anisopliae GAPDH. The transcription pattern of the M. anisopliae gpdh1 gene in response to different carbon sources (glucose, glycerol or ethanol as the sole carbon sources) was analyzed using Northern blots probed with the M. anisopliae gpdh1 cDNA-radiolabeled DNA. The gpdh1 transcript levels were considerably reduced in the presence of glycerol and ethanol as compared with glucose (Fig. 2a). The cognate protein levels were analyzed by immunodetection using 1- and 2-D gel electrophoresis of protein cell extracts from cultures in the same carbon sources (Fig. 2b–e). Similarly,

there was decreased accumulation of GAPDH protein in the presence of glycerol and ethanol as compared with glucose-containing cultures. Both the transcriptional and the protein expression patterns thus showed a direct response to substrate. selleck kinase inhibitor The gpdh1 transcripts from M. anisopliae cultivated in a medium containing tick exoskeleton and chitin as the sole carbon source were also analyzed (Fig. 3), showing a selleck monoclonal humanized antibody significant decrease in gpdh1 transcripts with chitin as compared with both glucose- and exoskeleton-containing cultures. To define the cellular localization of GAPDH in M. anisopliae cells, conidia, appressoria, mycelia and blastospores were examined using immunofluorescence microscopy. GAPDH was detected on the cell surface as well as in the

cytoplasm (Fig. 4a). The accumulation at blastospore poles was evidenced in 64-h incubation Adamek cultures. Moreover, most of the GAPDH migrated to the poles of germinating blastospores after 3 h of growth in CM medium (Fig. 4b and c). Fluorescent vesicular-shaped areas could be observed in the cytosol and on the cell surface. Triton X-100 cell washes substantially decreased the surface protein signals. The presence of GAPDH on the cell surface was Florfenicol also analyzed by measuring the GAPDH catalytic activity of intact conidia

in protein extracts from Triton X-100 washes. An increase in GAPDH activity was detected in a 20-min enzyme assay, indirectly indicating the presence of the protein on the cell surface (Fig. 5a). In order to quantify the GAPDH protein on the cell surface, the fluorescence of GAPDH immunolabeled with FITC was measured in intact conidia. Fluorescence corresponding to 2.4 times more GAPDH protein was detected in disrupted cells as compared with intact cells, indicating a markedly higher internal protein concentration (Fig. 5b). Adhesion assays showed that 71% (2279±246.0) of the WT conidia adhered to D. peruvianus fly wings could not be washed off with 0.05% Tween 20. When conidia were treated with anti-GAPDH serum before wing exposure, only 1.3% (30.07±4.959) (P<0.0001) adhered, showing that the antiserum efficiently blocked conidial binding to the wing.

The cFn comprises a large group of isoforms produced from splicing events that may or may not include the type III repeats called

extra domains, EDA and EDB, lacking in pFn (Pankov & Yamada, 2002). It is currently not known whether the presence of the extra domains or suprastructural organization is responsible for the selective binding of cFn to Scl1 protein. In addition to cFn, P176 also bound Lm. The cFn and Lm binding to P176 was concentration-dependent, indicating binding specificity (Fig. 1a, inset). The laminins comprise a family of A, B1, and B2 heterotrimeric glycoproteins that are constituents of basal lamina and are found in virtually all human tissues (Alberts et al., 1994). Various isoforms of laminin exist that are associated with characteristic see more tissue distribution. Early studies by Switalski et al. (1984) described GAS binding to Lm, although the GAS product responsible for this binding was not identified. Terao et al. (2002) identified a GAS Lm-binding protein, designated Lbp, which was recently characterized as primarily a zinc-binding protein with capacity to bind Lm (Linke et al., 2009). GAS interactions with Lm were also attributed to another streptococcal protein Shr that primarily binds human plasma hemoproteins (Fisher et al., 2008). Thus, unrelated surface proteins of GAS possess binding capacities toward ECM components Fn and Lm. Because both cFn and

Lm contain the collagen-binding Pexidartinib manufacturer domains (Alberts et al., 1994), we could not exclude a possibility that the CL region of Scl1 was responsible for ECM binding. Therefore, we constructed a chimeric recombinant protein by domain swapping consisting of the V-region of P176 and the CL-region of the ECM-binding negative protein P163. The resulting construct P181 bound cFn and Lm, indicating that ECM binding is mediated by the P176 V-region (Fig. 1a). We next devised a competition Dynein assay to investigate whether cFn and Lm binding is localized to the same site within the P176 V-region (Fig. 1b). First,

immobilized P176 was incubated with one of the primary ECM ligands, cFn or Lm, and then incubated with an alternate secondary ECM ligand. Sets of triplicate wells were immunoreacted with antibodies specific for both ECM ligands to assess the presence of cFn and Lm attached to P176. Immunoreactivities of the same amounts of P176-cFn and P176-Lm were considered as 100% binding (Fig. 1b; bars 1–2). Preincubation of P176 with cFn did not prevent Lm binding (Fig. 1b; bar 4); nor did Lm displace the cFn from P176 (Fig. 1b; bar 3). Likewise, preincubation with Lm did not prevent cFn binding to P176 (Fig. 1b; bar 5); nor was cFn able to displace the Lm from P176 (Fig. 1b; bar 6). Our data suggest that under these experimental conditions, the cFn and Lm did not compete for binding to P176. Binding between the rScl1.

The signalling molecules involved in growth stimulation were identified and domesticated variants emerged that were capable of independent growth after repeated cultivation in coculture with helper strains. It is likely that such combinatorial approaches will be required in the future to further

improve the range of bacterial life on Earth that can be cultured in the laboratory. “
“Transposon http://www.selleckchem.com/products/PF-2341066.html mutagenesis of Bacillus cereus ATCC 14579 yielded cold-sensitive mutants. Mutants of genes encoding enzymes of the central metabolism were affected by cold, but also by other stresses, such as pH or salt, whereas a mutant with transposon insertion in the promoter region of BC0259 gene, encoding a putative DEAD-box RNA helicase displaying homology with Escherichia coli CsdA and Bacillus subtilis CshA RNA helicases, was only cold-sensitive. Expression of the BC0259 gene at 10 °C is reduced in the mutant. Analysis of the 5′ untranslated region revealed the transcriptional start and putative cold shock-responsive elements. The role of this

RNA helicase in the cold-adaptive response of B. cereus is discussed. Bacillus cereus is a Gram-positive, endospore-forming bacterium that frequently causes emetic and diarrhoeal types of food-borne illnesses. The growth domains of strains of B. cereus sensu lato range from nearly thermophilic to psychrotrophic, LY2109761 and correlate with several phylogenetic clusters (Guinebretiere et al., 2008). The psychrotrophic strains are shared between two genetic groups: Group VI includes all Bacillus weihenstephanensis strains (Francis et al., 1998; von Stetten et al., 1998) having a low ability to cause gastroenteritis (Choma et al., 2000; Guinebretiere et al., 2008), whereas psychrotolerant strains of Group II have been associated with food poisoning outbreaks (Stenfors & Granum, 2001; Arnesen

et al., 2008; Guinebretiere et al., 2008). Bacillus cereus food-borne poisonings are the result of ingestion of foods supporting a high rate of multiplication of the bacterium and adaptation to low temperatures mafosfamide in case of refrigerated storage. Cold shock proteins are involved in B. cereus low-temperature adaptation, for instance in B. weihenstephanensis strains, where CspA protein appears to be strongly induced during low-temperature continuous growth and cold shock (Mayr et al., 1996). This protein may act as a chaperone to block the formation of RNA secondary structures at a low temperature. Bacillus cereus adapts membrane fluidity during low-temperature growth by increasing the proportion of branched-chain fatty acids and decreasing their equivalent chain length, and increasing the anteiso-/iso-branched ratio and the proportion of unsaturated fatty acids (Haque & Russell, 2004). Other mechanisms are likely involved as described for other bacteria. No functional evidence for the role of a gene in the cold adaptation of B. cereus has been obtained so far.

Lipodystrophy was evaluated according to this categorization in the face, arms, legs, buttocks, abdomen, neck and breasts. The sum of the values corresponding to each corporal zone indicated the degree of lipodystrophy: nonexistent (0), slight (1–6), moderate (7–12) and severe (13–18). In this study we included only moderate and severe cases in order to avoid an overlap between the LD+ and LD− subsets. The LD+ group comprised 26 patients with pure lipoatrophy and 106 patients with R428 price the mixed type. No cases of pure lipohypertrophy were recorded.

With respect to severity, 109 had moderate and 23 had severe lipodystrophy. After an overnight fast, 20 mL of blood obtained from a peripheral vein was collected in Vacutainer™ (Becton Dickinson Immunocytometry Systems, San Jose, CA, USA) ethylenediaminetetraacetic acid (EDTA) tubes. Five millilitres of whole blood was used to determine the CD4 T-cell count. Five hundred microlitres was used for DNA isolation with a MagNa Pure LC Instrument (Roche Diagnostics, PD0325901 in vivo Basel, Switzerland). Plasma and serum were obtained by centrifugation at 3500 g for 15 min at 4 °C and stored at −80 °C until use. HIV-1 infection and plasma HIV-1 viral load were assessed as described elsewhere [14]. The CD4 T-cell count was determined using a flow cytometer FAC Scan (Becton Dickinson Immunocytometry Systems). Data acquired were analysed using the multiset program

(Becton Dickinson Immunocytometry Systems). Plasma glucose, total cholesterol, HDL cholesterol and triglycerides were determined in an ADVIA 1200 (Siemens AG, Munich, Germany) auto-analyser using standard enzyme methods. Low-density lipoprotein (LDL) cholesterol was calculated using

the Friedewald formula [16]. Fasting plasma insulin was measured Methane monooxygenase using a specific immunoradiometric assay (Medgenix Diagnostics, Fleunes, Belgium) in which proinsulin did not cross-react. The intra- and inter-assay coefficients of variation (CVs) were 6% and 7%, respectively. The homeostasis model assessment of insulin resistance (HOMA-IR) as a marker for insulin resistance was calculated according to the formula [fasting glucose (in millimoles per litre) × fasting insulin (in microunits per millilitre)/22.5] [17]. Soluble tumour necrosis factor receptor 1 (sTNF-R1) and sTNF-R2 were assessed as previously described [18]. Adiponectin levels were measured using a standardized radioimmunoassay kit from Linco Research (Linco Research Inc., St. Charles, MO, USA). The kit has a sensitivity of 1 ng/mL. The intra- and inter-assay CVs were 8% and 12%, respectively. Plasma FABP-4 was measured using the Human Adipocyte FABP ELISA (BioVendor Laboratory Medicine, Palackeho, Czech Republic). The sensitivity was 0.1 ng/mL. The intra- and inter-assay CVs were 5.2% and 3.8%, respectively. The leptin concentration in plasma was determined with a Human Leptin ELISA kit (Assaypro, St Charles, MO, USA); the lowest detectable level was 0.15 pg/mL with an intra-assay CV of 4.

We recently managed a healthy 27-year-old French soldier returning from a 4-mo mission in Ivory Coast. He reported taking doxycycline 100 mg/d regularly during his stay but stopped the drug 1 wk after returning to France. One month after the last doxycycline dose, he started experiencing fever and fatigue. At admission 2 wk later, he had hypotension (85/45 mm Hg), thrombocytopenia (72 × 109/L), moderate renal failure (plasma creatinine,

152 µmol/L), moderate hepatic cytolysis (aspartate aminotransferase, 179 IU/L and alanine aminotransferase, R428 solubility dmso 128 IU/L), systemic inflammation (C-reactive protein, 86 mg/L and procalcitonin, 23.3 ng/mL), and a normal chest X-ray. A blood smear was positive only for Plasmodium malariae (0.2% parasitemia). Severe malaria and leptospirosis were suspected. Rapid fluid resuscitation, norepinephrine, intravenous quinine (loading dose, 1,400 mg over 4 h; then maintenance dose, 2,000 mg/d), and ceftriaxone were selleckchem given. The patient became comatose

and developed severe metabolic acidosis (lactate, 13 mmol/L; pH 6.97), requiring endotracheal mechanical ventilation. No other infections were identified by extensive microbiologic investigations including blood, cerebrospinal fluid, and urine cultures, and serological tests for hepatitis A, B, C, and E viruses, HIV, leptospirosis, Rickettsia conorii, Coxiella burnetti, and hemorrhagic fever viruses (including West Nile

and dengue viruses). Guidelines for treating severe falciparum malaria were followed,1 and the patient recovered fully. Nested polymerase chain reactions (PCRs) of the SSUrRNA gene with specific species primers were performed at the French Malaria Reference Center and were negative for both Plasmodium falciparum and Plasmodium knowlesi 3-mercaptopyruvate sulfurtransferase but positive for P. malariae.2 Nested PCRs with specific species primers followed by sequence analysis of the pLDH gene confirmed the diagnosis of P. malariae monoinfection.3 PCR testing found no evidence of a simian malaria species such as P. knowlesi. Before admission, the patient received no curative antimalarial drug that might have cleared a P. falciparum infection already responsible for organ dysfunction, as confirmed by the military medical personnel and by plasma antimalarial drug assays. Nevertheless, we cannot definitively rule out a bacterial coinfection because the first blood culture was drawn after administration of the first antimicrobial dose. As severe malaria due to pure P. malariae infection is infrequent, genetic polymorphisms associated with severe sepsis were investigated.

Our findings may provide a novel perspective on the pathogenic mechanism associated with biofilms

of F. oxysporum f. sp. cucumerinum. “
“The collection of 146 Staphylococcus epidermidis strains isolated from the nasopharynx of lung cancer patients has been studied for the ability of slime secretion and biofilm formation using the Congo red agar (CRA) test and the microtiter plate (MtP) method, respectively. The prevalence of the icaAD and the aap genes was also analyzed. Anti-infection Compound Library Some isolates (35.6%) were biofilm positive by the MtP method, while 58.9% of isolates exhibited a slime-positive phenotype by the CRA test. The sensitivities of the CRA test evaluated using the MtP method as a gold standard of biofilm production were 73.1%, 97.3% and 13.3% for all the strains screened, ica-positive and ica-negative strains, respectively. The genotype ica+aap+ was correlated with a strong biofilm-producer phenotype. Interestingly, some of the ica−aap− isolates could also form a biofilm. The correlation between the presence of icaAD genes and the biofilm-positive phenotype by the MtP method as well as

slime production by the CRA test was statistically BIBW2992 significant (P<0.0001). However, some S. epidermidis strains possess the potential ability of ica-independent biofilm formation; thus, further studies are needed to determine reliable, short-time criteria for an in vitro assessment of biofilm production by staphylococci. The coagulase-negative staphylococci (CoNS), including Staphylococcus epidermidis, Leukocyte receptor tyrosine kinase are a major component of the normal microbial communities of the human body, colonizing preferably the upper airways and skin. These opportunistic pathogens rarely cause

infections in a normal host; however, in recent years, CoNS have generally been accepted as important nosocomial pathogens, especially in patients with predisposing factors such as an indwelling or an implanted foreign body. The biomaterial-associated staphylococcal infections are more resistant to the host immune response and antimicrobial chemotherapy at a standard dosage. For these reasons, such infections are very difficult to eradicate (O’Gara & Humphreys, 2001; Hamilton, 2002; Cerca et al., 2005). The ability of S. epidermidis to form a biofilm represents the most important virulence determinant (Götz, 2002; Vuong & Otto, 2002). Several studies have been undertaken in order to determine the genetic and/or the environmental factors responsible for in vitro biofilm formation by S. epidermidis, the leading opportunistic pathogen involved in infections associated with biomaterials. A number of reports (Ziebuhr et al., 1997; Galdbart et al., 2000; McKenney et al., 2000; Mack et al., 2004, 2007; Maira-Litran et al., 2004; Rohde et al., 2005; Stevens et al., 2008) have highlighted that the ica and aap genes, known determinants of polysaccharide- and protein-mediated biofilm production, are widespread among clinically significant S.

An experimenter blind to the treatment groups performed all cell counts. Differences in these cell counts between groups and over circadian time were analysed using independent group two-way anovas, with ZT and genotype as the grouping variables, using Prism 5 for Mac OSX (v. 5.0c, 2009, GraphPad Software,

Inc., La Selleckchem Epacadostat Jolla, CA, USA). A total of 62 WT and GHSR-KO mice were transferred from the colony room to individual cages equipped with an activity wheel (Lafayette Instruments, Lafayette, IN, USA), and connected to a computer running Activity Wheel Monitor Software Running (Lafayette Instruments). Wheel activity was measured in 6-min bins throughout the experiment. Mice were housed in DD or LL for a minimum of 10 days, before being killed at one of four CT points (n = 3 or 4 animals per light cycle, genotype and time point) equally distributed over the rest–activity cycle. Circadian times were calculated using the last 10 days (2400 bins) of activity and producing an actogram, using Plot (R. Refinetti; http://www.circadian.org/softwar.html).

Period length and acrophase were calculated using the Tau (v. 6.5, Mar. 2006) and Acro (v. 3.5, Jan. 2004) programs (R. Refinetti; http://www.circadian.org/softwar.html), using a χ2 periodogram procedure and a fitted cosign wave function, respectively. These variables were used to produce an eye-fitted line projecting the time of activity offset (defined

as CT0), the midpoint of GSK J4 price the rest period (CT6), activity onset (CT12) or the mid-point of the active period (CT18). Whenever possible, pairs of animals consisting of one WT and one KO were killed at the same time by injection with an overdose of sodium pentobarbital and processed for immunocytochemistry as described above. All animals in this experiment were kept under DD or LL for at least 10 days, but some animals were kept for > 10 days due to the varying amounts of time required to assign animals to the appropriate eltoprazine CT time. Therefore, in order to standardise the behavioural analysis, calculations for activity levels (number of wheel revolutions), tau (Tau v. 6.5; Refinetti, 2006) and acrophase (Acro v. 3.5; Refinetti, 2004) were made on the first 2400 bins (10 days) of activity. A total of 22 GHSR WT and KO mice were individually housed in running wheel-equipped cages (Lafayette Instruments). All animals were allowed to acclimate to the equipment and lighting schedule under ad libitum feeding conditions for several days before beginning scheduled feeding (see below). A total of 10 animals (five WTs and five KOs) were exposed to an LD schedule (lights on at 02:00, lights off at 14:00 h) for 14 days followed by a 6-h delay of the LD (on at 08:00, off at 20:00 h), a few days of a 25-h day, and finally 24-h exposure to LL for ≈ 45 days (30 days ad libitum food access, followed by 16 days restricted feeding).

SSU-rDNA sequence buy NVP-BKM120 (GenBank accession no. EU710822) and used for the amplification of the nuclear SSU-rDNA, were designed from alignment of an orthologous gene from 10 fungal species. The PCRs were performed in a programmable thermal cycler GeneAmp® 2720 (Applied Biosystems). Amplifications were carried out in 50-μL reaction mixtures as described by Mouhamadou et al. (2008). Reactions were run for 40 cycles at 95 °C for 30 s (denaturation step), 4 °C below the Tm of both primers for 30 s (annealing step) and 72 °C for 2 min (elongation step). A final elongation for 10 min

at 72 °C was included at the end of the 40th cycle. PCR products were sequenced by Cogenics (Meylan, France). Comparisons with sequences of the GenBank databases were made using the blast search algorithm (Altschul et al., 1990). Alignments of nucleotide sequences were carried out using clustal w software (Thompson et al., 1994). Phylogenetic analyses were carried out with the entire sequences of the cox1 exonic sequences or the SSU-rDNA gene. The trees were obtained using the neighbor-joining method (Saitou & Nei, 1987), deriving from matrices of distances based on the

distance model proposed by Kimura (1983). The robustness of tree topologies was evaluated by performing bootstrap analysis of 1000 data sets using mega 3.1 (Tamura et al., 2007). In this article, we focused our study on the genera possessing multiple species to investigate the potential of the cox1 gene in their discrimination. All the isolates were first identified by their morphological characteristics buy KU-60019 using microscopic observations, and we chose isolates that were identified unambiguously. These isolates belong to four

and two genera of Ascomycota and Zygomycota, respectively (Table 1). We included in the Pseudogymnoascus genus, species belonging to Gymnostellatospora (Gy) that are phylogenetically related to Pseudogymnoascus and differ only by the forms of ascospores and species belonging to Geomyces, which are the anamorphs of Pseudogymnoascus. In the same way, Umbelopsis ramanniana was included in the phylogenetically remote genus Mucor. To determine the conserved primers for the amplification of the partial cox1 gene of fungal species, we chose nine complete cox1-coding sequences available in the GenBank Isotretinoin and representative of the Fungal Kingdom (Table 2). The alignment of these sequences has shown two regions possessing a high percentage of nucleotide identity (>70%) between them, allowing the design of two antiparallel oligonucleotides. The effectiveness of these primers was tested using a bioinformatic approach on the sequences of the GenBank database by setting a maximum size of PCR product of 2500  bp. The partial cox1 sequences of 25 species distributed among the phyla Ascomycota, Basidiomycota, Zygomycota and Chytridiomycota and for which the complete cox1 sequences are available could be amplified with sizes ranging from 626 to 2143 nt (Table 2).

The majority of axonal mitochondria were stationary for 3 h at both developmental stages, with a small number of appearance (red arrowheads) and disappearance (white arrowheads) events. The mitochondrial population identified at t = 0 min Alectinib solubility dmso progressively changed their positions with time. The fraction of mitochondria that remained at their initial positions was calculated as a position survival rate

P(t) (see ‘Materials and methods’). To examine the relationship between the proximity to presynaptic sites and mitochondrial dynamics, P(t) was measured from mitochondria near presynaptic sites (synaptic) and also away from presynaptic sites (non-synaptic; Fig. 3D and E). Because mitochondria found at t = 0 min included both stationary and mobile mitochondria, Δ(P(0) − P(180)) was not

an appropriate estimate of mitochondria that started to move during the 180 min observation period. PTC124 clinical trial Instead, we used Δ(P(30) − P(180)) as an index of the transition from stationary to mobile state (Fig. 3G and H). Using this index, we found that synaptic mitochondria were less likely to restart translocation than non-synaptic mitochondria at both developmental stages (2 weeks, t14 = 4.32, P < 0.001; 3 weeks, t12 = 3.57, P = 0.004; unpaired t-test; Fig. 3H). Both synaptic and non-synaptic mitochondria were less likely to transit to mobile state at 3 weeks than at 2 weeks (all, t13 = 9.65, P < 0.001; synaptic, t13 = 8.05, P < 0.001; non-synaptic, t13 = 4.89, P < 0.001; unpaired t-test; Fig. 3H). The treatment of neurons at 20 DIV with the sodium channel blocker TTX increased the transition probability to mobile state (3 weeks + TTX, 3297 mitochondria from n = 7 experiments; Erastin all, t12 = 4.72, P < 0.001; unpaired t-test; Fig. 3C,E,F and H). This effect was present in both synaptic and non-synaptic mitochondria (synaptic, t12 = 3.95, P = 0.002; non-synaptic, t12 = 3.88, P = 0.002; unpaired t-test; Fig. 3H). These results suggest that neuronal maturation, proximity

to synaptic sites and neuronal activity affect the stability of stationary mitochondria in the axon. We estimated the fraction of mobile mitochondria at t = 0 min [mobile fraction; calculated from P(t) at t = 0, 30 and 60 min; see Eqn (3) in 'Materials and methods'] (Fig. 3G). The mobile fraction at 3 weeks was smaller than at 2 weeks (t13 = 4.98, P < 0.001; unpaired t-test; Fig. 3I) and at 3 weeks with TTX (t12 = 3.82, P = 0.002; unpaired t-test; Fig. 3I). These results suggest that the ratio of mobile to stationary mitochondria in the axon was dependent on neuronal maturation and activity. In time-lapse imaging over 3 h, the majority of axonal mitochondria imaged at the initial time point remained stationary throughout the experiments (Fig. 3D–F), suggesting that the duration of stationary state is usually longer than several hours.

The majority of axonal mitochondria were stationary for 3 h at both developmental stages, with a small number of appearance (red arrowheads) and disappearance (white arrowheads) events. The mitochondrial population identified at t = 0 min www.selleckchem.com/products/pexidartinib-plx3397.html progressively changed their positions with time. The fraction of mitochondria that remained at their initial positions was calculated as a position survival rate

P(t) (see ‘Materials and methods’). To examine the relationship between the proximity to presynaptic sites and mitochondrial dynamics, P(t) was measured from mitochondria near presynaptic sites (synaptic) and also away from presynaptic sites (non-synaptic; Fig. 3D and E). Because mitochondria found at t = 0 min included both stationary and mobile mitochondria, Δ(P(0) − P(180)) was not

an appropriate estimate of mitochondria that started to move during the 180 min observation period. Metabolism inhibitor Instead, we used Δ(P(30) − P(180)) as an index of the transition from stationary to mobile state (Fig. 3G and H). Using this index, we found that synaptic mitochondria were less likely to restart translocation than non-synaptic mitochondria at both developmental stages (2 weeks, t14 = 4.32, P < 0.001; 3 weeks, t12 = 3.57, P = 0.004; unpaired t-test; Fig. 3H). Both synaptic and non-synaptic mitochondria were less likely to transit to mobile state at 3 weeks than at 2 weeks (all, t13 = 9.65, P < 0.001; synaptic, t13 = 8.05, P < 0.001; non-synaptic, t13 = 4.89, P < 0.001; unpaired t-test; Fig. 3H). The treatment of neurons at 20 DIV with the sodium channel blocker TTX increased the transition probability to mobile state (3 weeks + TTX, 3297 mitochondria from n = 7 experiments; Carbohydrate all, t12 = 4.72, P < 0.001; unpaired t-test; Fig. 3C,E,F and H). This effect was present in both synaptic and non-synaptic mitochondria (synaptic, t12 = 3.95, P = 0.002; non-synaptic, t12 = 3.88, P = 0.002; unpaired t-test; Fig. 3H). These results suggest that neuronal maturation, proximity

to synaptic sites and neuronal activity affect the stability of stationary mitochondria in the axon. We estimated the fraction of mobile mitochondria at t = 0 min [mobile fraction; calculated from P(t) at t = 0, 30 and 60 min; see Eqn (3) in 'Materials and methods'] (Fig. 3G). The mobile fraction at 3 weeks was smaller than at 2 weeks (t13 = 4.98, P < 0.001; unpaired t-test; Fig. 3I) and at 3 weeks with TTX (t12 = 3.82, P = 0.002; unpaired t-test; Fig. 3I). These results suggest that the ratio of mobile to stationary mitochondria in the axon was dependent on neuronal maturation and activity. In time-lapse imaging over 3 h, the majority of axonal mitochondria imaged at the initial time point remained stationary throughout the experiments (Fig. 3D–F), suggesting that the duration of stationary state is usually longer than several hours.