. RESULTS: Of 40 patients, there were 15 (37·5%) males and 25 (62·5%) females. Mean age at presentation was 26·7 ± 9·3 years. Visual field defects and/or cranial nerve palsies were found in 15 cases at presentation. Mean maximum tumour dimension was 20·83 ± 10·74 mm, and parasellar extension was seen in 25 (62·5%) patients. Plasma ACTH/maximum tumour dimension and 8 am serum cortisol/maximum tumour dimension decreased with increasing tumour size. Sixteen patients (40%) had remission (4: immediate, 12: delayed) after first transsphenoidal surgery (TSS). Larger tumour size and parasellar extension were predictors of failure to achieve remission. Four patients relapsed; noticeably all of them had delayed remission. Among the persistent and relapsed cases, second TSS was successful in two of eight patients, whereas 11 of 16 patients achieved remission after a mean duration of 12·14 ± 8·41 months postradiotherapy. CONCLUSION: Younger age at presentation and larger tumour size compared with previous series were distinctive features of our series. Large tumour size and parasellar extension were negative predictors of surgical remission. Delayed remission was seen in significant proportion of patients, but one-third later relapsed. Radiotherapy was an effective second-line treatment modality.

Hepatocyte-macrophage crosstalk via the PGRN-EGFR axis modulates ADAR1-mediated immunity in the liver.

ADAR1-mediated RNA editing establishes immune tolerance to endogenous double-stranded RNA (dsRNA) by preventing its sensing, primarily by MDA5. Although deleting Ifih1 (encoding MDA5) rescues embryonic lethality in ADAR1-deficient mice, they still experience early postnatal death, and removing other MDA5 signaling proteins does not yield the same rescue. Here, we show that ablation of MDA5 in a liver-specific Adar knockout (KO) murine model fails to rescue hepatic abnormalities caused by ADAR1 loss. Ifih1;Adar double KO (dKO) hepatocytes accumulate endogenous dsRNAs, leading to aberrant transition to a highly inflammatory state and recruitment of macrophages into dKO livers. Mechanistically, progranulin (PGRN) appears to mediate ADAR1 deficiency-induced liver pathology, promoting interferon signaling and attracting epidermal growth factor receptor (EGFR)+ macrophages into dKO liver, exacerbating hepatic inflammation. Notably, the PGRN-EGFR crosstalk communication and consequent immune responses are significantly repressed in ADAR1high tumors, revealing that pre-neoplastic or neoplastic cells can exploit ADAR1-dependent immune tolerance to facilitate immune evasion.

Pantothenate - kinase associated neurodegeneration.

Neurodegeneration with brain iron accumulation is a group of disorders, the commonest of which is PKAN (Pantothenate kinase associated neurodegeneration). We present here, a case of 18 year old boy with progressive dementia, pyramidal and extrapyramidal involvement, dysarthria, seizures and myoclonus. The patient was diagnosed as PKAN (formerly Hallervorden Spatz disease) after "eye of tiger" appearance on neuro-imaging.

Disseminated melioidosis presenting as septic arthritis.

Melioidosis is an infection caused by Burkholderia pseudomallei. The disease is known as a remarkable imitator due to the wide and variable clinical spectrum of its manifestations. Septic arthritis is rare but well-recognized manifestation of this disease. We report a case of melioidosis in a 52 year male with uncontrolled diabetes mellitus (DM) presenting with a rare combination of septic arthritis and abscesses in the chest wall, liver and subcutaneous tissue. The patient responded to prolonged treatment of intravenous ceftazidime followed by oral co-trimoxazole.

Mycotic aneurysm of the popliteal artery due to infective endocarditis.

Mycotic aneurysm (MA) is an infrequent complication of infective endocarditis (IE), reported in 3 to 15% of the patients with IE. The commonest site for such aneurysm is intracranial vessels (65%) followed by abdominal and then the peripheral vessels. We describe a case of 32 year old man with recently diagnosed rheumatic heart disease and mitral regurgitation. He had infective endocarditis (IE) and developed a large mycotic popliteal artery aneurysm (MPAA) and a small profunda femoris arterial aneurysm (PFAA) while he was on antibiotic therapy. The patient was successfully treated with prolonged antibiotic therapy and embolisation of the MPAA while PFAA was managed conservatively.

Rapid Mapping of Protein Binding Sites and Conformational Epitopes by Coupling Yeast Surface Display to Chemical Labeling and Deep Sequencing.

Delineating the precise regions on an antigen that are targeted by antibodies is important for the development of vaccines and antibody therapeutics. X-ray crystallography and NMR are considered the gold standard for providing precise information about these binding sites at atomic resolution. However, these are labor-intensive and require purified protein at high concentration. We have recently described [1] a rapid and reliable method that overcomes these constraints, using a panel of single cysteine mutants of the protein of interest and now provide protocols to facilitate its adoption. Mutants are displayed on the yeast cell surface either individually or as a pool, and labeled covalently with a cysteine specific probe. Binding site residues are inferred by monitoring loss of ligand or antibody binding by flow cytometry coupled to deep sequencing of sorted populations, or Sanger sequencing of individual clones. Buried cysteine residues are not labeled and library sizes are small, facilitating rapid identification of binding-site residues. The methodology was used to identify epitopes on the bacterial toxin CcdB targeted by twenty-four different monoclonal antibodies as well as by polyclonal sera. The method does not require purified protein or protein structural information and can be applied to a variety of display formats.

Isolation and purification of antibacterial compound from Streptomyces levis collected from soil sample of north India.

During the screening programme for microbial cultures producing antimicrobial agents, an active microbial strain of Streptomyces was isolated from the agricultural soil of Narnaul, Haryana India. Physiological, biochemical characteristics and 16S ribosomal RNA sequence homology studies revealed that it was similar to Streptomyces levis (sequence similarity 100%). The microbial strain was submitted to Genomebio Technologies Pvt. Ltd., Pune, Maharashtra, India under Accession No. EU124569. The isolated strain was found to produce extracellular active compound showing strong antimicrobial activity against Klebsiella pneumoniae MTCC 109, Pseudomonas aeruginosa MTCC 741 and Staphylococcus aureus MTCC 96. The antibacterial compound was successfully isolated and purified. Structure elucidation of antibacterial metabolite with EI-MS/ HRMS showed molecular ion peak at m/z 686 [M+H]+. Whereas, elemental analysis of the said compound showed C = 61.31, H = 8.61, N = 2.04 and O = 28.02, and indicated a molecular formula of C35H59NO12. The presence of 'chromone' nucleus in the compound's chemical structure was confirmed by using 1HNMR studies. The present study reports the purification of potential antibacterial compound from Streptomyces levis isolated from the unexplored soil of north India and warrants for further characterization of this potential compound for optimum utilization for antimicrobial purposes.

Mapping Protein Binding Sites and Conformational Epitopes Using Cysteine Labeling and Yeast Surface Display.

We describe a facile method for mapping protein:ligand binding sites and conformational epitopes. The method uses a combination of Cys scanning mutagenesis, chemical labeling, and yeast surface display. While Ala scanning is widely used for similar purposes, often mutation to Ala (or other amino acids) has little effect on binding, except at hotspot residues. Many residues in physical contact with a binding partner are insensitive to substitution with Ala. In contrast, we show that labeling of Cys residues in a binding site consistently abrogates binding. We couple this methodology to yeast surface display and deep sequencing to map conformational epitopes targeted by both monoclonal antibodies and polyclonal sera as well as a protein:ligand binding site. The method does not require purified protein, can distinguish buried and exposed residues, and can be extended to other display formats, including mammalian cells and viruses, emphasizing its wide applicability.

Long-term cardiac (valvulopathy) safety of cabergoline in prolactinoma.

BACKGROUND: Clinical relevance of association of cabergoline use for hyperprolactinemia and cardiac valvulopathy remains unclear. OBJECTIVE: The aim of the study was to determine the prevalence of valvular heart abnormalities in patients taking cabergoline for the treatment of prolactinoma and to explore any associations with the cumulative dose of drug used. DESIGN: A cross-sectional echocardiographic study was performed in patients who were receiving cabergoline therapy for prolactinoma. RESULTS: Hundred (61 females, 39 males) prolactinoma cases (81 macroprolactinoma and 19 microprolactinoma) were included in the study. The mean age at presentation was 33.9 ± 9.0 years (range: 16-58 years). The mean duration of treatment was 53.11 ± 43.15 months (range: 12-155 months). The mean cumulative dose was 308.6 ± 290.2 mg (range: 26-1196 mg; interquartile range: 104-416 mg). Mild mitral regurgitation was present in one patient (cumulative cabergoline dose 104 mg). Mild tricuspid regurgitation was present in another two patients (cumulative cabergoline dose 52 mg and 104 mg). Aortic and pulmonary valve functioning was normal in all the cases. There were no cases of significant valvular regurgitation (moderate to severe, Grade 3-4). None of the patients had morphological abnormalities such as thickening, calcification, and restricted mobility of any of the cardiac valves. CONCLUSION: Cabergoline appears to be safe in patients with prolactinoma up to the cumulative dose of ~300 mg. The screening for valvulopathy should be restricted to those with higher cumulative cabergoline exposure.

Gender differences in macroprolactinomas: a single centre experience.

Macroprolactinomas are the most common functional pituitary tumours. Hypotheses proposed to explain predominance of large tumours in males are: i) diagnostic delay, as hyperprolactinaemia remains under recognised in males and ii) gender-specific difference in tumour proliferation indices. Our study objectives are to compare gender differences in clinical, biochemical, radiological features, management outcomes and cabergoline responsiveness in macroprolactinomas. Drug resistance was defined as failure to achieve prolactin normalisation and >50% reduction in tumour volume with cabergoline (3.5 mg/week dose for minimum 6 months duration). The baseline characteristics of 100 patients (56 females and 44 males) with macroprolactinoma were analysed. Drug responsiveness was analysed in 88 treatment naive patients, excluding 12 post-primary trans-sphenoidal surgery cases. We found that females (30.29±10.39 years) presented at younger mean age than males (35.23±9.91 years) (P<0.01). The most common presenting symptom was hypogonadism (oligo-amenorrhoea/infertility) in females (96.15%) and symptoms of mass effect (headache and visual field defects) in males (93.18%). Baseline mean prolactin levels were significantly lower in females (3094.36±6863.01 ng/ml) than males (7927.07±16 748.1 ng/ml) (P<0.001). Maximal tumour dimension in females (2.49±1.48 cm) was smaller than males (3.93±1.53 cm) (P<0.001). In 88 treatment naïve patients, 27.77% females and 35.29% males had resistant tumours (P=0.48). On subgrouping as per maximum tumour dimension (1.1-2 cm, 2.1-4 cm and >4 cm), gender difference in response rate was insignificant. In conclusion, macroprolactinomas are equally prevalent in both sexes. Macroprolactinomas in males predominantly present with symptoms of mass effects, as against females who present with symptoms of hypogonadism. Males harbor larger tumours but are equally cabergoline responsive as those in females.

Multiple endocrine neoplasia type 1 syndrome: single centre experience from western India.

Multiple endocrine neoplasia type 1 syndrome (MEN1) is a rare autosomal dominant familial cancer syndrome affecting multiple endocrine glands. Published literature on MEN1 from Indian subcontinent is scarce. We report here a case series of MEN1 patients (n = 18) from 14 unrelated families. Retrospective study describing the clinical profile of MEN1 patients from endocrine unit of a tertiary care hospital from western India. Additionally clinical profile of primary hyperparathyroidism (PHPT) in MEN1 patients was compared with that of apparently sporadic PHPT cohort from our centre. Eighteen patients (10 males, 8 females) diagnosed as MEN1 were included. Mean age at diagnosis was 31.5 ± 10.6 years (range 17-54). Incidence of primary hyperparathyroidism (PHPT), pituitary adenoma (PA), and gastro-entero-pancreatic neuroendocrine tumor (GEP-NET) was 94.4, 72.2, and 72.2 %, respectively. GEP-NET was the commonest presenting lesion (33.3 %), followed by PA (27.7 %), PHPT (16.6 %), thymic carcinoid (5.5 %), while 16.6 % cases were identified on family screening. PHPT manifestations (clinical and biochemical) in MEN1 were less severe as compared to those of sporadic PHPT. Contrast enhanced computed tomography (CECT) and (68)Ga-DOTANOC PET/CT were equally sensitive (64.7 vs. 63.5 %) in identifying multiglandular parathyroid disease. Non functioning tumors (NFT) were the most common GEP-NET, followed by insulinoma (5/13, two were metastatic). (68)Ga-DOTANOC PET/CT had higher sensitivity in detecting GEP-NET lesions than CECT (100 vs. 62.5 %). The most common pituitary lesion was prolactinoma, and all were cabergoline responsive. Genetic analysis was available in 13 patients and 11 patients showed mutation in MEN1 gene. The clinical profile of MEN1 in Asian Indian patients is largely comparable to that reported in other cohorts. Peculiar findings of our cohort are predominance of GEP-NET as a presenting manifestation and relatively higher prevalence of insulinoma with higher occurrence of metastatic insulinoma. Clinical and biochemical profile of MEN1 associated PHPT is less severe than that of our sporadic PHPT.

Bilateral adrenal masses: a single-centre experience.

BACKGROUND: Bilateral adrenal masses may have aetiologies like hyperplasia and infiltrative lesions, besides tumours. Hyperplastic and infiltrative lesions may have coexisting hypocortisolism. Bilateral tumours are likely to have hereditary/syndromic associations. The data on clinical profile of bilateral adrenal masses are limited. AIMS: To analyse clinical, biochemical and radiological features, and management outcomes in patients with bilateral adrenal masses. METHODS: Retrospective analysis of 70 patients with bilateral adrenal masses presenting to a single tertiary care endocrine centre from western India (2002-2015). RESULTS: The most common aetiology was pheochromocytoma (40%), followed by tuberculosis (27.1%), primary adrenal lymphoma (PAL) (10%), metastases (5.7%), non-functioning adenomas (4.3%), primary bilateral macronodular adrenal hyperplasia (4.3%), and others (8.6%). Age at presentation was less in patients with pheochromocytoma (33 years) and tuberculosis (41 years) compared with PAL (48 years) and metastases (61 years) (P<0.001). The presenting symptoms for pheochromocytoma were hyperadrenergic spells (54%) and abdominal pain (29%), whereas tuberculosis presented with adrenal insufficiency (AI) (95%). The presenting symptoms for PAL were AI (57%) and abdominal pain (43%), whereas all cases of metastasis had abdominal pain. Mean size of adrenal masses was the largest in lymphoma (5.5cm) followed by pheochromocytoma (4.8cm), metastasis (4cm) and tuberculosis (2.1cm) (P<0.001). Biochemically, most patients with pheochromocytoma (92.8%) had catecholamine excess. Hypocortisolism was common in tuberculosis (100%) and PAL (71.4%) and absent with metastases (P<0.001). CONCLUSION: In evaluation of bilateral adrenal masses, age at presentation, presenting symptoms, lesion size, and biochemical features are helpful in delineating varied underlying aetiologies.

Comparison of 68Ga-DOTANOC PET/CT and contrast-enhanced CT in localisation of tumours in ectopic ACTH syndrome.

BACKGROUND: Localising ectopic adrenocorticotrophic hormone (ACTH) syndrome (EAS) tumour source is challenging. Somatostatin receptor-based PET imaging has shown promising results, but the data is limited to case reports and small case series. We reviewed here the performance of (68)Ga-DOTANOC positron emission tomography (PET)/computed tomography (CT) and contrast-enhanced CT (CECT) in our cohort of 12 consecutive EAS patients. MATERIALS AND METHODS: Retrospective data analysis of 12 consecutive patients of EAS presenting to a single tertiary care centre in a period between January 2013 and December 2014 was done. CECT and (68)Ga-DOTANOC PET/CT were reported (blinded) by an experienced radiologist and a nuclear medicine physician, respectively. The performance of CECT and (68)Ga-DOTANOC PET/CT was compared. RESULTS: Tumours could be localised in 11 out of 12 patients at initial presentation (overt cases), whereas in one patient, tumour remained occult. Thirteen lesions were identified in 11 patients as EAS source (true positives). CECT localised 12 out of these 13 lesions (sensitivity 92.3%) and identified five false-positive lesions (positive predictive value (PPV) 70.5%). Compared with false-positive lesions, true-positive lesions had greater mean contrast enhancement at 60s (33.2 vs 5.6 Hounsfield units (HU)). (68)Ga-DOTANOC PET/CT was able to identify 9 out of 13 lesions (sensitivity 69.2%) and reported no false-positive lesions (PPV 100%). CONCLUSION: CECT remains the first-line investigation in localisation of EAS. The contrast enhancement pattern on CECT can further aid in characterisation of the lesions. (68)Ga-DOTANOC PET/CT can be added to CECT, to enhance positive prediction of the suggestive lesions.