This may be of particular importance since previously published analysis of human

iliac crest biopsies from osteoporotic and non-osteoporotic (based on the classical clinical criteria) patients sustaining atraumatic or low trauma fragility fractures shows similar results as far as collagen cross-link ratio is concerned [17] and [18]. Additionally, the results were obtained in vertebrae, and the incidence of vertebral fractures in osteoporosis is twice that of hip fractures [70], although caution should be exercised as an animal model was employed in the present study. These results become even more important in view of the recent clinical reports, which have correlated plasma homocysteine levels and bone fragility [12], [13], [14] and [15] when it is noted that the mechanism by which homocysteine

and β-APN block collagen GSK269962 supplier cross-link formation is analogous. selleck chemicals llc None of the authors have any conflict of interest. The authors thank Gerda Dinst, Sabrina Thon, Phaedra Messmer, and Daniela Gabriel for careful sample preparations and qBEI measurements at the Bone Material Laboratory of the Ludwig Boltzmann-Institute of Osteology, Vienna, Austria. This study was supported by the Allgemeine Unfallversicherungsanstalt (AUVA), research funds of the Austrian workers compensation board; the Wiener Gebietskrankenkasse (WGKK), Viennese sickness insurance funds; and the Fonds zur Foederung der wissenschaftlichen Forschung (FWF); the Division of Periodontology, Ohio State University; a research grant from the Alliance for Better Bone Health (to EPP); NIH grant AR046505 (to EPP). “
“The incidence of vertebral fracture increases linearly with aging and is significantly correlated with declining bone mineral density mafosfamide (BMD). The incidence of hip fracture, on the other hand, rises exponentially

with aging, suggesting that age-related factors other than BMD contribute greatly to the fragility of the proximal femur. Hip fractures cause substantial disability and are associated with a high rate of death among elderly women [1]. Because vertebral fracture is the most common of osteoporotic fractures, the efficacy of anti-osteoporotic agents is judged in clinical trials by evaluating the incidence of vertebral fracture. The incidence of hip fracture is much lower than that of vertebral fracture, especially in elderly Japanese, and in clinical trials of anti-osteoporotic agents hip fracture is assessed as a secondary endpoint or as one of the non-vertebral fractures. However, in view of the increasing incidence of hip fracture in the Japanese population [2] and its consequences of seriously reducing quality of life (QOL) [3], measures to prevent hip fracture are of paramount importance.

The total percentage of identified saturated fatty acids was 40.53, 31.45 and 38.92% and for the unsaturated fatty acids was 37.29, 37.17 and 51.54% in the spring, summer

and autumn, respectively, with approximate ratios between the saturated and unsaturated fatty acids of 1.09, 0.85 and 0.76. For the individual fatty acids, the major saturated fatty acids were myristic acid (C13:0) and palmitic acid (C16:0) in both the spring and summer, whereas pentadecyclic acid (C15:0) and palmitic acid (C16:0) were the major saturated fatty acids http://www.selleckchem.com/products/dabrafenib-gsk2118436.html in autumn. By contrast, docosahexaenoic acid (C22:6) and pentadecenoic acid (C15:1) were the major unsaturated fatty acids during the different seasons. Table 3 shows the variation in total lipid content of U. linza in the spring, summer and autumn. The highest percentage was 4.14% of dry matter in the spring. Comparable percentages of 3.76 INK 128 and 3.20% were observed in

the summer and autumn, respectively. Table 3 also shows an overview of the fatty acid profiles of the alga. In this study, we identified several individual fatty acids during various seasons with different concentrations. The saturated fatty acids were primarily C16:0, with 56.13, 38.10 and 48.44% in the spring, summer and autumn, respectively. By contrast, the unsaturated fatty acids were mainly C22:6, with 9.16, 10.05 and 4.82%, and C15:1, with 4.92, 3.60 and 0.099% in the spring, summer and autumn, respectively. The sum of the saturated fatty acids of these seasons was 71.42, 51.20 and 63.63%, respectively, whereas the sum Inositol monophosphatase 1 of the unsaturated fatty acids was 18.31, 20.05 and 24.90%, respectively. The total lipid content of P. pavonica during different seasons is tabulated in Table 4. The lipid content

in terms of dry weight was 3.01, 2.18 and 1.82% in the spring, summer and autumn, respectively. The fatty acid composition varied among the different seasons ( Table 4). Autumn had the highest saturated fatty acid content as a percentage of the dry weight (74.26%), followed by summer (67.36%) and spring (58.38%). Moreover, similar results were obtained for the unsaturated fatty acid contents with a percentage of 22.02 in the autumn, 21.49 in the summer and 14.41 in the spring. The percentages of the saturated fatty acid C16:0 were 48.64, 45.59 and 42.61%, and the percentages of the unsaturated fatty acid C22:6 were 8.84, 6.12 and 5.99% from autumn to summer to spring, respectively. Principal component analysis of the total fatty acids data, sum of the saturated fatty acids and sum of the unsaturated fatty acids demonstrated a statistical distinction between the three seaweeds. These algae showed high factor loading on PCA1 and PCA2. A bi-plot of the total fatty acids data matrix (Fig. 1a) explained 98.5% of the variances (64.5% and 34%). When PCA was applied to the saturated fatty acids (Fig. 1b), the model explained 99% of the total variances (62.4% and 36.5%). For the unsaturated fatty acids (Fig.

To generate

a BSMV:TaWAK5 construct, a 298 bp sequence of TaWAK5 (from nucleotide position 1913 to 2211 in the TaWAK5 cDNA sequence) was amplified from the cDNA sequence for TaWAK5 selleck inhibitor from the genotype CI12633 with the primers TaWAK5-VIGS-F/TaWAK5-VIGS-R. PCR-amplified cDNA fragments were digested with Pac I and Not I, then ligated into the BSMV:RNAγ vector digested with Pac I-Not I, resulting in the recombinant construct RNAγ:TaWAK5-as. Following a previously described protocol [32], the tripartite cDNA chains of BMSV:TaWAK5, or the control virus BMSV:GFP genome, were separately transcribed into the RNAs, then mixed and used to infect CI12633 plants at the 2-leaf stage. At the same time, CI12633 plants were inoculated with only the buffer without virus. Hereafter, these plants treated only with buffer are referred to as mock treatments. The 4th leaves of the inoculated seedlings were collected and analyzed for the virus infection based on the RNA transcript presence of the BSMV coat protein gene using Tofacitinib primers BSMV-CP-F/BSMV-CP-R. These tissues were also evaluated for changes in TaWAK5 expression with primers TaWAK5-Q-F/TaWAK5-Q-R

at 10 days after BSMV infection. For R. cerealis inoculation, the fungus was cultured on potato dextrose agar at 25 °C for 10 days, then 1 cm2 plugs from the edge of R. cerealis colonies were placed into liquid PDA medium and cultured at 25 °C for 2 weeks, to develop the mycelia. The 4th base sheath of wheat plants was inoculated with 15 μL of the R. cerealis liquid culture at 20 days after BSMV virus inoculation. Inoculated plants were grown at 90% relative humidity for 4 days. Sharp eyespot symptoms were observed respectively at 14 days and 40 days after fungal inoculation. These are the times when sharp eyespot symptoms are normally present at the infected sheaths and stems, respectively, of the susceptible cultivar Wenmai 6. RT-PCR was performed with 20 μL reaction volumes from the TaKaRa Inc. kit containing 1 × PCR buffer, 2.0 μL 10 × first strand cDNA,

150 μmol L− 1 of each dNTP, and 1 U Taq polymerase, plus 0.25 μmol L− 1 of each primer. The program used was as follows: initial denaturation at 94 °C for 5 min; followed by 30 cycles Oxymatrine of 30 s at 94 °C, 30 s at 60 °C, and 30 s at 72 °C; and final extension at 72 °C for 5 min. The PCR products were detected on 2% agarose gels. In all the semi-quantitative RT-PCR experiments, wheat elongation factor 1 alpha-subunit (TaEF-1a) was used to normalize the cDNA contents among various samples. qRT-PCR was performed using SYBR Green I Master Mix from TaKaRa Inc. in a volume of 25 μL on an ABI 7300 RT-PCR system (Applied Biosystems Corp.). Reactions were set up with the following thermocycling profile: 95 °C for 5 min, followed by 41 cycles of 95 °C for 15 s and 60 °C for 31 s. The products were continuously examined with a melting curve analysis program.

The authors are grateful to J. Hutchings, CT Marshall and B Bogstad for comments and discussions on previous Selleck Lumacaftor versions of this manuscript, to P Sandberg and the Norwegian Fisheries Directorate for kindly providing the cost data and for discussions on the cod fishery, and to OR Godø for help with cod data. The authors are also grateful to the Research Computing Services at the University of Oslo for access to the computing resources required for this study. Funding was provided by the Norwegian Research Council (AME, DJD, MH, NCS), the European Commission

through the Specific Targeted Research Programme on Fisheries-induced Evolution (FinE, SSP-2006–044276) (AME, AR, MH, UD, NCS), the European Commission through the Marie Curie Research Training Network Selleck FDA approved Drug Library on Fisheries-induced Adaptive Change in Exploited Stocks (FishACE, MRTN-CT-2004–005578) (ESD, MH, UD), and through

the Marie Curie Programme (PIEF-GA-2010–274356) (AR), the Bergen Research Foundation (MH), the European Science Foundation (UD), the Austrian Science Fund (FWF: TECT I-106 G11, UD), the Austrian Ministry for Science and Research (UD), and the Vienna Science and Technology Fund (UD). Naturally, this article does not necessarily reflect the views of the European Commission and does not anticipate the Commission’s future policy in this area. “
“Marine spatial planning (MSP) is “a public process of analysing and allocating the spatial and temporal distribution of human activities in marine areas to achieve ecological, economic, and social objectives that are usually specified through a political process” [1]. MSP is often considered

Thiamet G a practical strategy to implement the ecosystem-based approach to the conservation and management of marine resources [2] and [3]. The policy landscape for MSP in Europe is still a young and emergent one. The concept of MSP is relatively new and some important policy drivers, such as the Marine Strategy Framework Directive (MSFD, Directive 2008/56/EC) and Integrated Maritime Policy (IMP, COM(2007) 575), came into force relatively recently. As an emergent policy landscape, it is also subject to on-going political and legislative changes that may significantly affect its future development. The European Union (EU) has recently adopted a new legislative procedure under the Lisbon Treaty (2009), which may affect the adoption of new policies or the revision of existing ones. A proposal for a new regulation under the Common Fisheries Policy (CFP) is currently being deliberated upon, following the new procedure as established in the Lisbon Treaty. New policy instruments on MSP are being explored by the European Commission (hereafter the ‘Commission’) as a means of promoting a common approach to MSP across Europe [4]. Such major policy reforms and new developments may significantly shape the vision and direction of MSP in Europe in the decades to come.

Krieger et al. have synthesised both transferrin-targeted and non-targeted PEGylated cisplatin-containing liposomes. Free CDDP displayed strong differences in cytotoxicity towards A2780 and resistant A2780cis breast cancer cells, whereas buy Idelalisib the liposomes all displayed comparable cytotoxicity in both cell lines. Thus, these liposomes are potential carriers to treat cisplatin-resistant tumours [ 30]. Liposomes encapsulating oxaliplatin and with bound human transferrin (23) show potency in vitro towards colon, pancreatic and neuroendocrine human

cancer cells [ 31]. The F3 peptide is a 31-residue fragment of the high mobility group protein (HMGN2) which has potential to bind to the nucleolin protein expressed on both the surface of endothelial and tumour cells. Winer et al. have encapsulated CDDP Nutlin-3a purchase in a polyacrylamide nanoparticle functionalised with the F3 peptide. Complex 24 displayed significant cytotoxicity towards tumour endothethial cells (TECs). Using a model of human tumour vasculature, it was shown that the F3-Cis-NPs bind to human

tumour vessels [ 32]. These results suggest the safety of F3-Cis-Nps and effectiveness for targeting TECs. Various peptide sequences can bind preferentially to tumour cells and thus can act as cancer targeting ligands. For example, chlorotoxin (CTX), a 36-amino acid peptide which blocks small-conductance chloride channels, binds to functional proteins such as matrix metalloproteinase-2 (MMP2) (overexpressed in glioma and related cancers) and chloride ion channels overexpressed in different types of cancers. CTX has been conjugated to a PtIV-succinato complex (25) as a prodrug for delivery of cisplatin. The cytotoxicity of 25 towards MCF-7 breast, A549 lung and HeLa cervical cancer cells was less than CDDP but greater than both the PtIV precursor and CTX alone. The kinetic inertness of the PtIV complex probably contributes to its reduced activity [33•].

Translocator proteins (TSPOs) are peripheral benzodiazepine receptors (PBRs) overexpressed L-NAME HCl in both human and rat glioma cells. Margiotta et al. have conjugated cis-PtII(NH3)(X)2 to TSPO-binding ligands ( Figure 2k). Such conjugates showed potency equivalent to that of cisplatin through apoptosis. The iodido complex 26 was slightly more potent than the chlorido derivative 27; however, unlike CDDP, both complexes were equally active towards sensitive A2780 and resistant A2780cis breast cancer cells [ 34•]. The targeting sequence NGR (Asn-Gly-Arg) can bind specifically to murine breast cancer cells. Two peptide conjugates of the type cyclic mPEG-CNGRC-Pt and cyclic mPEG-CNGRC-Pten (Figure 2l, 28 and 29) showed selective delivery and more effective destruction of PC-3 prostate (CD13 +ve) cells than the untargeted platinum complex, carboplatin [35].

Therefore, the other approach given by Hirst et al. (2005), which allows the use of such mean stage weight data, is included in our calculations. This correction of the ‘Moult Rate’ method (see equation (22) in their paper) is described by equation(3)

ln(Wi+1/Wi)/(Di+Di+1)/2=gi→i+1+[lnho(gi→i+1,Di+1)−lnho(gi→i+1,Di)]/(Di+Di+1)/2, where the function ho(g, D) is given by ho(g, D) = [exp(gD/2) − exp(−gD/2)]/(gD). click here Hence, this equation describes growth using arithmetic mean weights and stage durations of consecutive (moulting) stages ( Hirst et al. 2005). According to the data for Di at 15°C and excess food, the maximum growth rates of T. longicornis for nauplii, C1–C3 and C3–C5 were obtained by the numerical solution of equation (3), where Wi is the mean body weight for successive stages, Di is the stage duration and gi→i+1 is an unknown quantity. Equation (3) was solved by following the procedure below to give gi→i+1: step 1: read Wi, Wi+1, Di, Di+1; In this paper, the mean growth rate of T. longicornis for three developmental stages (N1–C1, C1–C3 and C3–C5) as a function of food concentration at 15°C is given by the equation: equation(4) gi=gmaxfte1−exp(−(Food−Foodo)kFood), Navitoclax where gmax (% of weight day−1) is the maximum growth rate at 15°C and excess food (see equation (3)), Food (mgC m−3) is the food concentration, Foodo

(mgC m−3) is the value of Food at which g = 0, and kFood (mgC m−3) is the half-saturation constant, since gmax/kFood for Food is slightly greater than Foodo, and fte is a function of

temperature. For each stage, Foodo = 0 and fte = 1 at T = 15°C; however, kFood lies in the 90–140 mgC m−3 range and is described by: kFood=(−0.0001(logFood)3+0.0016(logFood)2−0.0068logFood+0.0162)−1 for the naupliar stage (r2 = 0.9607), and kFood=(−0.0001(logFood)3+0.0019(logFood)2−0.0082logFood+0.0173)−1 for the copepodid stages (r2 = 0.9519). Growth rate values in the developmental classes at 15°C for different food supplies found by Klein Breteler et al. (1982) and computed here with equation (4) are shown in Figure Cell Penetrating Peptide 4. The dependence of the growth rate on temperature can be described by the equation: equation(5) fte=ft1ft2, where ft1=t1t2T,ft2=1T≤To1−(T−Tot3To)P1T≥To and fte = 1 for T = To. The function fte for temperatures over To is modified by part of ft2. In this paper, the influence of temperature on growth rate is described by equation (5) representing a Q10 value of 2.274 applicable to the temperature range of 5–15°C. The temperature coefficient Q10 was calculated according to the data given by Klein Breteler & Gonzalez (1986). The t2 coefficient was equal to 1.0856 based on Q10. Coefficient t1 was calculated so that fte was equal to 1 at 15°C; t1 was therefore equal to 0.292. Coefficients t1 and t2 were identical for all stages.

The chromosome damage observed in genotoxic assays performed with animal venoms showed that these toxins may possibly be used in the development of new therapeutic strategies for cancer control. There are some interesting examples with venoms from scorpions, bees and snakes (Zargan et al., 2011; Lee MK-2206 ic50 et al., 2007; Varanda et al., 1999; Wang et al., 2000; Wang and Groopman, 1999; Lerda et al., 2005; Brugger et al., 2006; Dönmez-Altuntas et al., 2007). The obtained results suggest that different toxins could induce breakages in DNA by different ways, which is corroborated by the results

obtained with BthTX-I, BthTX-II and BatxLAAO, which resulted in permanent breakages likely to be observed in the micronucleus assay. Conversely, the high rate of DNA breakage induced by BjussuMP-II is not maintained after the action of cell repair system, as observed in the micronucleus assay. Interesting, BthTX-I is an enzymatically inactive PLA2-like enzyme and showed similar mutagenic AZD6244 effect to BthTX-II, which is

catalytically active, suggesting that the genotoxicity is not related to the catalytic activity. The mechanisms of action of snake venom genotoxicity are not yet elucidated. The production of free radicals induced by some toxins is a valuable hypothesis that should be considered, since they participate in inflammatory processes and the mediators are intimately related to the oxidative stress. However, the apoptosis induction cannot be discarded considering the high number of published works describing this effect for different classes of toxins such as LAAOs,

metalloproteases and PLA2s (Iwanaga and Suzuki, 1979; Kang et al., 2011). Corroborating this hypothesis, the induction of oxidative stress has been described for some snake venoms and isolated toxins (Zhang and Cui, 2007; Yamasaki et al., 2008). This effect can also be associated with the DNA damage induced by venom toxins, through the formation of free radicals that could induce genotoxicity and, in high levels, even mutagenicity or cellular apoptosis. The induction also of micronuclei and DNA damage of lymphocytes observed after cell exposure to different concentrations of an LAAO from B. atrox showed in the present work are an indication that substantiates the hypothesis cited above. Future experiments using anti-oxidant agents, together with the toxins could elucidate the suggested mechanism, as showed for zearalenone ( Ouanes et al., 2003). The venoms from B. brazili and B. atrox did not induce DNA damage when assayed by the comet test, however, both showed genotoxic potential when assayed by the micronucleus test.

“
“Piano playing requires the accurate coordination of finger movements on both hands. Each finger movement has to be sequenced in the right order and executed with the right pace relative to finger movements on the same or the other hand. Skilled piano players can rapidly sequence these movements in MK-2206 mouse case of playing a familiar piece, however, in case of an unfamiliar piece, their movements become slower, less precise and seem to require more attention (Drake and Palmer, 2000 and Lotze et al., 2003). Previous studies suggest that different processes underlie the execution of familiar as compared to unfamiliar sequences of movements (e.g. Hikosaka et al., 1999,

Ivry, 1996 and Verwey, 2001). These processes can be studied by using so-called discrete movement sequences, which are relatively short sequences of movements usually consisting of three up to six

key presses with a clear start- and endpoint. The learning of these sequences has been described in several models, and is indeed thought to develop from an initial controlled attentive phase to a second automatic phase in which attention is no longer needed (e.g., Cohen et al., AZD6244 price 1990, Doyon and Benali, 2005 and Verwey, 2001). In our study, we examined whether these different processes underlying the execution of familiar and unfamiliar sequences of movements are already active while preparing these movements, by focusing on several measures derived from the electroencephalogram (EEG). Sequence learning can be studied by using the discrete sequence production (DSP) task. In a typical DSP task

discrete sequences are practiced by responding also to series of three to six key-specific stimuli. All stimuli, apart from the first stimulus, are presented immediately after the response to a previous stimulus. Since sequences have a limited length and a clear beginning and end, the DSP task is especially suitable for studying hierarchical control and segmentation (Rhodes, Bullock, Verwey, Averbaeck, & Page, 2004). Behavioral results of the DSP task show that execution gets faster with practice and that some keypresses within a sequence are executed consistently slower than other keypresses, which is assumed to index the segmentation of motor sequences (Verwey, 1996). As segments consolidate with practice, it is suggested that each segment involves the execution of a motor chunk (Verwey & Eikelboom, 2003). With practice, chunking can speed up the selection and initiation of familiar segments (Verwey, 1999). In motor sequencing tasks like the DSP task, anticipation and programming of the next motor response may already start while executing the previous response (Eimer, Goschke, Schlaghecken, & Sturmer, 1996).

g., prey and body condition studies, Dean et al., 2002; biomarker studies, Ballachey et al., 2002 and Miles et al., 2012). Survey data from throughout WPWS indicated a widespread decline in numbers in 2002 (Bodkin et al., 2011). The reasons for this decline are unknown, Ceritinib mw but it appears that numbers returned to previous levels the following year at most sites other than NKI. NKI may have taken longer to recover (Fig. 3b) because the regrowth of the small population there was limited by losses to killer whales or subsistence

harvests, or disturbance from the many scientists conducting studies in this area. Alternately (or additionally), population growth might have been restricted by relatively low pup survival due to limited shallow-water feeding habitat, even though food for adult otters is relatively plentiful (Dean et al., 2000 and Dean et al., 2002). Population growth at NKI might also have been constrained by diminished pup production (pup ratio, DNA Damage inhibitor Fig. 4b). This could have arisen as a result of

an altered sex ratio: a large group of males was observed in this area in 1996 (Garshelis and Johnson, 2001 and Bodkin et al., 2002), some of which may have taken up residence there. Such a slight perturbation in population composition could shift the dynamics of the small population in this area and render comparisons of population growth rates and pre- versus post-spill population sizes meaningless. Shifts in population composition might or might not have occurred as a result of the spill; such events have been documented in WPWS before the triclocarban spill (Garshelis et al., 1984 and Johnson, 1987). The biggest problem in weighing competing hypotheses to explain varying population trends in WPWS is that, despite many years of study, sea otter population dynamics are still poorly understood, even in the absence of major environmental perturbations. This is partly due to an incomplete understanding of otter behavior and partly due to the complexity of the ecosystem in which they live (Estes, 1990, Bodkin et al., 2000 and Harwell et al., 2010b). Additionally, significant environmental changes have

occurred in the Gulf of Alaska and PWS since sea otter research was initiated there in earnest in the early 1970s (Johnson, 1987). In 1964 the area was struck by the largest recorded earthquake in North America (Fig. 1), severely affecting (and possibly still affecting) habitat and food availability for sea otters (Garshelis and Johnson, 2001). Beginning in the mid-1970s, abrupt, large-scale changes in atmospheric conditions and ocean currents caused increased water temperatures in the northern Gulf of Alaska (including PWS), which altered the physical and biological processes of this region on a massive scale (Mundy, 2005 and Spies, 2007). This “regime shift” has been linked to marked changes in abundance of a number of marine species since that time (Anderson and Piatt, 1999, Benson and Trites, 2002 and Trites et al.

Our HIV clinic population is multiracial and international, with a high proportion of patients originating from Sub-Saharan Africa and significant numbers presenting

late with advanced HIV at diagnosis. We also sought to compare baseline characteristics of patients with and without cryptococcal antigenemia, in order to establish whether screening should be targeted at any specific groups. This was a retrospective cohort study conducted between April and October 2011 at Croydon University (previously Mayday) Hospital and St George’s Hospital in London. Newly diagnosed patients were identified from clinic and laboratory databases using the inclusion criteria: i) age ≥18 years; ii) new confirmed positive HIV see more serology diagnosed for the first time between January 2004 to October 2010, with stored serum or plasma available for testing; iii) CD4 count < 100 cells/μL; iv) not yet on ART at time of stored blood sample. The study was approved by the UK National Research Ethics committee and the Research and Development Office of St George's Hospital NHS Trust. St George's Hospital Virology laboratory stores serum for 2 years and plasma (HIV viral loads) for up to 10 years. Given the

use of retrospective stored samples, plus a requirement for samples to be at least 6 months old prior to testing (to allow patients to have become established on ART, such that any retrospective positive result would not impact current clinical Birinapant care), the requirement for informed consent was waived. Stored serum or plasma samples from time of initial HIV diagnosis were anonymised prior to testing. CRAG testing was performed on serum or plasma using the Cryptococcal Latex Agglutination test (Immuno-Mycologics Inc, USA), an antibody-agglutination reaction detecting the capsular polysaccharide antigen of C. neoformans with a specificity and sensitivity of >95%. Samples were incubated with Pronase(Roche) at 56 °C for 15 min and analysed according to manufacturers’

instructions. All samples were screened undiluted and at a 1:100 dilution. Any samples with a titre of ≥1:2 were defined as positive, and serially diluted twofold to determine the CRAG titre. Demographic and clinical data, including CD4 count at HIV diagnosis, age, sex, ethnic group, country Cyclin-dependent kinase 3 of origin and sexual orientation, were obtained from clinic databases by clinicians independent from the laboratory researchers. For any patients with cryptococcal antigenemia detected on retrospective testing of stored serum or plasma, clinical presentation at HIV diagnosis, results of relevant investigations, antifungal treatment, time to start of ART and development of incident or relapsed CM in the first 6 months on ART were obtained from medical notes and laboratory results review. Data were analysed using GraphPad Prism v5 (GraphPad Software, USA), using the t-test to compare continuous variables and the Fisher’s exact test for categorical variables.