The calibrated AZD6738 standard serum 89-SF was absorbed with 10 μg/mL CWPS. At least two internal controls were added to each plate. For serotypes 14 and 19F, a high-titre and a low-titre serum sample from

pneumococcal vaccine responders were used as internal controls, respectively. For serotype 23F, two high-titre and two low-titre serum samples from pneumococcal vaccine responders were used as internal controls. The coefficients of variation (CVs) for the high-titre controls were 19.53 and 16.55% for serotypes 14 and 19F and the CVs were 18.34 and 17.91% for serotype 23F. The CVs for the low-titre controls were 17.12 and 14.91% for serotypes 14 and 19F and the CVs were 15.39 and 14.23% for serotype 23F. Sera from persons with AIDS who had no antibodies to S. pneumoniae serotype 14, 19F, 23F or 6B capsules and <1 μg of antibody to CWPS per mL were used as negative controls. Capsular polysaccharides from S. pneumoniae serotypes 14 (catalogue number 197-X; lot #2038310), 19F (catalogue number 205-X; lot #2033178), 23F (catalogue number 217-X; lot #2048448) and 6B (catalogue number 225-X; lot #2045655) were obtained from the American

Type Culture Collection (ATCC; Manassas, VA, USA). These capsular polysaccharides were suspended in phosphate-buffered saline (PBS; pH 7.4) containing 0.02% NaN3 at a concentration of 10 μg/mL and used directly to coat wells

CDK inhibitor by incubation at 4 °C overnight. After washing of the antigen-coated plates, the pre-absorbed patient sera or controls were added to plates and incubated at room temperature for 2 h. After thorough washing to remove antibodies not bound to the wells, horseradish peroxidase (HRP)-conjugated goat antibody to human IgG (Zymed Laboratories Inc., San Francisco, CA, USA) at a dilution of 1:2000 was used to detect IgG, and the reaction was developed for 10 min in the dark by the addition of K-blue substrate (Neogen selleck chemical Corporation, Lexington, KY, USA), followed by the addition of 1N sulphuric acid to stop the reaction. Between incubations, all washings were performed with Tris buffered saline (TBS) buffer containing 0.1% Brij solution. Optical density was read in an ELISA reader (SpectraMAX 340; Molecular Devices, Sunnyvale, CA, USA) at a wavelength of 450 nm, with subtraction of the optical density of the appropriate blank. The antibody concentrations were calculated relative to the 89-SF control serum, using the US Centers for Disease Control and Prevention (CDC) program elisa for Windows (version 2.15) [28]. The detection limits for our assay for 14, 19F, 23F and 6B, determined using the control serum 89-SF, were 0.02, 0.015, 0.02 and 0.50 μg/mL, respectively.

, 1997), both of the pathways for nitrate reduction to ammonia are expressed only during anaerobic growth. Transcription of narGHJI and

nirBD is also activated by the NarX-NarL two-component regulatory system in response to moderate concentrations of nitrate; nirBD, and to a much lesser extent narGHJI, are also activated by the alternative two-component system, NarQ-NarP (Rabin & Stewart, 1993). Classical genetic approaches and more recent whole genome transcriptomic studies have indicated that the cytoplasmic pathway is physiologically more significant only in nitrate-rich environments that might occur in soil, in some highly contaminated sediments, and waste water treatment plants (Potter et al., 1999). In contrast, the transcription of genes for the periplasmic Nap-Nrf pathway Wnt inhibition is activated by NarQ-NarP in response to low concentrations of nitrate (< 100 μM) click here but are repressed by NarX-NarL when nitrate is abundant (Page et al., 1990).

This indicates that the periplasmic pathway confers a selective advantage for bacterial survival in the nitrate limited environment of the gastro-intestinal tract of humans and other warm blooded animals (Potter et al., 1999; Constantinidou et al., 2006). Based upon the accumulation of very small quantities of nitrous oxide during nitrite reduction, it was assumed that the rate of NO production was two to three orders of magnitude slower than the rate of nitrite reduction (Smith, 1983).

It was predicted that NO was a side product released during science nitrite reduction by either NirBD or NrfA. However, there is an extensive literature showing that the major source of nitrosative stress is NO generated by the interaction of the cytoplasmic nitrate reductase, NarG, with nitrite (reviewed in the accompanying paper by Vine et al., 2011). Realization that enteric bacteria can reduce nitrite to NO re-opened the question whether NO is generated by a single mechanism or by more than one pathway, depending on the conditions under which the bacteria are grown. Specifically, is more NO generated by the membrane-associated nitrate reductase, NarG, by one of the nitrite reductases, NirBD or NrfAB, or by other molybdoproteins that are active during anaerobic growth? The sensitive response of the transcription repressor, NsrR, to NO provides a method to detect the presence of NO in the bacterial cytoplasm (Hutchings et al., 2000; Corker & Poole, 2003; Bodenmiller & Spiro, 2006; Tucker et al., 2008). By coupling an NsrR-regulated E. coli promoter to lacZ expression during anaerobic growth in the presence of nitrite, it was shown that mutations in nirBD or nrfAB resulted in greater expression of lacZ, indicative of the increased accumulation of NO in the cytoplasm (Vine et al., 2011). Conversely, deletion of the narGHJI operon significantly decreased but did not eliminate lacZ expression, indicative of less accumulation of cytoplasmic NO.

Arousal was not formally assessed in our study, e.g. by scores or skin conductance responses. Therefore, we cannot make judgements regarding the level of arousal. However, the fact that there was a matching in the behavioural results of the tasks does aid the interpretation of the motor data in that any differences seen for the two behavioural conditions are a consequence of differences relating to underlying processes in performing them (presumably related to the differences in external and internal attention) rather than potentially a result of different associated difficulties. Whatever

the final explanation, the results are of relevance to a number of different disorders. As noted in the Introduction, focal dystonia often appears to be associated with the repeated performance of movements made under conditions of highly focussed attention, selleck chemicals such as occur in professional musicians. Indeed, attention is an important part of learning. However, too great a focus on one area may reduce inhibitory control in other areas and potentially contribute to an overflow of activity. In healthy individuals, this is often seen in the early phases of learning a see more new skill, but this is gradually reduced as learning progresses. It may that this natural process is defective in focal dystonia and leads to the persisting and unwanted activity characteristic

of the condition. It is remarkable how widespread is the range of disorders that involve abnormal SICI, e.g. dystonia (Sommer et al., 2002), Tourette’s syndrome (Orth & Rothwell, 2009), and first-episode schizophrenia (Wobrock et al., 2008). The interpretation tends to be that intracortical GABAA circuits per se are impaired. The

current study demonstrates a modulation towards a reduced amount of SICI when healthy participants pay attention to an internal or external locus. Therefore, the reduced inhibition seen in so many disorders might, in some cases, be explained by differences in cognitive states (attention state) rather than being a genuine physiological marker. A practical relevance of the present results seems more striking. High levels of attention are required for learning that interacts with synaptic plasticity processes (Ziemann et al., 2004). Behavioural data are supported by experimental methods that demonstrate the Idoxuridine interaction between attention and plasticity-inducing protocols (Stefan et al., 2004) that are facilitated by directing the subject’s attention to their own hand. This might be mediated via the fine tuning of inhibitory and excitatory circuits in the M1. A necessity of all goal-directed movements is the right balance between inhibiting and facilitating components. To reach an overall economical activation it is vital to be able to relax, for example, antagonistic muscles. The playing-related health problems of musicians are often the end-stage of suboptimal learning processes.

Arousal was not formally assessed in our study, e.g. by scores or skin conductance responses. Therefore, we cannot make judgements regarding the level of arousal. However, the fact that there was a matching in the behavioural results of the tasks does aid the interpretation of the motor data in that any differences seen for the two behavioural conditions are a consequence of differences relating to underlying processes in performing them (presumably related to the differences in external and internal attention) rather than potentially a result of different associated difficulties. Whatever

the final explanation, the results are of relevance to a number of different disorders. As noted in the Introduction, focal dystonia often appears to be associated with the repeated performance of movements made under conditions of highly focussed attention, Trametinib solubility dmso such as occur in professional musicians. Indeed, attention is an important part of learning. However, too great a focus on one area may reduce inhibitory control in other areas and potentially contribute to an overflow of activity. In healthy individuals, this is often seen in the early phases of learning a PF2341066 new skill, but this is gradually reduced as learning progresses. It may that this natural process is defective in focal dystonia and leads to the persisting and unwanted activity characteristic

of the condition. It is remarkable how widespread is the range of disorders that involve abnormal SICI, e.g. dystonia (Sommer et al., 2002), Tourette’s syndrome (Orth & Rothwell, 2009), and first-episode schizophrenia (Wobrock et al., 2008). The interpretation tends to be that intracortical GABAA circuits per se are impaired. The

current study demonstrates a modulation towards a reduced amount of SICI when healthy participants pay attention to an internal or external locus. Therefore, the reduced inhibition seen in so many disorders might, in some cases, be explained by differences in cognitive states (attention state) rather than being a genuine physiological marker. A practical relevance of the present results seems more striking. High levels of attention are required for learning that interacts with synaptic plasticity processes (Ziemann et al., 2004). Behavioural data are supported by experimental methods that demonstrate the click here interaction between attention and plasticity-inducing protocols (Stefan et al., 2004) that are facilitated by directing the subject’s attention to their own hand. This might be mediated via the fine tuning of inhibitory and excitatory circuits in the M1. A necessity of all goal-directed movements is the right balance between inhibiting and facilitating components. To reach an overall economical activation it is vital to be able to relax, for example, antagonistic muscles. The playing-related health problems of musicians are often the end-stage of suboptimal learning processes.

4%), C18:1 ω7c (19.8%), and C16:0 (17.0%). The DNA G + C content was 48.6 mol%. The 16S rRNA gene sequence analysis indicated that strain KU41ET is affiliated with the order Alteromonadales within the class Gammaproteobacteria and is most closely related to Pseudoteredinibacter isoporae SW-11T (93.6% similarity) and Teredinibacter turnerae T7902T (91.9% similarity). On the basis of physiological, chemotaxonomic, and phylogenetic data, strain KU41ET is suggested to represent a novel species of a new genus, for which the name Maricurvus nonylphenolicus gen. nov., sp. nov. is proposed. The type strain of M. nonylphenolicus is KU41ET (=JCM 17778T). Contamination of the marine

environment with alkylphenols is of great public concern because of their toxicity and endocrine disrupting activity in humans and marine organisms (David et al., 2009). A number of alkylphenol-degrading AZD9291 order bacteria have been isolated and characterized (Fujii et al., 2001; Ushiba et al., 2003), and the mechanism for alkylphenol degradation has been studied extensively (Corvini et al., 2006; Takeo et al., 2006; Porter & Hay, 2007). However, these Y27632 organisms have mainly been isolated from terrestrial or freshwater sites, and information regarding alkylphenol-degrading bacteria from marine environments is relatively scarce. Here, we report on the isolation and characterization of a novel

marine p-n-nonylphenol-degrading bacterium, strain KU41ET. Comparative 16S rRNA gene sequence analysis indicated that strain KU41ET forms an independent branch within Gammaproteobacteria. Accordingly, the aim of the present work was to determine the exact taxonomic position of strain KU41ET by a polyphasic characterization that included Bortezomib supplier phenotypic and chemotaxonomic properties and detailed phylogenetic analysis based on the 16S rRNA gene sequence. A p-n-nonylphenol-degrading bacterial strain

designated KU41ET was isolated from seawater collected from the coastal region of Ishigaki Island in Japan in December 2009. Marine bacteria were collected from 1 L of the seawater sample by filtration using the membrane filters (diameter 47 mm, pore size 0.45 μm; Nihon Millipore) and then suspended in 3 mL of the commercial artificial seawater medium Daigo’s IMK-SP, which was made by dissolving 252 mg of IMK medium in 1 L of Daigo’s Artificial Seawater SP (Nihon Seiyaku). A 1-mL suspension of the sample was inoculated into 4 mL of Daigo’s IMK-SP supplemented with 10 mM p-n-nonylphenol and incubated at 25 °C on a rotary shaker at 100 r.p.m. After 7 days of enrichment, 4 μL of the culture medium was transferred into a fresh medium and incubated for seven more days. The enriched culture was plated on the same medium solidified with 1.5% (w/v) agar, and the strain was purified by transferring the colony several times onto fresh agar plates. To completely isolate the p-n-nonylphenol-degrading bacterium, a colony was transferred onto a plate of Marine Agar 2216 (MA; Becton Dickinson).

1 and the possible significance of the histidine-rich C-terminal tail in selecting these polypeptide substrates. In

GroEL, the C-terminal tail is highly flexible and thus undefined in the crystal structures (Hartl & Hayer-Hartl, 2002; Machida et al., 2008). However, a detailed genetic analysis of the final 23 residues assessing the ability of C-terminal-truncated, double- and single-ring mutants to assist the refolding of rhodanese and malate dehydrogenase showed that this domain defines the environment within the central cavity and in particular its hydropathicity, features that would impact on both the size and nature of the substrate protein folded by the chaperonin (Tang et al., 2006; Machida et al., 2008). This is consistent with a role for the mycobacterial Cpn60.1 click here chaperonins in the folding EPZ-6438 supplier of a distinct class of proteins, possibly unique to mycobacteria or actinomyces. Although a distinct DNA-bound function in the assembly of the nucleoid has recently been proposed for Cpn60.1 (Basu et al., 2009) this is unlikely to involve the C-terminal tail sequence, as the mitochondrial Hsp60 chaperonin for which nucleotide binding has also been reported does not have a histidine-rich C-terminal tail (Kaufman et al., 2003; Basu et al., 2009). A database search with the histidine-rich C-terminal sequence of Cpn60.1 reveals highly homologous proteins across

all mycobacterial species, as well as Corynebacteria, Nocardia and Rhodococcus (C. Colaco, unpublished data). A common feature of all these Actinobacteria is their synthesis of a complex cell wall containing mycolic acid derivatives, and this suggests the intriguing possibility that the biological role of the mycobacterial Cpn60.1 may be to chaperone the folding of key enzymes involved in the synthesis Fossariinae of mycolic acid. Such a role for Cpn60.1 is also consistent with the defects

in mycolates and biofilm formation observed in the cpn60.1 knockouts in M. smegmatis, where the protein was also found to be associated with KasA and SMEG4308, both key enzymes implicated in biofilm formation and involved in fatty acid synthesis (Tang et al., 2006; Kumar et al., 2009). In this respect, it is interesting to note that the oligomerisation of Cpn60.1 has been shown to be facilitated by phosphorylation (Canova et al., 2009), which is thought to be mediated by the serine threonine protein kinases that have also been implicated in biofilm formation (Gopalaswamy et al., 2008). Finally, as KasA has been identified as an important drug target for the development of new drugs against TB (Brown et al., 2009), the most interesting implication of the suggested role of Cpn60.1 is that this novel mycobacterial chaperonin may present an upstream target for drug development. Thus, therapeutics that target Cpn60.

7% β-Gal activity was observed using 100 μM 2,2′-dipyridyl, an iron chelator) compared with high-iron conditions find more (50 μM FeCl3 and 50 μM haem, 34% and 26% β-Gal activity, respectively) (Fig. 4). The results suggested that repression of mbfA by IrrAt does not require iron or haem as a cofactor. To further identify amino acid residues that are important for the iron sensing of IrrAt in mediating the derepression of mbfA, the iron responsiveness of the mutant IrrAt proteins (pIRR38, pIRR45, pIRR65,

pIRR86, pIRR92, pIRR93, pIRR94, pIRR105, pIRR127, pIRRHHH and pIRRHHH86) in the iron regulation of mbfA-lacZ was compared with wild-type IrrAt (pIRR) (Fig. 4). A single mutation in IrrAt at H38, D86, H92, H93 or D105 led to a hyper-repressed phenotype in which the expression of mbfA-lacZ was low and was not derepressed in response to iron and haem (Fig. 4). These residues appeared to play a role in the iron responsiveness of IrrAt. Although single mutations at H45, H65 and H127 reduced the repressor activity of IrrAt, the mutant proteins still retained iron responsiveness (Fig. 4). In contrast, the H94 mutant protein showed a greater reduction in repressor activity, and its iron responsiveness was lost (Fig. 4). The results suggested that H45, H65, H94 and H127 may play a role in the DNA-binding Selleck Ibrutinib ability

of IrrAt. Moreover, H94 was involved in iron sensing by IrrAt. The iron responsiveness was also lost in the HHH mutant protein, which likely resulted from the mutation at H94 (Fig. 4). The HHH86 mutant protein showed a hyper-repressed phenotype (Fig. 4). In conclusion, site-directed mutagenesis analysis revealed that residues H45, H65, H94 and H127 and the HHH motif are important for the repressor function of IrrAt. Mutations at these key residues may cause changes in protein conformation, preventing the protein from functioning properly. Single mutations at H38, D86, H92, H93 and D105 led to a hyper-repressed phenotype

(Fig. 4), implying that these residues MycoClean Mycoplasma Removal Kit may be directly or indirectly involved in the iron or haem binding and the iron-responsive regulatory function of IrrAt. Interestingly, only mutation at D86 was able to restore the repressor function of IrrAt that lacked the HHH motif (Fig. 2b). Residue D86 of IrrAt is equivalent to E80 of FurPa (the structural zinc-binding site) and to E90 of FurHp (regulatory site S2) (Fig. 1). It is possible that the conformation of the HHH mutant protein may undergo further structural modifications due to the mutation of D86 such that the HHH86 mutant protein is readily able to interact with DNA and may lock the protein in its DNA-binding conformation, resulting in loss of iron responsiveness. Residue H94 of IrrAt is a part of the conserved HHH motif (Fig. 1), which is a domain involved in haem sensing and in the IrrBj and IrrRl regulatory switch through different mechanisms (Qi et al.

2a) with the IC50 value of 28 μM. Subsequently, the inducer concentrations for sensitizing PT44 clone against fusidic acid (which targets EF-G) were further optimized (Fig. 2b). Our results indicated that at 45 μM IPTG, the asRNA clone exhibits 12-fold increase (IC50 at 0 μM divided by IC50 at 45 μM) in MK-2206 mw sensitivity to the specific inhibitor (Fig. 2b). The optimized cell-based assay was performed against serial dilutions of nine other antibiotics (Fig. 2c). Results showed that the fusA asRNA clone was the most sensitive to

fusidic acid (12-fold), followed by erythromycin (fivefold) and tetracycline (fourfold), both are well-known antibiotics targeting protein synthesis (Fig. 2c). It was recognized that conditional silencing by introduced asRNAs in Gram-negative

bacteria is less efficient than in Gram-positive bacteria (Wagner & Flardh, 2002). Specifically, while global essential genes in S. aureus (Ji et al., 2001; Forsyth et al., 2002) and S. mutans (Wang & Kuramitsu, 2005) have Crizotinib datasheet been identified by regulated asRNAs, the adoption of such approach in Gram-negative bacteria has not been reported (Good & Stach, 2011). Although the reasons for such discrepancy are not well defined, one possible explanation lies in the reduced stability of plasmid-borne artificial asRNAs in E. coli probably due to the presence of RNase E in this bacterium (Xu et al., 2010), but not in S. aureus. For this reason, Nakashima and colleagues (Nakashima et al., 2006) designed a series of E. coli plasmid vectors which produce RNA molecules with paired-termini to increase the asRNA stability G protein-coupled receptor kinase and conditional gene silencing. Targeted antisense fragment cloning using such paired-termini vectors has produced asRNA constructs which have shown to knock-down

or silence the expression of a number of essential genes in E. coli (Nakashima et al., 2006). In this communication, we report a genome-wide application of regulated asRNA expression in E. coli using the vector pHN678. Here, we demonstrated that employing this paired-termini vector indeed identified a large number of asRNA constructs targeting E. coli essential genes and, to a lesser extent, some nonessential genes which share operons with essential genes. While asRNA constructs targeting essential genes of a number of cellular processes in E. coli were identified (Table 1 and Table S1), particularly striking was the observation that the asRNAs predominately silence the expression of essential genes (77% of total genes) involved in protein synthesis processes (tRNAs, tRNA synthetases, transcription, ribosomal proteins, and translation factors) (Table S1). We speculate that this bias may have been caused by high basal level (leaky) promoter (Ptrc) activity from the vector in the absence of IPTG (Nakashima & Tamura, 2009) during the library transformation process.

4A]. Neurons were without any obvious damage to the axonal, mitochondrial and synaptic selleck screening library morphology during observation. Although the mitochondrial distribution was rearranged,

the density of mitochondria did not change (normalised by 4 day average: day 1, 99.0 ± 1.4%; day 2, 97.4 ± 5.9%; day 3, 101.0 ± 1.4%; day 4, 102.6 ± 1.4%, eight experiments). This further supported the absence of damage to the imaged neurons. With a longer imaging duration, the rearrangement of mitochondrial distribution increased (Fig. 4A). To quantify the long-term stability of axonal mitochondria, we measured P(t) in the same way as we did in the time-lapse imaging for 3 h (Fig. 4B). Synaptic mitochondria again showed higher stability than non-synaptic mitochondria. P(t) was fitted by the single exponential decay equation (Eqn (2) in ‘Materials and methods’). By this curve fitting, we could obtain both the time constant for P(t) decrease and an offset value (Table 1). An offset indicates the size of a mitochondrial fraction immobile on time scales of several days. The time constants and offsets that we obtained by curve fitting should be consistent with the results from the time-lapse imaging for 3 h. We used the time constants and offsets to calculate

estimated Δ(P(30) − P(180)) and compared them with the experimentally obtained Δ(P(30) − P(180)) (Table 1). All three estimated Δ(P(30) − P(180)) this website matched reasonably well with the actual data from time-lapse imaging for 3 h. Although statistically insignificant, there was a small tendency for the estimated Δ(P(30) − P(180)) to be smaller than the experimental data for all conditions. This may reflect the reappearance of mitochondria at the same position within a day (Fig. 4A, arrowheads), which causes underestimation of the P(t) decrease with time. We therefore concluded that 57% of synaptic mitochondria were considered to be ‘potentially mobile’ with an expected duration of prolonged pause of approximately 2.4 days. The remaining 42% of synaptic mitochondria were immobile on time scales of several

days. The expected duration of stationary mitochondria that were localised near Bupivacaine synaptic sites (approximately 2.4 days) was twofold longer than that of non-synaptic mitochondria (approximately 1.0 days in 78% of total non-synaptic mitochondria). To determine whether the stability of synaptic mitochondria was related to the size of nearby synapses, the relationships between the fluorescence intensities of EGFP-VAMP2 puncta and mitochondrial localisation frequency near synaptic sites were examined (Fig. 4C). Only presynaptic sites that existed for 4 days were analysed and the total or maximum consecutive number of days in which mitochondria were co-localised was examined. Stationary mitochondria near presynapses with higher EGFP-VAMP2 fluorescence intensity showed higher stability.

In summary, long-term follow-up data from the MONARK trial show that LPV/r monotherapy was able to maintain sustained viral suppression in 38 of the 56 patients who had already achieved HIV RNA <50 copies/mL at week 48. These results confirm those from previous studies which indicated that boosted PI monotherapy has

the ability to induce and maintain viral suppression in most patients [16]. However, first-line LPV/r monotherapy appeared somewhat less active than standard triple Metformin therapy, and some patients showed persistent low-level viraemia. Higher levels of adherence may be required for constant suppression with LPV/r monotherapy than with LPV/r-containing combination therapy. Persistence of low-level viraemia with LPV/r monotherapy may increase the risk of selection

Sirolimus research buy of drug-resistant viruses, whereas combination therapy with LPV/r is considered to rarely select for PI resistance in antiretroviral-naïve patients [17,18]. Long-term follow-up data from the MONARK study indicate that major PI-associated resistance mutations emerged in five of 83 patients after 40–90 weeks on treatment. Of note, however, three of five patients who were found to harbour selected major PI resistance mutations remained on LPV/r and underwent treatment intensification with NRTIs and achieved resuppression to HIV RNA <50 copies/mL, suggesting that control of viral replication could still be achieved with a PI/r-containing regimen. An important concern regarding LPV/r monotherapy is the possible lack of efficacy

in reservoirs, particularly the central nervous system (CNS) and male genital tract. LPV/r is highly protein bound to plasma proteins in blood. Poor penetration RNA Synthesis inhibitor of LPV/r through the blood–brain and blood–testis barriers has therefore been expected. Recently, a preliminary analysis reported an unexpected high failure rate on LPV/r monotherapy, which may be related to residual replication in the CNS compartment [19]. However, other data suggest significant activity for LPV/r monotherapy, at therapeutic concentrations, in the CNS [20]. In the context of a triple-drug regimen, low concentrations of LPV/r were found to inhibit HIV replication in this compartment, as the concentrations reached in the cerebrospinal fluid exceeded the 50% inhibitory concentration (IC50) for wild-type virus [21,22]. Of note, no neurological event was reported throughout the 96-week follow-up period in patients randomized to first-line LPV/r monotherapy in the MONARK trial. Furthermore, in a substudy of MONARK analysing the impact of 1 year of LPV/r monotherapy in the male genital tract, no local viral production was evident in the semen, despite undetectable local drug concentrations [23]. Limitations of this analysis include its open-label design. An additional limitation is the noncomparative analysis at week 96 because of the higher rate of premature terminations in the triple combination arm.