mutans, which is one of the principle causative agents of dental caries, has to be elucidated. MicroRNAs (miRNAs) are small noncoding RNAs that are c. 22 nt long. They are found in various species of plants, animals and viruses (Bushati & Cohen, 2007), and normally act as regulators in every major cellular event through inhibitory

mechanisms (He & Hannon, 2004). It has long been known that bacteria contain noncoding small RNAs (sRNAs) that have regulatory functions, other than miRNAs (Gottesman, 2005; Waters & Storz, 2009). sRNAs are usually between 50 and 200 nt in length and have been predicted by computational searches in a variety of bacterial species (Livny & Waldor, 2007). Like miRNAs, sRNAs usually act as post-transcriptional regulators by interacting with the target mRNAs through a variety of mechanisms, including changes in RNA conformation and modulation of the stability

of the specific targets (Waters & Storz, find more check details 2009). Smaller RNAs that have size similar to miRNAs are not well understood in bacteria, although many of them may be found among sequence reads registered in the transcriptome of Escherichia coli (Dornenburg et al., 2010). Streptococcus mutans is the major causative agent of human dental caries and is considered to be the most cariogenic of all of the oral streptococci (Ajdić et al., 2002). The disease occurs when ecologically driven changes in oral biofilms are perturbed and S. mutans is mainly responsible for the formation of the oral biofilms (Burne, 1998). The genome of S. mutans has been fully sequenced and contains c. 2 Mb and 1963 ORFs (Ajdić et al., 2002). This study examined the existence of small (c. 26 nt) RNAs in S. mutans that we subsequently isolated. For this purpose, a deep sequencing (next-generation sequencing) approach was used and more

than 19 million sRNA clones were read. To differentiate these very sRNAs from the bacterial sRNAs (50–250 nt) and well-studied eukaryotic miRNAs, we suggest the term ‘miRNA-size, PLEKHB2 small RNA’ (msRNA). Their origin and putative functional significance are discussed. Streptococcus mutans (ATCC 25175) were inoculated into brain heart infusion broth (three independent cultures) and total RNA was extracted from the cultured S. mutans after pooling using the miRNeasy Mini kit (Qiagen, CA) according to the manufacturer’s protocol. RNA was processed and used for deep sequencing by LC Sciences (Houston, TX). An sRNA library was generated from the S. mutans RNA according to Illumina’s sample preparation instructions for Illumina Genome Analyzer IIx (Ilumina Inc., San Diego, CA). The following gives a brief summary of the procedures performed. The total RNA sample was size-fractionated on a 15% Tris–borate–EDTA (TBE)–urea polyacrylamide gel. The RNA fragments of c. 15–50 nt in length were eluted and ethanol-precipitated.

Eight hospital health care providers and 35 committed PCC staff members were

involved in the unit. With the aim of improving the detection of HIV infection in the general population, we undertook a prospective study in PCCs to compare the outcomes of two HIV testing strategies. The proportion AZD8055 datasheet of PCC attendees who were offered screening, the acceptance rate, the HIV prevalence and the numbers and characteristics of newly diagnosed individuals were evaluated. One of the approaches was based on screening patients attending the PCCs with four selected indicator diseases/conditions (ICs) [herpes zoster (HZ), seborrhoeic eczema (SE), mononucleosis syndrome (MNS) and leucopenia/thrombocytopenia (L/T)] (the IC approach) and the other approach was to test nontargeted patients

attending these centres for any other reason [the non-indicator condition (NIC) approach]. From October 2009 to February 2011, a NVP-BKM120 multicentre, prospective study was carried out, without the involvement of additional staff, in four PCCs in Barcelona, Spain, which were identified as C1, C2, C3 and C4. Centres C1, C2 and C3 serve populations of medium to high economic status with low rates of immigration, while centre C4 serves a population of low to very low socioeconomic status with a high rate of immigration. If the patients presented with any of the four selected ICs (HZ, SE, MNS or L/T) they were allocated to the IC group. If they presented with any other medical condition, they were randomly selected (one in every 10 patients) to participate in the NIC group. Patients recruited to the IC group were also included in the international pilot study HIV Indicator Diseases across Europe Study I (HIDES I) to evaluate the prevalence of HIV in patients with ICs [7]. Consecutive patients between 18 and 65 years old who were not already known to be HIV positive, attending any of the four selected PCCs, L-gulonolactone oxidase were offered an HIV test.

If they gave written consent, they were interviewed using a standardized questionnaire (with questions about their demographic characteristics, previous HIV testing behaviour and relevant past medical history) and a rapid HIV test was performed at the same time. The study was approved by the ethics committees of the different centres. The HIV test used for screening was a fourth-generation rapid test (Determine® HIV-1/2 Ag/Ab Combo; Alere Medical Company, Chiba, Japan) that analyses a blood sample extracted by finger-stick. The cost for every rapid test performed was €6. Before the beginning of the study, PCC staff members were trained in how to perform the rapid test. Patients with an HIV-positive diagnosis were referred to a specialized centre for further assessment.

The EZ::Tn5 carrying plasmid pMOD-3 < R6Kγori/MCS> (EPICENTRE® Biotechnologies) was modified for use in BF638R. The erythromycin resistant gene (ermF) along with its promoter was PCR-amplified with ermF

F SacI and ermF R SacI primers (Table 1) using the Bacteroides shuttle vector pFD288 as template DNA (Smith et al., 1995) and ligated into pGEM®-T Easy. Escherichia coli Top 10 chemically competent cells were Lenvatinib mouse transformed with the ligation mix, and transformants were selected on LB-Amp agar plate, yielding plasmid pT-ermF-4. The ermF was retrieved from pT-ermF-4 by Sac I digestion and ligated into Sac I-digested pMOD-3 < R6Kγori/MCS > . Escherichia coli Top10 competent cells were transformed with the ligation mix, and transformants were selected on LB-Amp agar plate, yielding pYV01. The kanamycin gene (km) along with its promoter was PCR-amplified with Km F EcoRV and Km R EcoRV primers PF-562271 (Table 1) using pET-27B(+) as template DNA. The amplified PCR product (0.95 kb) was purified and ligated into pGEM®-T Easy. Escherichia coli Top 10 cells were transformed with the ligation mix, and transformants were selected on LB-Km agar plate, yielding plasmid pT-Km-2. The km gene was retrieved from pT-Km-2 by EcoRV digestion and ligated into

SmaI-digested pYV01. Escherichia coli Top10 competent cells were transformed with the ligation product, and transformants selected on LB-Km agar plate, yielding plasmid pYV02; this plasmid was used for transposome preparation (see below). pYV02 was passed through BF638R, so that the transposon would be properly modified by the host methylation system to avoid subsequent degradation. For this purpose, repA (for replication in BF) was PCR-amplified using primers pFKRepAF Thymidylate synthase and pFKRepAR using pKF12 as template DNA (Haggoud et al., 1995). The amplified PCR product (1.68 kb) was purified, digested with SmaI/Eco RV, and ligated into SmaI site of pYV02. BF638R was transformed with the ligation mix by electroporation, and transformants selected on BHI-Erm agar plate, yielding pYV03. Transposomes were prepared according to manufacturers’ protocol with the following modifications. EZ::TN5 transposon DNA was retrieved from either pYV02 or pYV03 (BF-R/M vector) following

PvuII digestion. The resulting 2.6-kb fragment was gel-purified and column eluted (Qiaquick Gel Extraction Kit; Qiagen, Inc., Valencia, CA) with TE buffer [10 mM Tris–HCL (pH7.5), 1 mM EDTA]. For transposome preparation, 2 μL of EZ::TN5 transposon DNA (100 ng μL−1) was mixed with 4 U (4 μL) of En-Tn5™ transposase (EPICENTRE® Biotechnologies) plus 2 μL of glycerol (100%) and incubated for 1 h at room temperature. The resulting transposon–EZ::TN5 transposase mixture (transposome) was stored at −20 °C and used for mutagenesis of BF. A single colony of BF638R grown on BHI was inoculated in 5 mL BHI broth and incubated anaerobically overnight (16 h) at 37 °C. Cultures were diluted (1 : 100) in 100 mL BHI broth and allowed to grow to an OD600 nm of 0.3–0.4.

1), but in many TA operons the antitoxin and toxin learn more genes overlap, indicative of translational coupling between the two cistrons (Gerdes et al., 2005). The sequence of the Ps-Antox protein also shares high identity values with other reported antitoxins, specifically with the well-described VapB and VagC antitoxins (Table 1).

Additionally, the Ps-Antox contains a putative SpoVT/AbrB domain, which is present in toxins of the VagC family. Bacillus subtilis SpoVT/AbrB domain proteins are transcriptional regulators, which are expressed during the transition state between vegetative growth and the onset of stationary phase and sporulation (Robertson et al., 1989). The presence of a SpoVT/AbrB domain in the Ps-Antox protein could be explained

by the fact that all the Type II TA operons are autoregulated at the level of transcription by the antitoxins, which bind to the TA locus promoters (Gerdes et al., 2005). The best reported example of this issue is the E. coli YefM–YoeB system, which is transcriptionally autoregulated (Kedzierska et al., 2007). We have not explored this possibility in this report. The FgeneB analysis of the putative sequence of the Ps-Tox protein was submitted to blastp analysis, yielding high identity with members of the VapC family proteins (Table 1). The sequence alignment of Ps-Tox with VapC homologues showed a high level of conservation Selleckchem GSKJ4 (see Table S1), indicating that it does correspond to a toxin coded in a bacterial TA module. The Ps-Tox protein contains a PIN domain, which is another distinctive feature of the toxins from the VapC and ChpK families (Arcus et al., 2005; Miallau et al., 2008). The PIN domains (homologues of the pilT N-terminal domain) are small protein

domains of ∼140 amino acids (Arcus et al., 2005). In eukaryotes, PIN-domain proteins function as ribonucleases with activity linked to RNAi and nonsense-mediated RNA degradation (Clissold & Ponting, 2000). In prokaryotes, the majority of PIN-domain proteins are the toxic components (by virtue of their ribonuclease activity) of chromosomally encoded eltoprazine TA operons (Arcus et al., 2005). Because the Ps-Tox toxin does display endoribonuclease activity (Fig. 5) as do other VapC homologues and also contains a PIN domain, we could speculate a putative action similar to the Mycobacterium tuberculosis VapC-5 product, which specifically blocks protein translation via mRNA cleavage (Ramage et al., 2009). In fact, the structural model of Ps-Tox (Fig. 4b) shows that the secondary structure elements of the toxin are preserved in comparison with the M. tuberculosis VapC-5 toxin, with some helix and beta sheet shorter residues in Ps-Tox. Notably, the active site defined by VapC-5 from M. tuberculosis and shared by PIN domains (Miallau et al., 2008) is conserved in the Ps-Tox protein (Fig. 4c).

We conclude that despite

the failures and variability in synaptic delay that are present at the calyx of Held synapse, their contribution to tone adaptation is relatively small compared with upstream factors. “
“Lesion and electrophysiological studies in rodents have this website identified the amygdala and hippocampus (HPC) as key structures for Pavlovian fear conditioning, but human functional neuroimaging studies have not consistently found activation of these structures. This could be because hemodynamic responses cannot detect the sparse neuronal activity proposed to underlie conditioned fear. Alternatively, differences in experimental design or fear levels could account for the discrepant findings between rodents and humans. To help distinguish between these alternatives, we used tissue oxygen amperometry to record hemodynamic responses from the basolateral

amygdala (BLA), dorsal HPC (dHPC) and ventral HPC (vHPC) in freely-moving rats during the acquisition and extinction of conditioned fear. To enable BIBF 1120 mw specific comparison with human studies we used a discriminative paradigm, with one auditory cue [conditioned stimulus (CS)+] that was always followed by footshock, and another auditory cue (CS−) that was never followed by footshock. BLA tissue oxygen signals were significantly higher during CS+ than

CS− trials during training and early extinction. In contrast, they were lower during CS+ than CS− trials by the end of extinction. dHPC and vHPC tissue oxygen signals tuclazepam were significantly lower during CS+ than CS− trials throughout extinction. Thus, hemodynamic signals in the amygdala and HPC can detect the different patterns of neuronal activity evoked by threatening vs. neutral stimuli during fear conditioning. Discrepant neuroimaging findings may be due to differences in experimental design and/or fear levels evoked in participants. Our methodology offers a way to improve translation between rodent models and human neuroimaging. “
“A large forebrain circuit, including the thalamus, amygdala and frontal cortical regions, is responsible for the establishment and extinction of fear-related memories. Understanding interactions among these three regions is critical to deciphering the basic mechanisms of fear. With the advancement of molecular and optogenetics techniques, the mouse has become the main species used to study fear-related behaviours. However, the basic connectivity pattern of the forebrain circuits involved in processing fear has not been described in this species. In this study we mapped the connectivity between three key nodes of the circuit, i.e.

The major protein translocation pathway in bacteria, the general secretory (Sec) pathway, transports proteins across both the thylakoid membranes and the cytoplasmic membrane, as demonstrated for Synechococcus PCC 7942 (Nakai et al., 1993). Synechococcus PCC 7942 has just a single gene for each of the Sec translocase components (secA, secY, secE and secG) and so an identical translocase must be operating in both membrane locations (Nakai et al., 1993). The Tat pathway also operates in both membrane systems in Synechocystis sp. (Aldridge et al., 2008) and this raises the question of how Tat substrates are targeted to a particular membrane. Two main hypotheses have been proposed: one hypothesis is that proteins are sorted

before translocation occurs. This would require the same translocation machinery recognizing a specific subset of proteins in different membrane systems, and there is some limited evidence in favour of this model. GW-572016 cell line Thus, the signal sequences of noncytoplasmic proteins have different chemical properties depending on the final localization of the cargo protein (Rajalahti et al., 2007). This would suggest that membrane targeting is at least in part dictated by the signal peptide. Furthermore, the signal peptide of Tat substrates interacts with membranes as an early step in the translocation process (Hou et al., 2006; Bageshwar et al., 2009). Differences in the composition of the two membranes could provide one possible

mechanism for this pretranslocation sorting.

An alternative SB203580 chemical structure hypothesis is that proteins are sorted post-translocation and again isothipendyl some evidence suggests that this might be the case. For example, cyanobacterial photosystems have been found to partially assemble within the plasma membrane before being translocated to the thylakoid membrane in a mechanism that might involve vesicular transport (Zak et al., 2001; Nevo et al., 2007). The actual mechanism of sorting is likely to be complex and may even involve both of the presented models to some extent. Approximately one in three cellular proteins are predicted to use metal ions for either a structural or functional role (Holm et al., 1996). Amongst the so-called trace-metals, iron and zinc are the two most frequently utilized (Maret, 2010). Cyanobacteria are likely to have played a major role in the bioavailability of metal ions through the evolution of oxygenic photosynthesis and the consequent oxygenation of the Earth’s atmosphere, roughly 2.75 billion years ago (Saito et al., 2003). Once soluble forms of Fe(II) were oxidized to more insoluble Fe(III) compounds, this is thought to have resulted in the evolution of sophisticated iron acquisition systems. Other metals, such as copper and zinc were liberated from insoluble sulphides and whilst this would have initially presented a challenge because of toxicity, it also presented cells with an opportunity to acquire and utilize ‘new’ metals (Cavet et al.

The mean number of months per year was 10.6 (median Selleck Buparlisib 12). The dependent variable was the number of distinct in-patient bacteraemia/septicaemia episodes in a calendar year. We calculated incidence rates for bacteraemia in each year. Multivariate analyses used the person-year as the unit of analysis, with the number of months of exposure in each year incorporated in the model as an offset. To incorporate the correlation between multiple observations for most patients, we used generalized estimating equations (GEEs), with an exchangeable correlation matrix and robust standard errors clustered on each patient. Because 86–90% of patients with a bacteraemia

episode in a year had only one episode, we used logistic regression to analyse any episode (none vs. one or more) in a year. For comparison, we also report a negative binomial regression selleck inhibitor of number of episodes in a year. We first examined each demographic and clinical factor separately. In further multivariate analyses, we used logistic regression to estimate the adjusted odds ratios for age, sex, race, HIV transmission risk factor,

CD4 cell count, HIV-1 RNA, HAART and insurance. To assess trends over time, dummy variables for each year (2001–2008) were included in the model. In all models, the first CD4 cell count and HIV-1 RNA of each calendar year were used. Age, CD4, HIV-1 RNA, insurance and HAART were treated as time-varying covariates. To account for geographic differences

in HIV care, all models were also adjusted for HIV care site. All reported P-values are two-tailed. Statistical analyses were performed using stata 9.0 (Stata Corporation, College Station, TX, USA). We classified bacteraemia episodes on the basis of the type of organism producing the infection, and we examined trends over time in the types of organisms. A subanalysis was performed at one large academic hospital where all ‘bacteraemia, not otherwise specified’ (NOS) cases were evaluated by manual abstraction by searching hospital laboratory records to determine the organism of interest. This large hospital constituted 42% of all bacteraemia NOS cases. Because of Institutional Review Board issues, hand searching was not possible at other participating study sites. Between January 2000 and December 2008, 39 318 patients were followed for a total PAK5 of 146 289 PY at 10 HIVRN sites. The sample was 71% male, 48% Black and 21% Hispanic, with a median age of 39 years (range 18–94 years) (Table 2). The major HIV risk factors included MSM (36%), HET (34%) and IDU (22%). During the study period, 57% of the patients received HAART. Most patients had Medicaid (32%) or were uninsured (27%). The median CD4 count was 321 cells/μL and the median HIV-1 RNA was 7760 copies/mL. During the study period, 2025 episodes of bacteraemia occurred, for an incidence rate of 13.8 events per 1000 PY. A total of 1538 patients (3.9% of 39 318) had one or more episodes.

, 2011). In humans, the default mode network not only http://www.selleckchem.com/products/Vorinostat-saha.html consists

of mPFC areas but also medial parietal areas (including midline anterior and posterior cingulate cortices; Raichle et al., 2001). Recent investigations in macaques have identified electrophysiological correlates of default mode processing in both mPFC and posterior cingulate cortices (Hayden et al., 2009; Kojima et al., 2009). The positron emission tomography imaging study of Kojima et al. (2009) in awake unanaesthetized monkeys clearly demonstrated a default mode of cortical activity with higher rest-related activity in mPFC areas compared with working memory tasks. The activity in macaque mPFC reported here before and during eye-closure may therefore represent in part alterations in the activity of mPFC areas associated with the default mode network in monkeys. It is of interest that Rudolph et al. (2007)

reported that a significant proportion (~45%) of presumed pyramidal (broad spike/regularly spiking) neurons in parietal association cortex also discharged during SWS and were silent during waking. In relation to these default mode network studies, the value of the present investigation is that it shows electrophysiologically that the firing rates of a significant selleck products number of mPFC neurons (those of cell Type 1 representing about 28% of sampled neurons) in the monkey were low in the awake state (mean 3.1 spikes/s) and increased significantly during sleep (mean 10.2 spikes/s). The firing rates of the neurons involved in default mode network activities, and exactly how they may change, is not directly measured in human neuroimaging studies. Given the increase in the human BOLD (blood oxygen level-dependent) response during operation of the default mode network, it is tempting to speculate that some of the neurons whose firing rates increased during periods of ‘eye-closure’ may have intracortical axonal arbors instrinsic to the mPFC that innervated nitric oxide (NO)-producing cells (Gabbott and Bacon, 1996). The activity of such cells would lead to local vasodilatation

(through NO-mediated mechanisms) and thus increased blood flow in specific mPFC regions with raised metabolic demands during periods of augmented information processing Neratinib clinical trial (Duchemin et al., 2012). The data from the present study have implications for the generation of sleep activity in humans, both in health and in disease. Many neuropsychiatric and neurodevelopmental disorders, for example depression, schizophrenia and autism, which include functional modifications of the default mode network, have symptoms that include poor sleep architecture (Drevets et al., 1997; Wichniak et al., 2000; Vogt, 2009; Gregory et al., 2011; Vukadinovic, 2011; Price & Drevets, 2012). Patterns of abnormal sleep structure (narcolepsy, sleep inertia, parasomnias, non-REM and REM sleep behaviour disorders, etc.

Such conditions may favor mutations that help these bacteria adapt to a hostile environment (Galhardo et al., 2007). The prevalence of strong mutators, which are characterized by an increased frequency of spontaneous mutations, ranges from about 1% among pathogenic strains of Escherichia coli (Baquero et al., 2004) to more than 30% among Pseudomonas aeruginosa stains isolated from cystic fibrosis patients (Oliver et al., 2000). The role of the

strong mutator phenotype in pathogenic bacteria has been discussed at great length (Jolivet-Gougeon et al., 2011), but the link between this phenotype and virulence is not yet well understood. However, a strong mutator phenotype is expected to drive adaptation to a hostile environment (Taddei et al., 1997). Strong mutators are detected easily by enumeration

of antibiotic-resistant mutants on culture media containing rifampicin, fosfomycin, nalidix FGFR inhibitor acid, streptomycin, or spectinomycin (LeClerc et al., 1996; Matic et al., 1997). Polymorphisms in rifampicin resistance genes have been studied by Baquero et al. (2004), who arbitrarily defined four categories of E. coli strains according to their mutation frequencies (f) as follows: hypomutator Nutlin-3a clinical trial (f ≤ 8 × 10−9), normomutator (8 × 10−9< f < 4 × 10−8), weak mutator (4 × 10−8 ≤ f < 4 × 10−7), and strong mutator (f ≥ 4 × 10−7). In most cases, the mutator phenotype is due to a defective methyl mismatch repair (MMR) system (LeClerc triclocarban et al., 1996), which plays a key role in the correction of base–base mismatches and insertion/deletion mispairs that appear during DNA replication. MutS, MutL, and MutH are three bacterial proteins that are essential for initiation of methyl-directed DNA mismatch repair (Li, 2008). The objectives of this study were to determine the prevalence of mutators among human clinical isolates of Salmonella by prospective screening and to characterize the detected strong mutators by sequencing the MMR genes to find short tandem repeats (STRs). This study included all strains of Salmonella (n = 130) collected from clinical samples between the 1st of March 2009 and the 30th of April 2010 in seven French hospital laboratories. The hospitals were located in Angers,

Brest, Lorient, Quimper, Rennes, Saint-Brieuc, and Vannes. In cases of outbreaks, only the first isolated strain was included. The great majority of strains were isolated from stool samples (n = 119). The remaining strains were isolated from blood (n = 7), intestinal biopsies (n = 2), urine (n = 1), and hematoma (n = 1) (Table 1). Rifampicin and fosfomycin resistance mutation frequencies were determined as described previously (LeClerc et al., 1996; Denamur et al., 2002). Briefly, a single colony of the bacterial strain was suspended in 10 mL LB broth (AES Laboratory) and incubated at 37 °C for 24 h. One hundred microliters of this culture were spread onto LB agar plates with and without rifampicin (Sigma Aldrich) at 100 μg mL−1 or fosfomycin (Sigma Aldrich) at 30 μg mL−1.

4%), followed by decompression sickness (2/47, 4.3%) and barotrauma (1/47, 2.1%). Two divers died of natural causes (heart failure and heart

attack) (2/47, 4.3%). All the divers Nintedanib ic50 who died from natural causes and decompression sickness were tourists. Some of the drowning victims died because of unfavorable sea conditions [high waves (2/42, 4.8%)], while others owing to underwater obstacles disabling the diver from ascending to the surface (concrete blocks, shipwreck) (4/42, 9.5%), and one diver died of drowning after being hit by a speedboat (1/42, 2.4%). Even though it was not the direct cause of death, another drowning victim showed signs of decompression sickness and embolism that probably triggered drowning (1/42, 2.4%). A section of the divers suffered from a preexisting health problem while engaged in diving. Fifteen victims (31.9%) showed signs of acute, chronic, or congenital diseases. In six divers more than one pathologic condition was found (6/15, 40%). The pathology ranged from heart and blood vessel diseases (12/15, 80.0%; myocarditis,

pericarditis, severe atherosclerosis, congenital narrowness of the aorta, hypertrophy, etc.) to lung diseases (3/15, 20.0%), renal diseases (4/15, 26.7%), and hepatic diseases (2/15, 13.3%). Preexisting Selleck Z-VAD-FMK health-disrupting conditions were found in 10.5% of resident divers and 46.4% tourist divers. Alcohol intoxication was absent from all recorded victims, except for the oldest victim who drowned during snorkeling. The study evidenced a continuous

increase of diving-related deaths in the studied regions, especially among free-divers. The majority of victims were foreign citizens (59.6%) most of whom fell victim to scuba diving (70.4%). Seventy-nine percent of resident divers succumbed during free-diving. The victims usually belonged to younger age groups with tourist divers being significantly older than local divers; 31.9% of divers, mostly tourists, showed signs of acute, chronic, or congenital pathological conditions. Rucaparib ic50 Even though diving has a small overall mortality and accident rate, the growing number of divers and the development of diving tourism have caused a volume-related increase in the number of diving injuries and deaths.[10] Such trends have also been recorded in the Primorje-Gorski Kotar County where the numbers of diving-related deaths, especially of tourists, show a continuous increase during the 31-year period, with 46.8% of the deaths occurring during the last decade (2001–2010). Although in Croatia there is no law that fully regulates diving activities, up to now activities related to scuba diving have been normatively controlled directly or indirectly by a number of regulations and articles scattered in different laws.[11, 12] These do not include regulations on free-diving activities, in turn making scuba diving a better monitored and controlled underwater activity.