These efforts routinely consisted of microscopic

observations of diseased coral tissues, all of which revealed the presence of various bacteria and fungi. The photosynthetic and heterotrophic bacteria (such as Phormidium corallyticum) were proposed as potential agents of coral black band disease (Frias-Lopez et al., 2004); and the bacterium Vibrio charcharii was associated with coral white band disease (Richardson et al., 1998). In addition, a few studies found that fungi Aspergillus sydowii and Aspergillus versicolor were causal agents of the coral aspergillosis (Nagelkerken et al., 1997; Geiser et al., 1998; Fabricius & Alderslade, 2001; Sakayaroj et al., 2006). However, some microbes that had been identified as potential selleck chemical Tanespimycin order agents of coral diseases have been found in healthy corals (Koh et al., 2000; Toledo-Hernandez et al., 2007), which suggested that these microbes were part of the normal microbial communities. Furthermore, some coral diseases were believed to be caused by microbial communities instead of a single pathogenic microbe

(Zuluaga-Montero et al., 2010). These findings highlight our ignorance of the basic microbial ecology of corals. Most of our limited knowledge of microbes in corals comes from stony and soft corals. From recent studies of coral microbial ecology, it is known that microbes in stony corals are distinct from those in the water column, and there appear to be coral species-specific microbial communities (Rohwer et al., 2001, 2002; Johnston & Rohwer, 2007). Stony

coral-associated microbes clearly represent one of the most complex and important components of the biodiversity of coral communities (Frias-Lopez et al., 2002; Yakimov et al., 2006). Moreover, many studies indicated that microbial communities occupy a range of niches in stony corals, from within the surface mucus layer not (Bourne & Munn, 2005; Ritchie, 2006) to on and within the coral tissue layers (Banin et al., 2000; Frias-Lopez et al., 2002). In addition, microorganisms in soft corals might be saprophytic or pathogenic, or may provide other important functions for corals (Santavy & Peters, 1997; Harvell et al., 1999). Microorganisms found in soft corals may help the host by protecting them against pathogens and/or may supply nutrients (Shnit-Orland & Kushmaro, 2009). Although our understanding of the microbial communities and their role in stony and soft corals is evolving, the microbial diversity of black corals (order: Antipatharia) is still poorly understood. This is mainly due to the paucity of field studies that have focused on these black corals, which can be found in all oceans at depths ranging from those of shallow waters to 2000 or more meters (Lapian, 2009).

25 (Liu & Muse, 2005). Sequences were deposited in the GenBank database (JQ901106–JQ901377,

Supporting Information, Table S1). Cross-priming was tested for amplification on 10 other strains belonging to 10 Agaricus species (Table 2). PCR conditions were the same as previously described. Zhao et al. (2011) [JF797194] this study [JQ824135] Zhao et al. (2012) [JN204430] this study [JQ824134] Zhao et al. Epigenetics inhibitor (2011) [JF797195] This study [JQ824136] Zhao et al. (2012) [JN204434] Zhao et al. (2011) [JF797188] Kerrigan et al. (2005) [AY899263] A total of 61 757 reads with an average 283 bp were obtained (NCBI SRA accession number SRA050786). Of them, 866 (1.4%) qualified sequences which were non-redundant, longer than 80 bp and containing at least one microsatellite motif with flanking region suitable for primers design, were released. The design of primer pairs was successful for 305 candidate microsatellites (258 perfects, 47 compounds, 0.49% of the starting number of reads) in 272 sequences. This result was lower than those observed in the foundation paper (Malausa et al., 2011) reporting between 1 and 8% of theoretically amplifiable markers in the obtained sequences. This percentage was clearly species-dependent.

We have little hindsight on the efficiency of such an approach on fungi. Only two fungal species were studied in Malausa et al. this website (2011), a basidiomycete Armillaria ostoyae and an oomycete Phytophtora alni mafosfamide for which 0.93 and 0.7% of amplifiable markers in the obtained sequences were described, respectively. Our results were slightly higher than those obtained for

arbitrary 454 shotgun library (Abdelkrim et al., 2009; Gardner et al., 2011) and may suggest some failure in the enrichment process. However, regarding the distribution of the patterns observed among the 866 qualified sequences, the most commonly found were in agreement with those expected according to the library enrichment with, for example, 36.3 and 27.6% of (AG)n and (AC)n motifs, respectively (Table S2). About 18% of motif types did not match any used for enrichment, but this number was in the same order of magnitude as those described in Malausa et al. (2011). Focusing on AG and AC motifs, the average number of repeats was 6.9 for AG and 6.7 for AC. Whatever the length of the motif, 90.5% of the microsatellite showed a number of repeats lower than 10. This was consistent with previous reports on fungal microsatellite with, for example, 6.2 repeats per locus in A. bisporus (Foulongne-Oriol et al., 2009). The shortness of microsatellite loci in fungi, together with their weak representation in fungal genomes render their isolation arduous (Dutech et al., 2007) and may explain our results. An adaptation of the enrichment protocol with shorter probes could enhance the efficiency of the technique.

Fibrobacter succinogenes S85 was incubated in medium containing rice straw as the sole carbon source for 48 h and centrifuged (2300 g, 4 °C, 10 min), and the supernatant was filtered through a sterile filter (0.22 μm; Millipore, Billerica, MA) in the anaerobic chamber

(Coy, Grass Lake, MI) maintained at the atmosphere of 95% CO2 and 5% H2. A cell suspension of strain R-25 with OD660 nm = 0.2 was inoculated to the obtained culture supernatant of F. succinogenes S85 and grown to mid-log phase. The control (rice straw medium without inoculation of APO866 nmr F. succinogenes S85) was processed as above. In addition, cultures of strain R-25 incubated in basal medium containing 0.5% (w/v) cellooligosaccharides (SEIKAGAKU BIOBUSINESS, Tokyo, Japan) or xylooligosaccharides (Wako, Osaka, Japan) as the sole carbon source was used for comparative study. Extracellular and intracellular enzyme assays were performed following the protocol described above. Rice straw particles in the monoculture and coculture were sampled at 48 h. The samples were washed three times with 50 mM potassium phosphate buffer (pH 6.8) and fixed with 3% glutaraldehyde in the same buffer for 1 h at room temperature. After fixation, the samples were washed four times with 50 mM potassium phosphate buffer and postfixed

for 30 min in 1% osmic acid (OsO4) in the same buffer. After washing four times, the samples were dehydrated by graded ethanol solution series [50, 70, 90, 99.5% (v/v), 10 min at each concentration] AZD0530 nmr and exposed to isoamyl acetate for 20 min twice. Isoamyl acetate was removed with a critical point dryer using liquid CO2 (HCP-2; Hitachi, Tokyo, Japan) in eight 15-min treatments. The samples were coated with gold in an ion sputter (E101; Hitachi) and

observed in a JSM-6301 low vacuum scanning electron microscope (JEOL, Tokyo, Japan) at an accelerating voltage of 5 kV. The means of DM digestion, metabolites, 16S rRNA gene copy number, and enzyme activity for each CYTH4 treatment were analyzed by one-way analysis of variance of spss ver. 16.0 J (IBM, Tokyo, Japan). P < 0.05 was regarded as significant. DM digestion of rice straw by F. succinogenes S85 was 32.8%, while strain R-25 did not digest rice straw (Table 1). DM digestion with coculture of strains R-25 and F. succinogenes S85 was 1.13-fold higher (P < 0.05) than that of monoculture of F. succinogenes S85 (36.9% and 32.8%, respectively). The extracellular CMCase and xylanase activities in monoculture of strain R-25 or F. succinogenes S85 and their coculture are shown in Table 1. For both CMCase and xylanase, the activities in coculture were higher than those of the F. succinogenes S85 monoculture (P < 0.05). Changes in 16S rRNA gene copy number for strains R-25 and F. succinogenes S85 in monoculture and in their coculture are presented in Table 2. At the beginning of incubation, the copy numbers of 16S rRNA gene for strain R-25 and F. succinogenes S85 were 8.1 × 106 mL−1 and 9.0 × 106 mL−1, respectively.

Number of patients with an undetectable VL on current regimen and documented previous NRTI resistance who have switched a PI/r to either an NNRTI or INI as the third agent. Number of patients on PI/r monotherapy as ART maintenance strategy in virologically suppressed patients and record of rationale. Record in patient’s notes of resistance result

at ART initiation (if available) and at first VL >400 copies/mL and/or before switch. Record in patient’s notes of adherence assessment and tolerability/toxicity to ART, in patients experiencing Staurosporine cell line virological failure or repeated viral blips. Number of patients experiencing virological failure on current ART regimen. Proportion of patients experiencing virological failure switched to a new suppressive MG-132 nmr regimen within 6 months. Proportion of patients on ART with previous documented HIV drug resistance with VL <50 copies/mL. Record of patients with three-class virological failure with or without three-class resistance referred/discussed in multidisciplinary team with expert advice. Proportion of patients with TB and CD4 cell count <100 cells/μL started on ART within 2 weeks of starting TB therapy. Proportion of patients with active TB on anti-TB therapy started on ART containing EFV, TDF and FTC. Proportion of patients with a CD4 cell count ≥500 cells/μL and an HBV DNA ≥2000 IU/mL and/or evidence of more than minimal

fibrosis commencing ART inclusive of anti-HBV antivirals. Proportion of patients with a CD4 cell count <500 cells/μL receiving TDF/FTC or TDF/3TC as part of a fully suppressive combination ART regimen. Proportion of patients receiving 3TC or FTC as the sole active drug against HBV in ART. Proportion of patients with a CD4 cell count <500 cells/μL commencing ART. Among patients receiving DAAs for HCV genotype 1 with ART for wild type HIV, the percentage on a recommended regimen,

i.e. RAL with TDF plus FTC with boceprevir; or RAL or boosted ATV with standard dose telaprevir; or EFV with increased dose 1125 mg tds telaprevir. Proportion of patients with an AIDS-defining malignancy on ART. Proportion of patients with a non-AIDS-defining malignancy on ART. Record in patient’s notes of potential pharmacokinetic drug interactions HAS1 between ARVs and systemic anticancer therapy. Proportion of patients with symptomatic HIV-associated NC disorders on ART. Proportion of patients with HIV-associated NC disorders on ART containing two NRTIs and one of the following: NNRTI, or PI/r or INI. Proportion of patients with HIVAN started on ART within 2 weeks of diagnosis of CKD. Number of patients with CKD stages 3–5 on ARVs that are potentially nephrotoxic and record of rationale. Record in patient’s notes of the calculated dose of renally cleared ARVs in patients with CKD stage 3 or greater.

Number of patients with an undetectable VL on current regimen and documented previous NRTI resistance who have switched a PI/r to either an NNRTI or INI as the third agent. Number of patients on PI/r monotherapy as ART maintenance strategy in virologically suppressed patients and record of rationale. Record in patient’s notes of resistance result

at ART initiation (if available) and at first VL >400 copies/mL and/or before switch. Record in patient’s notes of adherence assessment and tolerability/toxicity to ART, in patients experiencing PLX4032 virological failure or repeated viral blips. Number of patients experiencing virological failure on current ART regimen. Proportion of patients experiencing virological failure switched to a new suppressive Dabrafenib regimen within 6 months. Proportion of patients on ART with previous documented HIV drug resistance with VL <50 copies/mL. Record of patients with three-class virological failure with or without three-class resistance referred/discussed in multidisciplinary team with expert advice. Proportion of patients with TB and CD4 cell count <100 cells/μL started on ART within 2 weeks of starting TB therapy. Proportion of patients with active TB on anti-TB therapy started on ART containing EFV, TDF and FTC. Proportion of patients with a CD4 cell count ≥500 cells/μL and an HBV DNA ≥2000 IU/mL and/or evidence of more than minimal

fibrosis commencing ART inclusive of anti-HBV antivirals. Proportion of patients with a CD4 cell count <500 cells/μL receiving TDF/FTC or TDF/3TC as part of a fully suppressive combination ART regimen. Proportion of patients receiving 3TC or FTC as the sole active drug against HBV in ART. Proportion of patients with a CD4 cell count <500 cells/μL commencing ART. Among patients receiving DAAs for HCV genotype 1 with ART for wild type HIV, the percentage on a recommended regimen,

i.e. RAL with TDF plus FTC with boceprevir; or RAL or boosted ATV with standard dose telaprevir; or EFV with increased dose 1125 mg tds telaprevir. Proportion of patients with an AIDS-defining malignancy on ART. Proportion of patients with a non-AIDS-defining malignancy on ART. Record in patient’s notes of potential pharmacokinetic drug interactions for between ARVs and systemic anticancer therapy. Proportion of patients with symptomatic HIV-associated NC disorders on ART. Proportion of patients with HIV-associated NC disorders on ART containing two NRTIs and one of the following: NNRTI, or PI/r or INI. Proportion of patients with HIVAN started on ART within 2 weeks of diagnosis of CKD. Number of patients with CKD stages 3–5 on ARVs that are potentially nephrotoxic and record of rationale. Record in patient’s notes of the calculated dose of renally cleared ARVs in patients with CKD stage 3 or greater.

Streptococcus suis isolates were examined for their ability to autoaggregate buy Entinostat according to the protocol of Basson et al. (2008). Bacteria were grown overnight in THB medium, washed, and resuspended in sterile distilled water to an OD660 nm of 0.3. The degree of autoaggregation of all isolates was determined using the equation: % autoaggregation=(((OD660 nm at T0−OD660 nm at T60 min)/OD660 nm at T0) × 100). OD660 nm was recorded following

a low-speed centrifugation at 400 g for 2 min. Assays were run in triplicate and the means ± SD of three independent experiments were calculated. The relative surface hydrophobicity of S. suis cells was determined by measuring their absorption to n-hexadecane according to the procedure described by Rosenberg et al. (1980). Assays were run in triplicate and the means ± SD of three independent experiments were calculated. The subtilisin-like and dipeptidyl peptidase IV (DPP IV) activities of S. suis cells were measured using the chromogenic substrates succinyl–Ala–Ala–Pro–Phe–p-nitroanilide (p-Na) (Sigma-Aldrich Canada Ltd, Oakville, ON, Canada) and Gly–Pro–p-Na (Sigma-Aldrich

Canada Ltd), respectively. For both proteolytic assays, 100 μL of a cell suspension at OD660 nm=2 (in 50 mM Tris-HCl, pH 8, containing 5 mM CaCl2) was added to 20 μL of substrate (2 mg mL−1 in 50% dimethyl sulphoxide), and the mixtures were incubated at 37 °C for 4 h. The release of p-Na, indicative of substrate E7080 supplier degradation, was determined visually by the appearance Phosphoprotein phosphatase of a yellow colour. The culture broth medium used to investigate biofilm formation by S. suis contained 0.5% glucose, 2% peptone (Proteose Peptone no. 3, Difco, Detroit, MI), 0.3% K2HPO4, 0.2% KH2PO4, 0.01% MgSO4·7H2O, 0.002% MnSO4·6H2O, and 0.5% NaCl. Biofilm formation was measured in 96-well polystyrene microplates (Nunc-Immuno® MaxiSorp;

Nalge Nunc International) and crystal violet staining as described previously (Grenier et al., 2009). Assays were run in triplicate and the means ± SD of two independent experiments were calculated. The adhesion property of 13 S. suis strains (six of serotype 2 and seven nontypeable) to fibronectin immobilized onto polystyrene plate wells was investigated. The results presented in Table 2 indicate that none of the S. suis strains could adhere to BSA, which was used as a control protein. However, the seven nontypeable isolates of S. suis (1078212, 1079277, 1097925, 1185293, 1148795, 1077009, and 1079506) showed a marked capacity to adhere to the fibronectin-coated surface. Under the conditions used in our study, all strains of S. suis serotype 2 attached poorly to the fibronectin-coated surface. The adherence properties of three nontypeable strains of S. suis were further investigated by evaluating their attachment to brain microvascular endothelial cells. As shown in Fig.

Here, we investigated the effects of the neurotransmitter serotonin and antidepressant fluoxetine (a selective serotonin reuptake inhibitor) on the modulation of adaptation-induced orientation plasticity. We show that serotonin and fluoxetine promote mostly attractive shifts. Attractive shifts augmented in magnitude towards adapter, whereas repulsive neurons reversed their

behavior in the direction of the forced orientation. Furthermore, neurons which retained their original preferred orientation expressed plasticity by shifting their tuning CB-839 in vivo curves after drug administration mostly towards adapter. Our data suggest a pre-eminent role of fluoxetine by inducing and facilitating short-term plasticity in V1. “
“The suprachiasmatic nucleus (SCN) is the principal pacemaker driving circadian rhythms of physiology and behaviour. Neurons within the SCN express both classical and neuropeptide transmitters which Bortezomib order regulate clock functions. Cholecyctokinin (CCK) is a potent neurotransmitter expressed in neurons of the mammalian SCN, but its role in circadian timing is

not known. In the present study, CCK was demonstrated in a distinct population of neurons located in the shell region of the SCN and in a few cells in the core region. The CCK neurons did not express vasopressin or vasoactive intestinal peptide. However, CCK-containing processes

make synaptic contacts with both groups of neurons and some CCK cell bodies were innervated by VIPergic neurons. The CCK neurons received no direct input from the three major pathways to the SCN, and the CCK neurons were not light-responsive as evaluated by induction of cFOS, and did not express the core clock protein PER1. Accordingly, CCK-deficient mice showed normal entrainment BCKDHA and had similar τ, light-induced phase shift and negative masking behaviour as wild-type animals. In conclusion, CCK signalling seems not to be involved directly in light-induced resetting of the clock or in regulating core clock function. The expression of CCK in a subpopulation of neurons, which do not belonging to either the VIP or AVP cells but which have synaptic contacts to both cell types and reverse innervation of CCK neurons from VIP neurons, suggests that the CCK neurons may act in non-photic regulation within the clock and/or, via CCK projections, mediate clock information to hypothalamic nuclei. “
“Ernest Gallo Clinic and Research Center at UCSF, Suite 200, Emeryville, CA, USA Intense fearful behavior and/or intense appetitive eating behavior can be generated by localized amino acid inhibitions along a rostrocaudal anatomical gradient within medial shell of nucleus accumbens of the rat.

You can’t really imagine it until you see it Most of the pharmacists were running their own clinics and they were very up close and personal with the patients so it was interesting to see the role directly with patients When we were on the ward round she asked 3-Methyladenine manufacturer us questions like what does this mean or what could be causing this. I thought that was really good because you could then be like oh I actually know

this. This study has achieved its aim of exploring MPharm undergraduates’; views on this targeted optional placement in a specialist oncology setting. The placement was recognised as a valuable learning experience, despite its short duration, by students and staff from the university Fluorouracil solubility dmso and hospital. Other optional placements in a variety of settings are now being introduced

in the pharmacy programme and evaluated using a similar approach. 1. Braun V, Clarke V. Using thematic analysis in psychology. Qualitative Research in Psychology. 2006, 3(2), 77–101 I. Stupansa, S. McAllisterb, C. Cliffordc, J. Hughesd, I. Krasse, G. Marchf, S. Owenf, J. Woulfeg aUniversity of New England, NSW, Australia, bFlinders University of South Australia, SA, Australia, cUniversity of Western Australia, WA, Australia, dCurtin University, WA, Australia, eUniversity of Sydney, NSW, Australia, fUniversity of South Australia, SA, Australia, gUniversity of Technology Sydney, NSW, Australia Prior to this project Australian pharmacy programmes have had a number of curriculum influences including those of accreditation, the profession and individual university practices, but no nationally agreed learning outcomes for graduates. A collaborative project, focussing on the development and endorsement of learning outcomes was undertaken. Application of these learning outcomes and exemplar

standards will ensure that all graduates of all pharmacy programmes will have achieved at least the same threshold regardless of the university from which they graduate. Contemporary practices in higher education, including Morin Hydrate practices in pharmacy education, have moved from a focus on “inputs” to assuring graduate outcomes with increased attention on robust and reliable assessment of those outcomes. This project was guided by the understanding that learning outcomes are explicit definitions of all essential domains of learning at the point of graduation. Exemplar standards for each of the domains specify expected levels of achievement, indicating the dimensions of breadth, depth, utility and application to practice and proficiency. Thus exemplar standards operationalise learning outcomes for curriculum development and assessment.

Recent successes in the identification of schizophrenia common allele associations

can largely be attributed to the Schizophrenia Working Group of the Psychiatric Genomics Consortium (PGC), which was created with the aim to maximise sample size by combining GWAS data from multiple international research groups [ 49]. The latest data from the PGC identified 128 linkage disequilibrium (LD)-independent genome-wide significant associations in 108 distinct loci [ 45••]. The most significant allelic association in schizophrenia is in the extended Selleck LDK378 major histocompatibility complex (MHC) on the short arm of chromosome 6 [ 45••]. Identifying candidate genes from this association is a major challenge as the existence of strong LD across this region of about 8Mb makes it difficult to localise the association find more to one, or even a few, of the hundreds of genes at the locus. The MHC’s involvement in immunity suggests that immune dysfunction

might play a role schizophrenia, although non-immune genes are also found in this region [ 50]. Additional genome-wide significant associations are found in genes long believed to play a major role in schizophrenia, such as the dopamine receptor D2 gene, which encodes the therapeutic target of most antipsychotic drugs [ 45••]. This suggests that biological insights gained from other novel common allele associations have the potential to identify new drug targets. Gene-set analyses have not yet shown any biological

pathway to be significantly enriched for the 128 schizophrenia genome-wide significant associations after correction for multiple testing, and a definitive analysis is awaited [ 45••]. However, the associations are enriched for enhancers expressed in brain, and also for enhancers in tissues involved with immunity [ 45••]. Schizophrenia has been shown to share common risk alleles with other psychiatric Idoxuridine disorders, such as bipolar disorder (BP), major depressive disorder (MDD), ASD and ADHD [51]. The most powerful demonstration of this comes from the en masse effects of SNPs which have revealed a high genetic overlap between schizophrenia and BP, a moderate overlap between schizophrenia and MDD, and a small but significant overlap between schizophrenia and ASD [ 46 and 48••]. Combining GWAS data from schizophrenia and BD has proved fruitful in identifying common risk alleles [ 52 and 53], although polygenic risk scores have also been able partly to distinguish between these disorders, suggesting that some risk alleles may confer more specific effects at the level of the psychiatric phenotype [ 53].

Under physiological conditions, B2 receptor knockout mice (B2−/−) present normal development [9], renal hemodynamics and salt balance [2], [26] and [35]. Nevertheless, data regarding the effects of B2 receptor deletion on blood pressure regulation are controversial. Some authors have demonstrated that B2−/− are

normotensive [1], [2], [3], [11], [12], [26], [35], [37] and [39] while other groups observed a slight but significant increase in blood pressure levels [15], [16], [21] and [22]. Considering that both B1 and B2 receptors are located in the endothelium and in vascular smooth muscle cells [7] and [19], and that resistance vessels are the most important sites for determining peripheral vascular resistance [38], the present study SB431542 was addressed to investigate the vascular reactivity of mesenteric arterioles of B1−/− RG7204 mouse and B2−/− in response

to endothelium-dependent and -independent agonists. In parallel, plasma NO levels, vascular NO release and NOS activity in the mesenteric vessels were also analyzed in order to provide information about NO bioavailability in these mice strains. C57Bl/6 male knockout B1 (B1−/−), B2 (B2−/−) and wild type (WT) mice, aged 10–14 weeks were obtained from the breeding stock of Centro de Desenvolvimento de Modelos Experimentais para Medicina e Biologia (CEDEME – UNIFESP). Mice were kept in a temperature-controlled room on a 12 h light/day cycle, 60% humidity, standard mice chow and water ad libitum. In B1−/− and B2−/−, the absence of the kinins receptors was shown by undetectable level of mRNA encoding for the

B1 or B2 receptor, respectively, using a semi-quantitative RT-PCR technique. All procedures were approved and performed in accordance with the guidelines of the Ethics Committee of the UNIFESP (protocol number 0928/05), conformed with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). Isolated mesenteric vascular beds were prepared as previously described for the rat preparation [24], with slight adaptations for the mouse. The mesenteric vascular bed was perfused with Krebs-Henseleit solution, pH 7.4, 37 °C, gassed with 95% O2 Rolziracetam and 5% CO2, at a constant rate of 2 mL/min using a peristaltic pump. Vascular responses were evaluated by changes in the perfusion pressure (mmHg) measured by a data acquisition system (PowerLab 8/S, ADInstruments Pty Ltda, Australia). To confirm the viability of tissues, preparations were perfused with KCl (90 mmol/L) added to the Krebs solution for 5 min. After 30 min of stabilization, increasing doses of norepinephrine (NE) (5–100 nmol), acetylcholine (ACh) (0.1–10 nmol) and sodium nitroprusside (SNP) (0.1–10 nmol) were injected in bolus, in a volume range of 30–100 μL, with a 3-min interval between each dose.