We used a freely available algorithm to perform spectral rotation on the musical stimuli (http://www.fil.ion.ucl.ac.uk/~jcrinion/rotation/blesser3.m). This method has been described in previous works (Blesser, 1972; Scott et al., 2000; Warren et al., 2006; Abrams et al., 2012). The center frequency for spectral rotation was 5512 Hz. This center frequency was chosen so that the rotated frequencies would be within the frequency response range of the fMRI-compatible headphones (20–10 000 Hz). Phase-scrambling was performed by applying

a Fourier transform to each of the four symphonies that constitute the Natural Music stimulus and then randomizing its http://www.selleckchem.com/products/Bleomycin-sulfate.html phase response by adding a random phase shift at every frequency

(Prichard & Theiler, 1994). The phase shifts were obtained by randomly sampling in the interval (0, 2π). This process preserves the power spectrum of each of the four symphonies. Note that, by design, the Phase-Scrambled control stimulus preserves spectral density but not time-dependent fluctuations. We preferred this design as it facilitates a simple and interpretable result: brain structures that show greater ISS for Natural Music compared with the Phase-Scrambled condition are sensitive to the temporal structure of music. Our design therefore forms a necessary starting point for future investigations of more complex time-dependent attributes of musical structure that lead to synchronized responses among subjects, perhaps using a wavelet transform that preserves

both the www.selleckchem.com/products/azd9291.html spectral density and the time-dependent fluctuations in that density. Brain images were acquired on a 3T GE Signa scanner using a standard GE whole head coil (software Lx 8.3). For the Natural Music, Spectrally-Rotated and Phase-Scrambled conditions, images were acquired every 2 s in two runs that lasted 9 min 42 s. The sequence of these stimulus conditions was consistent across listeners: the Natural Music condition was presented first, the Phase-Scrambled condition Thiamine-diphosphate kinase was presented second and the Spectrally-Rotated condition was presented third. While it would have been preferable to have randomized the stimulus presentation order across subjects to control for attention and fatigue, we do not believe that this had a significant effect on the results given that there was vastly greater ISS for the final stimulus condition (Spectral-Rotation) relative to the penultimate stimulus condition (Phase-Scrambled), which would not have occurred had fatigue and attention negatively affected ISS results. Subjects were instructed to attend to all the music and music-like stimuli. To allow for a natural listening experience, we did not provide any additional instructions to the subjects. A custom-built head holder was used to prevent head movement. Twenty-eight axial slices (4.0 mm thick, 0.

The emergence of pandemic influenza A(H1N1) a few months before the 2009 Hajj season was the most recent of these pandemics.9 The lack of an available, effective, and publicly acceptable vaccine in time was just one of

the challenges.10 Restricting age groups at higher risk of complications from attending Hajj activities, as happened for the 2009 Hajj because of the H1N1 influenza threat,10 or even applying Ganetespib cost a ban on individuals from certain countries during the maximum incubation period as happened with severe acute respiratory syndrome (SARS),11 was considered at length, though not fully implemented, and may be imperative for a future Hajj or other mass gathering events to combat future epidemics. This study was conducted to better document whether the several recommendations that were put into practice before the launch of 2009 Hajj season10 were effective in reducing the spread of pandemic influenza A(H1N1) and other viruses among pilgrims. The study’s primary objective

was to determine whether pilgrim attendance at Hajj venues increased risk of acquiring influenza (or other respiratory viruses). An additional AG-014699 cell line objective was to assess compliance with influenza immunization and other recommended preventive measures. Our study uses data collected from pilgrims participating in the 2009 (1430H) Hajj. The main religious activities of the 2009 Hajj season started on November 25, 2009 and continued for 5–6 days, according to each pilgrim’s plan. The 2009 Hajj, similar to the Hajj in other years, included Muslims from all over the world, was one of the world’s largest yearly mass gatherings, and was culturally very diverse, including males and females of different ages, races, educational levels, and socioeconomic levels. Two cross-sectional surveys were conducted at the King Abdulaziz International Airport in Jeddah. It is the main airport used by pilgrims and the Hajj terminal is only used by pilgrims. The first survey was conducted during the week before Hajj activities began on November 25, 2009. As the survey was conducted during a declared influenza A(H1N1) pandemic, all pilgrims arriving at the King Khalid international

Hajj terminal were screened by thermal cameras Dimethyl sulfoxide and questioned about flu symptoms. This was documented in the incoming survey, and results were included in the final analysis; however, departing pilgrims were not questioned about flu symptoms. The primary sampling units were incoming flights. It proved impractical to select flights by probability sampling; instead, survey teams, after finishing one flight, generally selected the next arriving flight. After deplaning, pilgrims waited in an arrival room (a separate one for each flight) before immigration formalities began, grouped around rows of seats. Interviewers randomly selected a row, a person around the row, and a clockwise or counterclockwise direction, and then interviewed pilgrims successively until the room cleared.

no change in the placebo group [14]. Lo et al. showed, in an 18-month placebo-controlled study in which 52 HIV-infected relatively GH-deficient patients received learn more a mean dose of 0.33 mg rhGH/day, that trunk mass and VAT decreased by −0.5 kg and −22 cm2 in the GH group vs. 0.2 kg and −4 cm2 in the placebo group, corresponding to a treatment

effect of a reduction of approximately 5% in trunk fat and 8% in VAT. Notably, rhGH therapy in this setting was accompanied by minor deterioration of glucose tolerance [15]. In the present study, a slightly higher dose of rhGH compared with the regimen used by Lo et al. produced a more pronounced effect on abdominal fat distribution, without a concomitant change in 2-h post-challenge glucose level. Whether or not these results were attributable to counteracting of the glucose metabolic deterioration frequently caused by rhGH therapy, facilitated by a more beneficial effect on fat distribution, as demonstrated in the present study, remains elusive and requires further research. Recently, in a large 26-week placebo-controlled selleck kinase inhibitor study of 404 HIV-infected patients with an accumulation of abdominal fat,

who received a synthetic GH-releasing factor analogue (Tesamorelin), Falutz et al. reported that trunk fat mass and VAT decreased by −1.0 kg and −28 cm2 in the Tesamorelin group vs. 0.4 kg and 5 cm2 in the placebo group, respectively, corresponding to a net treatment effect of an 11% reduction

in trunk fat and a 20% reduction in VAT, which is comparable to the results of the present study. Tesamorelin did not seem to affect glucose metabolism but 23% of the patients discontinued the study, 9% because of adverse events [21]. Patients in the GH group in the present study showed significantly greater increases in lean mass and maximal oxygen uptake compared with patients in the placebo group. This finding is consistent with data from previous studies of pharmacological 3-oxoacyl-(acyl-carrier-protein) reductase rhGH dose regimens in HIV-infected patients, in which subjects showed increases in muscle power, endurance and maximum work output [22–24]. A possible mechanism for the more pronounced effect in the present study, compared with studies in which a comparable dose was used, could relate to the timing of the dose. In healthy individuals, as in HIV-infected patients without fat redistribution, the mean concentration of GH from 12 am to 8 pm is low, compared with the remaining 16 h of the day [25,26]. We found the same to be true of HIV-infected patients with HALS (SB Haugaard, unpublished data). By administering rhGH at the time when endogenous GH secretion is likely to be low, we may have increased the diurnal mean level of GH. In this study, the effect of rhGH on HIV-infected patients regardless of the presence of HALS was investigated. This offered the opportunity to evaluate not only a possible effect of rhGH in patients with HALS vs.

Recent studies in HIV-uninfected persons showed that nonalcoholic fatty liver disease (NAFLD) was independently associated with the presence and extent of coronary disease [19,20]; however, a single study in HIV-infected persons did not find a significant relationship [21]. Given that liver test abnormalities and fatty liver disease are common among HIV-infected persons [22], determining their relationship with coronary artery atherosclerosis may be helpful in the development of screening guidelines selleck compound and risk stratification for underlying cardiovascular disease in this population [14]. Therefore, we evaluated the potential relationship between subclinical

coronary atherosclerosis (as measured using CAC scores) and

fatty liver disease among HIV-infected persons. We enrolled in a cross-sectional study 223 HIV-infected adults who underwent screening CT scans for CAC and fatty liver disease between 9 December 2008 and 1 March 2010. The primary study objective was to examine the association between fatty liver disease and CAC scores among HIV-infected persons, with secondary objectives of evaluating other factors, including metabolic and morphological measures, associated with subclinical coronary atherosclerosis. Inclusion criteria for study participation included documented HIV infection [enzyme-linked immunosorbent assay GSI-IX molecular weight (ELISA) confirmed by western blot], age ≥18 years and a negative pregnancy test among women. Patients with a history of coronary vessel stents were excluded as CAC scores are unreliable in this setting. Participants were military beneficiaries, including active duty members,

retirees and family members. All participants provided written informed consent; the oxyclozanide study was approved by the governing institutional review board and registered at http://ClinicalTrials.gov (NCT00889577). Data collected for this study including imaging, questionnaires, body measurements and blood specimens were collected during the same visit. All participants underwent imaging using a single, multidetector CT scan (Siemens Definition Dual Source CT Scanner; Siemens Medical Solutions, Forsheim, Germany). Prospectively gated axial 3-mm images were obtained at 120 kV during a single breath hold. The scanning protocol captured images with a 330-ms gantry rotation time, an individual detector width of 0.6 mm with a reconstructed section width of 3 mm, and a temporal resolution of 165 ms. No contrast media were administered. CAC scoring was performed on an Aquarius workstation (TeraRecon, San Mateo, CA, USA) and scores were calculated as the sum of all lesions in each of the coronary arteries using Agatston units, as previously described [23]. A CAC score of >0 was considered positive for detectable calcium and a score of >100 was considered clinically significant.

Most often, the interaction occurs within the 5′-noncoding region of the mRNA target or at the beginning of the message’s coding sequence. In many cases, these interactions are facilitated by the highly conserved bacterial sRNA chaperone protein Hfq (Valentin-Hansen et al., 2004). A homologue of Hfq is present in almost half of all sequenced Gram-negative and Gram-positive species, and in at least one archaeon (Sun et al., 2002; Nielsen et al., 2007; Soppa et al., 2009; Straub et al., 2009). At least 15 of 46 known sRNAs in E. coli interact with Hfq (Zhang et al., 2003). In Pifithrin-�� cost E. coli, the Hfq chaperone is critical for the stability, function,

and base pairing of the iron-responsive RyhB sRNA. The 90-nucleotide long RyhB downregulates a set of iron-storage and iron-using proteins when iron is limiting; RyhB is itself negatively regulated by the Fur (ferric uptake regulator) protein (Masse & Gottesman, 2002; Tjaden et al., 2006; Desnoyers et al., 2009). Analysis of the N. europaea genome revealed that, like other bacteria, it contains a homologue of hfq denoted as NE1287 (Chain et al., 2003). This may suggest the existence of a similar mechanism utilizing sRNAs in N. europaea. In this study, computational analyses of the N. europaea genome and N. europaea microarray data were used to search for evidence of sRNA genes in this bacterium (Tjaden, 2008a, b). Fifteen psRNAs were identified.

We experimentally confirmed the transcription Navitoclax in vivo of two psRNAs under selected treatments and analyzed the transcriptional profiles of possible target genes that may be under their regulation. This is the first experimental evidence for expression of sRNA

genes in an ammonia-oxidizing bacterium. Batch cultures of wild-type N. europaea were grown to the late log phase as described (Wei Guanylate cyclase 2C et al., 2006a, b). Treatments with chloromethane and chloroform have been reported in our previous research (Gvakharia et al., 2007). The N. europaea fur-deficient mutant strain (fur:kanP) was created with a kanamycin-resistance cassette insertion in the promoter region of the fur homologue encoded by NE0616. Construction of the fur:kanP mutant of N. europaea is described elsewhere (N. Vajrala, L. Sayavedra-Soto & D. Arp, unpublished data). Iron-replete and iron-depleted conditions were used to grow wild-type N. europaea and the N. europaea fur:kanP strain to the late log phase as described previously (N. Vajrala, L. Sayavedra-Soto & D. Arp, unpublished data). Total RNA was extracted and purified with RNeasy® Mini Kit (cat. no. 74104) from Qiagen (MD) according to the manufacturer’s recommendations. cDNA was synthesized with the IScript™ cDNA Synthesis Kit (Bio-Rad Laboratories Inc., Hercules, CA) with RNA extracted from cells that were exposed to chloroform or chloromethane, or from cells that were grown in iron-replete or iron-depleted media. Transcript levels were measured by real-time PCR with IQ™ SYBR Green Supermix (Bio-Rad).

The downregulation of the aflatoxin cluster at higher temperatures may be explained by the selleck chemicals llc low levels of AflR as well as by inhibitor binding due to reduced levels of AflS. This is in contrast to previous microarray studies (OBrian et al., 2007), which reported that the aflR and aflS transcripts were expressed at about the same level under both temperature conditions. This discrepancy may be due to the lower sensitivity

associated with microarray gene expression studies. Unlike the aflatoxin cluster, cluster #55, which controls the biosynthesis of CPA (Chang et al., 2009), was expressed under both conditions, although the expression levels were much higher at the lower temperature (Table 2). This indicates that the two adjacent clusters are regulated by slightly different mechanisms. No putative transcription factor genes have been found in this cluster. CPA is typically produced under the same conditions that favor aflatoxin production. CPA is known to be produced at both high and low growth temperatures, although the 24-h time point may not be its peak production time. Further studies with multiple time points may be needed to elucidate the mechanism of transcriptional regulation of this cluster. Traditionally, researchers relied on microarray technology to

reveal genes required for toxin biosynthesis and regulation in Aspergillus species (OBrian et al., 2007; Wilkinson et al., 2007a, b). However, due to the sensitivity Obatoclax Mesylate (GX15-070) problem, PLX4032 in vivo microarrays are not the best technology to detect expression levels of regulatory genes, such as aflR and aflS. This study demonstrates that the RNA-Seq approach can profile a cell’s entire transcriptome with almost infinite resolution. The obtained data defined conclusively the complete aflatoxin cluster consisting of 30 genes, which are coordinately regulated. Having the accurate measurement of the aflR and aflS transcript abundance levels allowed us to conclude that high temperature negatively affects aflatoxin production by turning

down transcription of aflR and aflS. We would like to thank Yan Yu, Sana Scherbakova and Karen Beeson from JCVI for their superb technical assistance during library preparation and sequencing. J.Y. and N.D.F. contributed equally to this work. Table S1. Illumina read statistics. Table S2. Gene expression of the 55 predicted secondary metabolism gene clusters in Aspergillus flavus at temperature 30 vs. 37°C. Please note: Wiley-Blackwell is not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article. “
“Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) represents a simple reliable approach for rapid bacterial identification based on specific peptide/protein fingerprints.

An environmental sample of mixed free-living eukaryotes suspended in f/2 media from Glebe Harbour, Sydney, Australia, was used in probe specificity studies. A sample of Symbiodinium sp. (Dinophyceae) was from Acropora tenuis (Cnidaria) obtained from a coral store (Kim’s Aquatic

World, Ashfield, Australia). Approximately 106 cells of C. velia (from 2-week-old mid-exponential cultures), resuspended in 1 mL of PBS (pH 7.2), were used throughout this study. Cells were fixed in freshly made 6% paraformaldehyde at 4 °C for 1 h. The paraformaldehyde was removed by centrifugation and cells resuspended with PBS. Permeabilization was achieved by resuspending the C. velia cell pellet Dasatinib solubility dmso in 1 mL of 5% DTAB/PBS and incubating at 80 °C for 30 min. This was followed by another washing step with PBS to remove all traces of DTAB from the cells. The pelleted cells were resuspended

in 500 µL of PBS, from which 20 µL aliquots were placed on glass microscope slides. All centrifugation steps were performed in an Eppendorf 5415D microcentrifuge at 16 000 g. Roxadustat concentration Cell aliquots were allowed to dry, following which hybridizations were conducted directly on the slide within a humidifying chamber. The hybridization buffer [0.9 M NaCl, 20 mmol-1 Tris–HCl, 0.05% sodium-dodecylsulphate (SDS), pH 7.2] contained a final probe concentration of 1 pmol mL-1. The overall hybridization signal after 1-, 1.5-, 4-, and 15-h incubations at 48 °C with the probe was evaluated. Following hybridization, all slides were rinsed with

2 mL of prewarmed PBS to wash away any unbound probe. Prior to microscopic examination, Fluoroshield (Sigma-Aldrich, Australia) was added to the sample slides. Fluorescent emissions from all treated and untreated control samples were observed using an Olympus BX60 (Olympus, Australia) equipped for FITC detection (excitation maximum 488 nm and emission maximum 525 nm; filter cube U-MWIB, excitation range 460–490 nm, dichroic mirror 505 nm, and a long pass emission range of > 515 nm). UV autofluorescence was detected using filter cube U-MWU (excitation 330–385 nm, dichroic mirror 400 nm, emission > 420 nm). Photographs of the FITC fluorescence signals Protein kinase N1 were taken with an Olympus DP70 colour camera. Three criteria were used to classify cells as FISH-positive. Firstly, the characteristic bright green fluorescence signal of FITC had to be observed in positive cells. Secondly, higher signal intensities had to be detected in probed samples compared to un-probed samples, and this signal had to originate from the cell’s cytoplasm. Thirdly, we assessed the difference in fluorescence pattern between probed and un-probed cells, because C. velia emits natural autofluorescence. Presence of C. velia in the samples was confirmed using bright microscopy as well as by UV autofluorescence (filter cube U-MWU). A USB2000 + UV-VIS spectrometer (Ocean Optics, Inc.

The V. cholerae strain MCV09 characterized in the present investigation was isolated from a patient who died due to severe dehydration in the Medical College Hospital, Trivandrum, India. The strain was maintained as peptone agar stab cultures at room temperature and stocked in tryptic soy broth with 30% glycerol at −70 °C till further use. Initial biochemical screening was performed to identify the strain, followed by serological analysis (Polyclonal O1, monospecific Ogawa

and Inaba antisera supplied by the World Health Organization (WHO), Regional Office of South East Asia, New Delhi, India). The strains of V. cholerae, 569B (O1 classical Inaba) and VC20 (O1 El Tor Ogawa) were used as controls for agglutination. The V. cholerae O1 El Tor Ogawa strain,

check details TV107, served as a control for detection of int and drug resistance genes. The V. cholerae O139 strain, MO10, was used as a control for amplification of attP attachment sites. The O1 El Tor Ogawa strain, MCV08 (Trivandrum, India), and environmental Toxigenic O1 El Tor Ogawa strain, A880 (Alappuzha, India), were also used for amplification of attP sites. The rifampicin-resistant Escherichia BTK inhibitor coli strain (DH5α) was used as a recipient for the conjugation experiment. The test strain was examined for resistance to 15 major antibiotics using commercial discs (Himedia, Bombay, India) according to the interpretation criteria recommended by the WHO (1993). Escherichia coli ATCC 25922 was used for quality control for the antibiotic resistance assay. The MIC value for ciprofloxacin, nalidixic acid, tetracycline and trimethoprim was determined using the E-test (AB-BIODISK). The conjugation experiment was carried

out on Luria–Bertani (LB) agar plates as described previously (Waldor et al., 1996). For all PCR assays except detection of attP sites, cell lysates were used as template DNA. acetylcholine For the amplification of attP sites, a single bacterial colony was picked from an LB agar plate and directly added to the PCR mixture. The lists of primers used and their sources are given in Table 1. The presence of int was detected using the method of Ahmed et al. (2005). The PCR cycle for the attP site consisted of an initial denaturation at 94 °C for 4 min, followed by 30 cycles of denaturation for 30 s at 94 °C, primer annealing for 30 s at 50 °C, extension for 45 s at 72 °C and a final extension at 72 °C for 10 min. The associated drug resistance genes viz dfrA1, strB and sul2 were examined by specific PCR (Falbo et al., 1999; Hochhut et al., 2001; Ramachandran et al., 2007). The amplification of the QRDR of gyrA, gyrB, parC and parE was performed as described by Baranwal et al. (2002). The amplified products were separated on a 1% agarose gel, stained with ethidium bromide and visualized using a Fluor-S-MultiImager (Bio-Rad).

In agreement with previous studies (Schenberg et al., 2000; Schimitel et al., 2012), these data add fresh evidence of the separate processing of DPAG-evoked somatic (freezing and flight) and pelvic (micturition and defecation) responses. Interestingly, urges for micturition and defecation are neither experienced by patients during panic attacks (Goetz et al., 1994, 1996) nor recognised as symptoms typical of clinical panic (WHO, 1993;

APA, 2000). Lastly, comparisons of the thresholds of FS, ES and IS groups are validated by the remarkable similarity of stimulated sites. Indeed, electrodes were mostly localised in DPAG (76.9%) and nearby regions of superior Cabozantinib solubility dmso colliculus (21.5%) that cannot be discriminated by electrical stimulation with sine-wave pulses (Bittencourt et al., 2004; Schenberg et al., 2005). Evidence amassed over recent decades suggests that subjects exposed to uncontrollable stress develop a depression-like syndrome see more characterised by a decrease in motivation to respond to the same or other aversive stimuli, a cognitive deficit (learned helplessness) that interferes with the learning of a new escape task in a heterotypical context, and emotion and mood effects, including the early increase in anxiety and the late development of depression upon prolonged exposure to uncontrollable stress. Data

from yoked experiments presented compelling evidence that these effects result from the subject’s learning that stress is beyond control and not from the stressor aversiveness on its own (Maier & Seligman, 1976; Maier, 1984; Maier & Watkins, 1998, 2005). Similarly, the FST is a widespread procedure for screening of potential antidepressants (Porsolt et al., 1991) that is based on the assumption that floating is an expression of a depressed mood brought about by inescapable stress. Although these models are both based on learning, they differ in other respects. Thus, whereas the learned helplessness appears to be the

Loperamide result of the subject’s associative learning that responses are equally rewarded or punished (Seligman & Beagley, 1975; Maier & Seligman, 1976), the FST is an extinction-like non-associative learning whereby the subject learns that swimming is a futile effort in successfully cope with stress (i.e., escape from the water tank). Consequently, floating has also been interpreted as an energy-sparing tactic (West, 1990). Regardless of whether or not uncontrollable stress produces a true depressed mood, IS inhibition of escape responses to foot-shock and intracranial stimulus implicates the DPAG as a likely substrate of both responses. Indeed, although most researchers associate the outcome of uncontrollable stress with putative changes in hippocampus (Leshner & Segal, 1979; Petty et al., 1993, 1994; Amat et al., 1998; Joca et al., 2003, 2006; Malberg & Duman, 2003; Zhou et al., 2008), amygdala (Maier et al., 1993; Amat et al.

This is a case report of a young man who presented as an emergency with type 1 diabetes, adrenal failure and primary hypothyroidism. It highlights the importance of considering the diagnosis of adrenal failure in an individual presenting with type 1 diabetes who does not respond as expected to initial treatment, and of looking for other autoimmune conditions at the initial presentation. JL, a 35-year-old gentleman, presented at emergency with a three-week history of feeling generally unwell. Specifically he had symptoms of malaise, tiredness and feeling faint. He had also noticed increased thirst, drinking more than 3L/day, urinary frequency, leg cramps and reduced exercise tolerance. For the preceding week

he had been troubled Seliciclib by nausea and vomiting to the extent that he was unable to eat but had been able to drink. The day prior to admission he had developed abdominal pain and diarrhoea. During this time ABT-199 molecular weight period he had lost weight but there were no other associated symptoms or signs. He had recently visited his GP for the treatment of oral thrush but a capillary blood glucose was normal at that

time. He had a past medical history of mild asthma and used Ventolin infrequently. There was a family history of autoimmune disease; a cousin with type 1 diabetes and an aunt with a ‘thyroid problem’. He worked as an engineer, was a non-smoker and drank six units of alcohol per week. On examination, he was noted to be thin, dehydrated with extensive oral thrush. He was tanned but there was no pigmentation of the buccal mucosa or palmar creases. His temperature

was 35.5oC. He was cardiovascularly stable with a pulse of 90bpm and blood pressure 141/61mmHg but he was unable to stand without feeling dizzy. Cardiovascular and respiratory examination was normal and his abdomen was soft but tender to light palpation with normal bowel sounds and no rebound or guarding. A capillary blood glucose was 20.7mmol/L. Arterial blood gases were done and were normal (pH 7.43, pCO2 4.2kPa, pO2 10.6kPa, BE -2.6). Urine dipstick was positive for ketones (+++) and glucose (+++). After the initial assessment the impression was that he had newly diagnosed diabetes but had not developed diabetic ketoacidosis, he was dehydrated and that his abdominal Thymidine kinase symptoms may have been related to his diabetes but that a polyendocrine syndrome should be considered. An insulin infusion and intravenous fluids were commenced. Blood was sent for urea and electrolytes, glucose, thyroid function, liver function, calcium, amylase and cortisol. He was reviewed four hours later. At this time despite his glucose normalising and fluid resuscitation having occurred his pulse had increased and his blood pressure had dropped (Figure 1). He looked worse and did not feel any better despite appropriate treatment. An intravenous venous short synacthen test was performed with a baseline ACTH.