Please note: Wiley-Blackwell is not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article. “
“One of the major successes in the management of HIV-positive patients has been the PMTCT of HIV-1. With the widespread implementation of routine antenatal screening for HIV-1, transmission of HIV-1 from mother

to child is now a rare occurrence in the UK. Despite few recent RCTs regarding the use of ART in pregnancy or obstetric intervention, practice continues to evolve. This is largely informed by observational data, theoretical considerations and expert opinion. At the outset, the aim of the Writing Group was to make these guidelines as clinically relevant and as practical

as possible. The Writing Selleck VE822 Group drew up a list of questions reflecting day-to-day practice and queries. It was acknowledged that the level of evidence for many of these topics was poor but recognized that there was a need to provide guidance. These guidelines have expanded on all areas relevant to the clinical care of HIV-positive pregnant women. The guidelines are intended to inform and aid healthcare workers in the management of pregnant women with HIV. They are not intended to be prescriptive or restrictive and it is recognized that situations will arise where the optimum management may deviate from these recommendations and new data will emerge to better inform practice. A particular FK506 research buy focus has been obstetric management. An increasing number

of women are aiming for and achieving a vaginal delivery but the rate of emergency CSs has increased. It is hoped that the recommendations contained within these guidelines will enable a further increase in the proportion of vaginal deliveries and a reduction in the number of emergency CSs. Linked to this is the proposed starting gestation for women temporarily taking HAART in pregnancy, which has been brought forward depending on baseline VL. It is anticipated that this will result in a larger proportion of women achieving a VL <50 HIV RNA copies/mL by 36 weeks' gestation, thereby allowing them to plan for a vaginal Thymidylate synthase delivery. Additional guidance has been provided with regard to conception on HAART, the choice of specific drugs or drug classes and the management of women with HBV or HCV coinfection. For the first time these guidelines have addressed the issue of continuation of HAART post delivery in women with a baseline CD4 cell count >350 cells/μL. The paediatric section provides further guidance on infant PEP, drug dosing and safety. It is clear that there exists an urgent need for paediatric syrup preparations for a wider variety of ARV drugs because the current options, particularly in the case of maternal viral resistance, are limited.

Among the uncultured EPZ5676 order Prevotella, 60 clones (43.2%) had 92–96% similarity to previously reported sequences (Table 4). The Chao1 and Shannon indices predicted more diversity in the hay library (Table 4), and libshuff comparison showed significant (P=0.001) differences in the composition of the two libraries (data not shown). Of the 17 clones that showed ≥97% sequence similarity with known Prevotella species, 16 clones were retrieved from concentrate-fed

sheep (Table 4) and 11 clones were related to P. ruminicola, while five were related to P. bryantii. Only a single clone from the hay diet was related to P. ruminicola at 97% sequence similarity. No sequences having ≥97% similarity with P. brevis and P. albensis were found. The results of phylogenetic analysis of 16S rRNA gene sequences from the two libraries are shown in HDAC inhibitor Fig. 2. Although the bootstrap values were <50%, we divided the phylogenetic tree into seven sections to show the distribution of the clones. Sixty-six out of 79 clones from the concentrate library were found in sections 1 and 3; meanwhile, sections 4–7 contained 42 clones from the hay library. Hay clones were distributed in all sections of the tree. Application of molecular biological tools in the analysis of several environmental microbial communities revealed that only a small fraction of the microbiota is represented by cultured species (Janssen, 2006) and the rumen microbial community is no exception. A previous

study indicated that from only 11% of OTU detected in the rumen contain cultured representatives (Edwards et al., 2004). We focused on the population dynamics, ecology and diversity of Prevotella in order to estimate the contribution of this genus to digestion of feed in the rumen. Real-time PCR quantification revealed that the proportion of two representative Prevotella species (P. ruminicola and P. bryantii) was one-quarter of that of the genus (4.4% vs. 19.7% for concentrate-fed sheep). This result indicates

that Prevotella is abundant in the rumen and the majority of members of this genus are yet to be cultured. It was reported that the abundance of the other two ruminal Prevotella spp. (P. brevis and P. albensis) was negligible (Stevenson & Weimer, 2007). Similar to the other reports on rumen bacterial clone library analysis (Whitford et al., 1998; Tajima et al., 1999; Koike et al., 2003), we did not find the sequences of these two species in our clone libraries. Therefore, P. brevis and P. albensis seemed to be minor in the rumen, and they were not quantified. The high proportion of Prevotella observed in the present study agrees with the report of Wood et al. (1998), who estimated the combined Prevotella/Bacteroides ribotypes in the rumen in the range of 12–62%. The numerical dominance of Prevotella spp. reported in different experiments (Van Gylswyk, 1990; Wood et al. 1998; Stevenson & Weimer, 2007) suggests their importance in the ruminal digestion of feed.

, 1998). All E. coli strains were grown overnight in LB broth at 37 °C with aeration. Twenty microliters of cultures were mixed with or without 0.5% BE. The mixtures were then spread onto nematode growth media

agar plates (Hope et al., 1998). The plates were dried at 25 °C and immediately utilized for the assays. Twenty nematodes previously synchronized on the L4 stage were transferred to each plate and incubated at 25 °C. After every 24 h, live worms were scored. When the worms did not respond to being touched by a platinum wire pick, they were considered dead. Data are expressed as mean±SD. An unpaired Student’s learn more t-test was used to analyze the data. To compare differences among more than three groups, one way anova was used. A P-value of <0.05 was considered statistically significant. All the experiments were repeated for reproducibility. AI-2-mediated QS plays a major role in the virulence of E. coli O157:H7 (Sperandio et al., 2001; Sircili et al., 2004). To investigate the specific effect of the

BE on QS, we measured the level of AI-2 secreted by E. coli O157:H7 in response to the treatment with BE. When assayed using V. harveyi AI-2 reporter strain BB170, a decreasing level of AI-2 was detected in culture supernatants of E. coli O157:H7 Selumetinib cell line grown with increasing concentrations of BE. Figure 1a shows a dose-dependent decrease in AI-2 level upon treatment with BE. It is of note that AI-2 level was almost undetectable in the presence of 5% BE. AI-2 level at each treatment normalized to that obtained from growth with no BE (Fig. 1a). We then tested C. violaceum strain CV026, which produces violacein, a violet pigment, as a result of QS through its autoinducer N-hexanoyl homoserine lactone (McClean et al., 1997). Violacein production in the presence of BE was also gradually decreased in a dose-dependent manner (Fig. 1b), suggesting that BE is also capable of inhibiting QS of C. violaceum CV026. To rule out the possibility that reduced production of AI-2 is a consequence of decreased bacterial growth, we examined whether or not BE exhibited any adverse effects on bacterial growth. Figure 1c compares

the growth curves of E. coli O157:H7 during 8 h cultures in LB without or with 5% BE. In our experiments, stationary phase was achieved after ∼6 h of culture. Interleukin-2 receptor Growth of E. coli O157:H7 was elevated by the addition of BE (Fig. 1c). The bacterial culture reached OD600 nm of ∼5.0 after 6 h of growth in plain LB, whereas bacterial cell density reached OD600 nm of ∼5.7 in LB media amended with BE. Taken together, these results demonstrate that suppressed AI-2 production was not due to any secondary effects associated with retarded bacterial growth and occurred rather efficiently even at higher cell density. It has been reported that swarming motility is dependent on AI-2 signaling in E. coli O157:H7 (Sperandio et al., 2002). To test whether the reduced AI-2 synthesis by BE treatment is reflected in bacterial motility, a swarming motility assay was performed.

, 2007). These signaling pathways do not function independently but influence each other through a complex network of synergistic and antagonistic Ixazomib in vitro interactions (Koornneef & Pieterse, 2008). Trichokonins upregulated the expression of SA-responsive PR gene acidic NtPR1a, ethylene-responsive gene basic NtPR3 and the key player in activating the JA signaling pathway, NtCOI1 (Fig. 4b). These results suggested

that multiple defense pathways are involved in Trichokonin-induced resistance in tobacco against TMV. Likely, cross-talk between the different defense pathways occurs. In summary, we studied the antiviral effect of Trichokonins against TMV infection and the mechanism involved. Trichokonins from T. pseudokoningii screening assay SMF2 can induce tobacco systemic resistance against TMV via activation of multiple plant defense pathways. The results imply the potential of peptaibols in plant viral disease control. This work was supported by Hi-Tech Research and Development program of China (2007AA091504),

National Natural Science Foundation of China (30870047) and Foundation of State Key Lab of Microbial Technology, Shandong University, China. Table S1. Primers used for RT-PCR analysis in tobacco plants. Please note: Wiley-Blackwell is not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article. “
“Berberine,

a natural isoquinoline alkaloid found in many medicinal herbs, has been shown to be active against a variety of microbial infections. To examine the potential effects of berberine on Shigella flexneri, a whole-genome DNA microarray was constructed and a transcriptome analysis of the cellular responses of S. flexneri when exposed to berberine chloride (BC) was performed. Our data revealed that BC upregulated a group of genes involved in DNA replication, repair and division. Intriguingly, the expression of many genes related to cell envelope biogenesis Histone demethylase was increased. In addition, many genes involved in cell secretion, nucleotide metabolism, translation, fatty acid metabolism and the virulence system were also induced by the drug. However, more genes from the functional classes of carbohydrate metabolism, energy production and conversion as well as amino acid metabolism were significantly repressed than were induced. These results provide a comprehensive view of the changes in gene expression when S. flexneri was exposed to BC, and shed light on its complicated effects on this pathogen. Shigella is a gram-negative, facultative, intracellular pathogen responsible for endemic shigellosis, which remains a major worldwide health problem, particularly in developing countries. The estimated annual incidence of this disease is 160 million individuals, most of whom are children, and the annual mortality is 1.1 million (Kotloff et al., 1999).

These DNA polymerases perform translesion DNA synthesis (TLS) when a replication fork has collapsed at a blocking lesion, whereas the DNA synthesis by specialized Y-family polymerases Pol IV and Pol V is considered error prone (Sutton & Walker, 2001; Goodman, 2002; Nohmi, 2006). Each of

these three DNA polymerases is specialized for polymerization through different structural classes of DNA damage (Wagner et al., 2002; Nohmi, 2006). Pol II is a high-fidelity enzyme possessing proofreading activity (Cai et al., 1995). However, it can perform error-prone TLS across various types of damage in CDK inhibitor review template DNA (Nohmi, 2006). On the other hand, although Y-family polymerases Pol IV and Pol V are APO866 concentration typically viewed as low-fidelity DNA polymerases, recent studies suggest that they can perform proficient and moderately accurate bypass of particular types of DNA damage (Jarosz et al., 2007). For example, Pol IV is involved in the error-free bypass of cytotoxic alkylating DNA lesions (Bjedov et al., 2007). Escherichia coli cells exposed to UV irradiation have increased mutation frequency that is dependent on Pol V. It was noted already in the late 1970s that E. coli strains lacking genes umuD and umuC are modestly sensitive to UV irradiation and do

not express the UV mutagenesis phenotype (Kato & Shinoura, 1977; Steinborn, 1978). The next studies of the DNA damage-induced mutagenesis and tolerance mechanisms favored the idea that UmuC and UmuD modulate the replicative DNA polymerase, Pol III, and allow it to bypass base damage (Echols & Goodman, 1990; Rajagopalan et al., 1992). Then, in 1999, it was demonstrated that UmuD2′C is a DNA polymerase that provides mutagenic TLS across DNA damage (Reuven et al., 1999; Tang et al., 1999). In the classic model of the E. coli SOS response, the LexA protein represses a set of genes whose products are involved in

a number of different cellular processes, such as inhibition of cell division, nucleotide excision repair (NER), homologous recombination or error-prone replication (Courcelle et al., 2001). The SOS response is a tightly regulated process, and it is temporally divided into an early, relatively accurate DNA repair phase and a later, mutagenic damage-tolerance phase (Opperman et Methisazone al., 1999). Pol V (encoded by umuDC genes) is the most error-prone SOS-inducible DNA polymerase of E. coli, and this is a reason why Pol V is induced only about 45 min after the DNA damage and if the damage is not fully repaired by high-fidelity pathways such as NER and homologous recombination (Tippin et al., 2004). The products of the umuD gene play key roles in coordinating the switch from accurate DNA repair to mutagenic TLS. The uncleaved UmuD2 dimer, which appears early after SOS induction, together with UmuC, delays the recovery of DNA replication and cell growth after DNA damage (Opperman et al., 1999).

However, the C- and N- terminal regions were conserved. Except for a region on the flagellum surface, structural predictions of type I and II flagellins revealed that Regorafenib the two flagellin types were strongly correlated with each other. Phylogenetic analysis of the 115-amino acid N-terminal sequences revealed that the Actinoplanes species formed three clusters, and type II flagellin gene containing three type strains were phylogenetically closely related each other. The genus Actinoplanes (Couch, 1950; Stackebrandt & Kroppenstedt, 1987) is a member

of the family Micromonosporaceae (Krasil’nikov, 1938; Zhi et al., 2009), and is characterized by the presence of spherical, subspherical, cylindrical or very irregular sporangia (Lechevalier et al., 1966). The motile sporangiospores move by means of polar or peritrichous flagella (Couch, 1950). The flagellated spores exhibit chemotactic properties and are attracted to a variety of substrates, including those that contain bromide or chloride ions (Palleroni, 1976), fungal conidia, chlamydospores, sclerotia, or exudates of these (Arora, 1986), γ-collidine, d-Xylose, and pollen (Hayakawa et al., 1991a, b). Phylogenetic analyses based on the 16S rRNA gene sequences of members of the family Micromonosporaceae revealed that motile genera, such

as the Actinoplanes, do CAL-101 order not form coherent clusters or linaeages (Inahashi et al., 2010). Similarly, other motile actinomycetes were phylogenetically distributed among at least 20 families in the order Actinomycetales. Indeed, these findings indicate that the relationship between phylogeny and the propagation of the gene(s) encoding the flagellar system in prokaryotic organisms, including actinomycetes, is unclear. Bacterial flagella are considered to be composed of three parts: a basal body, a hook, and a filament (Macnab, 1992). The filament is composed of the flagellin protein,

which enough is synthesized internally and transported through the cell membrane to an external site for flagellum assembly (Snyder et al., 2009). The flagellin-encoding gene, fliC, has been used previously as a biomarker in studies of the taxonomy, epidemiology, and virulence of Burkholderia cepacia, Borrelia spp., and Clostridium difficile (Fukunaga & Koreki, 1996; Hales et al., 1998; Tasteyre et al., 2000). However, few studies have been conducted to date on the flagellar protein (Vesselinova & Ensign, 1996; Uchida et al., 2011) of motile actinomycetes. Vesselinova & Ensign (1996) reported that flagellins show two different sizes (32–43 and 42–43 kDa) in Actinoplanes spp. Recent advances in whole genome sequence analysis have facilitated examinations of bacterial flagellar diversity. Snyder et al. (2009) reported the distribution of flagellar genes and the predicted nucleotide sequences of the genes responsible for synthesis of flagellar systems using blastp in a mutual-best-hit approach (e-value < 0.

Similarly, bacteria express a variety of regulatory RNA species ranging from trans-acting RNAs (sRNA), cis-acting RNAs (riboswitches), antisense RNAs and protein-interacting RNAs (6S RNA, CsrB-like RNAs) (Waters & Storz, 2009), and while our knowledge on these species is currently mostly based on E. coli, this is likely to change with the advent of sequencing-based transcriptomics. When combined with Navitoclax in vitro the latest developments in microarray technologies,

like high-density tiling microarrays (Rasmussen et al., 2009; Toledo-Arana et al., 2009), we now have the ability to investigate transcription at single-nucleotide resolution. This is likely to enrich our knowledge of microbial diversity, and will undoubtedly show us the many different approaches used by bacteria to solve the problems encountered in their respective niches. The author thanks the members of the research group and the collaborators, as well as three anonymous reviewers for helpful comments and suggestions. Research at the author’s laboratory is supported by the BBSRC Institute Strategic Programme Grant to the IFR. “
“Carotenoids

are a structurally diverse class of terpenoid pigments that are synthesized by many microorganisms and plants. In this study, we identified five putative carotenoid biosynthetic click here genes from the ascomycete Gibberella zeae (GzCarB, GzCarO, GzCarRA, GzCarT, and GzCarX). HPLC showed that the fungus produces two carotenoids: neurosporaxanthin and torulene. We deleted

the five genes individually to determine their functions. GzCarB, GzCarRA, and GzCarT were required for neurosporaxanthin biosynthesis, but the deletion of GzCarX or GzCarO (ΔgzcarX or ΔgzcarO) failed to alter the production of neurosporaxanthin check details or torulene. ΔgzcarRA and ΔgzcarB did not produce neurosporaxanthin or torulene. ΔgzcarB led to the accumulation of phytoene, which is an intermediate in carotenoid biosynthesis, but ΔgzcarRA did not. ΔgzcarT produced torulene but not neurosporaxanthin. Based on these functional studies and similarities to carotenoid biosynthesis genes in other fungi, we deduced the functions of the three genes and propose the carotenoid biosynthetic pathway of G. zeae. Carotenoids are important natural terpenoid pigments produced by many microorganisms and plants, but not animals. Of the >700 natural carotenoids that have been identified, most are C40 terpenoids that vary in the number of conjugated double bonds, end-group structures, and oxygen-containing functional groups (Britton et al., 2004). The interesting properties and human health benefits of carotenoids have received much attention. Carotenoids exhibit significant anticarcinogenic and antioxidant activity, and play an important role in preventing chronic disease (Landrum & Bone, 2001). Carotenoids are derived from the isoprenoid biosynthetic pathway (Umeno et al., 2005).

The DNA fragment containing the 6× his-tagged-irr

fusion was amplified from pQE30IRR using primers BT3157 and BT3158. The PCR product was digested with EcoRI and then cloned into pBBR1MCS-4 (Kovach et al., 1995), which was digested with EcoRI and SmaI, generating a plasmid named pHIRR. The full-length A. tumefaciens manganese uptake Galunisertib in vitro regulator gene (mur, Atu0354) (Wood et al., 2001) without the start codon was amplified by PCR using primers BT3321 and BT693. A similar protocol as described above was used to construct pQE30MUR and pHMUR. The plasmids pHIRR and pHMUR were transferred to A. tumefaciens cells to produce the 6× His-tagged fusion proteins His-Irr and His-Mur. Site-directed mutagenesis was performed on the irr coding sequence using pIRR or pHIRR as the template and a QuikChange XL mutagenic PCR kit (Stratagene) following the manufacturer’s instructions. Amino acid residues in the candidate metal- and haem-binding sites of Irr protein, including H38, H45, H65, D86, H92, H93, H94, D105 and H127, were mutated to alanine individually or in combination (Table 1). The primers for site-directed mutagenesis are listed in Supporting Information, Table S1. The

mTOR inhibitor mutations were confirmed by DNA sequencing. Exponential growth phase cells were washed and resuspended in minimal Agrobacterium (AB) medium (Cangelosi et al., 1991). The cells were grown for another 1 h and were then harvested. Crude bacterial lysates were prepared as previously described (Kitphati et al., 2007). Protein concentrations were determined using the Bradford Bio-Rad protein assay. The total protein (75 μg) from lysate samples was separated on 12.5% SDS-polyacrylamide gels and transferred onto Hybond-P PVDF membranes (Amersham Pharmacia Biotech) using a Bio-Rad semi-dry blotting apparatus. The recombinant 6× His-tagged proteins were detected using mouse anti-RGS-His monoclonal antibody (Qiagen) and sheep anti-mouse

IgG-HRP conjugate (Qiagen). Proteins were visualized using the Lumi-Lightplus chemiluminescent peroxidase (POD)-substrate (Roche). DNA fragments (378 bp) containing the promoter region of mbfA (Atu0251) GNE-0877 (Wood et al., 2001) were amplified by PCR with primers BT1707 and BT1665. The PCR products were cloned into a promoter probe vector pUFR027lacZ, as previously described (Kitphati et al., 2007), to generate plasmid pPNLZ01. Exponential phase cells were washed and resuspended in minimal AB medium to an OD600 nm of 0.1. Cells were untreated or treated with 50 μM FeCl3, 100 μM 2,2′-dipyridyl (Dipy) or 50 μM haem. The cells were incubated at 28 °C with shaking for 18 h. The cells were harvested and β-galactosidase activity was measured as described previously (Miller, 1972). Specific activity is defined as the units per mg of protein (U mg protein−1) and is expressed as the mean of triplicate samples ± SD. Cells grown on LA for 48 h were washed and resuspended in fresh LB medium.

Stocks of CHIR-99021 datasheet bacterial strains were kept in 20% glycerol at −70 °C. Columbia blood agar (Difco) supplemented with 5% whole human blood was used for routine culture of bacteria. His-tagged recombinant zoocin A was produced from E. coli zooA and purified as described previously (Lai et al., 2002). Two gene targets essential for cellular function were selected because their downregulation

by PS-ODNs would result in inhibition of bacterial growth. FABM (5′-AATTTCCTTAAAATCCAT-3′), FBA (5′-TGCTGAAACGATTGCCAT-3′), and ATS (5′-TCGAATACCGGCGCAACG-3′) were 18-nucleotide PS-ODNs with all internucleotide linkages phosphorothioated. FABM and FBA were designed to complement the ATG start codons of fabM, a gene encoding an see more enoyl-CoA hydratase (Fozo & Quivey, 2004), and fba,

a gene encoding a fructose-bisphosphate aldolase shown to be essential for growth in Streptococcus pneumoniae (Song et al., 2005), mRNA, respectively. FABM was designed to the fabM sequence of S. mutans UA159 (GenBank accession no. AE014133) and FBA was designed to the fba sequences of S. pneumoniae R6 (GenBank accession no. AE007317). ATS was a randomly generated sequence such that there was no extensive complimentary sequence within the UA159 genome. PS-ODNs were synthesized by Shanghai Sangon Biological (China). Stock solutions (100 μM) were prepared in sterile MilliQ water and stored in siliconized tubes (Sigma Chemical Co., St. Louis, MO) at −20 °C until required. The presence or absence of the FABM and FBA target sequences within each bacterial strain were established before examining the inhibitory effect of each PS-ODN on growth. Bacterial chromosomal DNA was extracted 4��8C using a DNeasy Tissue kit (Qiagen, Hilden, Germany) as per the manufacturer’s instructions. PCR was performed using KOD Hot Start DNA polymerase (Novagen, Merck KGaA, Germany) in accordance with the manufacturer’s instructions and primers FfabM (5′-ATGGATTTTAAGGAAATT-3′) and RfabM (5′-ATCATTTGTAAATGCTAA-3′) targeted to fabM or Ffba (5′-ATGGCAATCGTTTCAGCAGA-3′)

and Rfba (5′-TCAGGAATACCTGAACCACCGTG-3′) targeted to fba. PCR products were sequenced by the Allan Wilson Center (Massey University, New Zealand) using a prism ready reaction DyeDeoxy terminator cycle sequencing kit (Applied Biosystems Inc., Warrington, UK). The nucleotide sequences were analysed using editseq (DNASTAR Inc.) and the program blastn (National Centre for Biotechnology Information, Los Alamos, NM). PS-ODNs and zoocin A were serially diluted in THB in siliconized tubes to attain the desired concentrations and 10-μL volumes dispensed into the wells of a 96-well low cell binding microtiter plate (Nalgene NUNC International, Denmark). A 5% inoculum of an overnight culture of the bacterial strain being tested was dispensed into the wells and the total volume of each well was made up to 200 μL with THB.

, 2007). For example, in Fig. 1A, the delay R1 was ∼40 ms and the Gaussian curve peaked at ∼60 ms, thus ∼100 ms after the previous motor unit discharge, i.e. a discharge rate of ∼10 Hz (Bawa & Lemon, 1993). The delay R1 was adjusted according to the

motor unit firing rate, so that TMS was delivered within the recovery phase of the after-hyperpolarization. Thus, when the computer triggered a single TMS pulse at check details delay R1, the effects on the membrane potential of the motoneuron are optimized, and peak(s) appeared in the PSTH (from an FDI unit) 20–35 ms after TMS (Fig. 1B). The(se) peak(s) reflect(s) the arrival of corticospinal input(s) at motoneuron level, and indicate(s) that the resulting corticospinal excitatory post-synaptic potential(s) [EPSP(s)] were sufficient to advance the Selleckchem Carfilzomib discharge of the motoneuron, as compared with its firing rate during voluntary contraction, by shortening the after-hyperpolarisation duration (Fig. 1A and B). The peak in the PSTH is correlated to the ascending phase of the underlying EPSP at motoneuron level (Kirkwood & Sears, 1978; Ashby &

Zilm, 1982). Therefore, the TMS-induced peak in PSTH can be used to estimate the corticospinal EPSP produced at the motoneuron level. The hot spot for FDI and the RMT were determined at the beginning of the experiment. The intensity of both test and conditioning pulses influences the level of SICI (Chen et al., 1998; Sanger et al., 2001; Orth et al., 2003; Roshan et al., 2003; Garry & Thomson, 2009; Lackmy & Marchand-Pauvert, 2010). Therefore, the test pulse intensity was changed so as to evoke a peak in the PSTH of different size (normalized to the number of stimuli, see PSTH analysis below), reflecting corticospinal EPSPs of different size. It was necessary to adjust the intensity of the conditioning pulse to produce SICI without evoking a peak in the PSTH, to prevent possible Bay 11-7085 summation of corticospinal volleys (induced by the test and conditioning pulses) at the motoneuron level. As a consequence, the conditioning pulse could only be set to 0.6 RMT, an intensity at which TMS did not

produce a peak in the PSTH (Fig. 1C) but was sufficient to activate SICI (Fisher et al., 2002). At 0.65 RMT, a peak occurred in the PSTH of some motor units (see Results). A recording session consisted of sequential alternation (0.3 Hz) of isolated test and paired pulses (conditioning + test pulses with a 2-ms interval), to deliver as many test pulses (test peak) as paired pulses (conditioned peak). To avoid muscular fatigue (which can develop rapidly in FDI), 30–50 single and 30–50 paired pulses were delivered during each recording session; the session was stopped when the subjects developed fatigue or had difficulty in maintaining a steady motor unit discharge. Care was taken to ensure that the same motor unit was studied in each session, based on the shape of the potential, its firing rate, the hand position and the movement performed by the subject, and the peak latency in the PSTH.