Search

Background: Telehealth can improve access to evidence-based care at a lower cost for patients, especially those living in underserved and remote areas. The barriers to the widespread adoption of telehealth have been well documented in the literature. However, the barriers may not be the same for pediatric patients, who must rely on their parents or guardians to make healthcare decisions. This paper presents some of the leading barriers parents or guardians of pediatric patients report in using telehealth to meet their children's healthcare needs. Methods: This cross-sectional survey was conducted in a tertiary care pediatric Emergency Department (ED) at a children's hospital in Alabama between September 2020 to December 2020. The parents or guardians of pediatric patients were asked about their reasons for not using telehealth despite having healthcare needs for their children, whether they canceled or rescheduled healthcare provider visits and facility visits, and whether the child's health conditions changed over the past three months. Descriptive analyses were conducted that explored the distribution of telehealth use across the variables listed above. Results: Five hundred ninety-seven parents or guardians of pediatric patients participated in the survey, and 578 answered the question of whether they used telehealth or not over the past three months. Of them, 33.1% used telehealth, 54.3% did not, and 12.6% did not have healthcare needs for their child. The leading reason for not using telehealth was that the doctor or health provider did not give them a telehealth option, the second main reason was that they did not know what telehealth is, and the third leading reason was that the parents did not think telehealth would help meet healthcare needs for their child. Conclusions: This study highlights the telehealth utilization barriers among underserved pediatric populations, including the need for physicians to proactively offer telehealth options to parents or guardians of pediatric patients. Improving health literacy is of paramount importance, given that a substantial proportion of parents were not familiar with telehealth. Policymakers and healthcare organizations should raise awareness about the benefits of telehealth which can improve healthcare access for underserved pediatric patients. [ABSTRACT FROM AUTHOR]

Integrated disease management is the best and most environmentally friendly technique for managing of a plant disease. Among the nine fungicides tested in vitro against Botrytis cinerea, which causes grey mould in Gladiolus, hexaconazole and a combination of iprodione+carbendazim exhibited complete inhibition of the mycelia growth at all the tested concentrations. In the evaluation of 15 different plant oils against B. cinerea, oils of Mentha piperita, Cymbopogon martini, Pelargonium graveolens, Cymbopogon sp., and Oreganum vulgare resulted in 100% mycelial growth inhibition of B. cinerea at all concentrations ranging from 200 to 1000 ppm. Aqueous extract of cloves of Allium sativum resulted best among the different botanicals and bioproducts tested in vitro with a maximum average mycelial growth inhibition of 57.39%, followed by the leaves of Azadirachta indica (45.47%) at 20, 50, 75, and 100 ppm. In integrated management of the grey mould of gladiolus under in vivo conditions, the combination of quintal + A. sativum + neem oil exhibited the maximum reduction in disease (94.40 %), followed by the combination of contaf + A.sativum + neem oil, which showed 93.19% disease reduction. The treatments viz., quintal +A.sativum + neem oil also resulted in the most superior treatment in enhancing growth and yield parameters of gladiolus with significantly improved plant height (70.34 cm), yield of corms (38.00), spike length (53.17 cm), number of florets (15.65), and number of flowers (32.67). These results indicate that the integrated management approach by using fungicides, botanicals and essential oils could be used for the control of grey mould diseases caused by Botrytis pathogens. [ABSTRACT FROM AUTHOR]

Mycobacteria are intrinsically resistant to a variety of stresses including many antibiotics. Although a number of pathways have been described to account for the observed resistances, the mechanisms that control the expression of genes required in these processes remain poorly defined. Here we report the role of a predicted anti-sigma factor, MSMEG_6129 and a predicted eukaryotic like serine/threonine protein kinase, MSMEG_5437, in the intrinsic resistance of Mycobacterium smegmatis to a variety of stresses including the genotoxic agent mitomycin C, hydrogen peroxide and at least four different antibiotics - isoniazid, chloramphenicol, erythromycin and tetracycline. We show that MSMEG_5437 influences the phosphorylation state of MSMEG_6129. Further, MSMEG_6129 controls the expression of a plethora of genes including efflux pumps, ABC transporters, catalases and transcription factors, either directly or via regulators like WhiB7, which account for the observed multi-drug resistance phenotypes. MSMEG_6129 in turn phosphorylates a contiguously located putative anti-anti-sigma factor, MSMEG_6127. We therefore propose that MSMEG_5437, MSMEG_6129 and MSMEG_6127 are components of a master regulatory network, upstream of whiB7, that controls the activity of one or more of the 28 sigma factors in M. smegmatis. Together, this network controls the expression of a regulon required for resistance to several unrelated antibiotics. [ABSTRACT FROM AUTHOR]

Phage-encoded serine integrases mediate directionally regulated site-specific recombination between short attP and attB DNA sites without host factor requirements. These features make them attractive for genome engineering and synthetic genetics, although the basis for DNA site selection is poorly understood. Here we show that attP selection is determined through multiple proofreading steps that reject non-attP substrates, and that discrimination of attP and attB involves two critical site features: the outermost 5–6 base pairs of attP that are required for Int binding and recombination but antagonize attB function, and the “discriminators” at positions −15/+15 that determine attB identity but also antagonize attP function. Thus, although the attachment sites differ in length and sequence, only two base changes are needed to convert attP to attL, and just two more from attL to attB. The opposing effect of site identifiers ensures that site schizophrenia with dual identities does not occur. [ABSTRACT FROM AUTHOR]

At present ΦC31 is the only phage integrase system available for directionally regulated site-specific DNA integration in the Drosophila genome. Here we report that mycobacteriophage Bxb1 integrase also mediates targeted DNA integration in Drosophila with high specificity and efficiency. By alternately using Bxb1 and ΦC31, we were able to carry out multiple rounds of successive and targeted DNA integrations in our genomic engineering founder lines for the purpose of generating complex knock-in alleles. [ABSTRACT FROM AUTHOR]

Bacteriophage-encoded serine-integrases are members of the large family of serine-recombinases and catalyze site-specific integrative recombination between a phage attP site and a bacterial attB site to form an integrated prophage. Prophage excision involves a second site-spedfic recombination event, in which the sites generated by integration, attL and attR, are used as substrates to regenerate attP and attB. Excision is catalyzed by integrase but also requires a phage-encoded recombination directionality factor (RDF). The Bxbl recombination sites, attP and attB, are small (<50 bp), different in sequence, and quasi-symmetrical, and they give rise to attL- and attR-recombinant products that are asymmetric but similar to each other, each being composed of B- and P-type half-sites. We show here that the determination of correct excision products is a two-step process, with a presynaptic ROF-dependent step that aligns attL and attR in the correct orientation and a postsynaptic step in which the nonpalindromic central dinucleotide confers identity to attL and attR and prevents each from recombining with itself. [ABSTRACT FROM AUTHOR]

Mycobacteriophage Bxb1 integrates its DNA at the attB site of the Mycobacterium smegmatis genome using the viral attP site and a phage-encoded integrase generating the recombinant junctions attL and attR. The Bxb1 integrase is a member of the serine recombinase family of site-specific recombination proteins and utilizes small (<50 base pair) substrates for recombination, promoting strand exchange without the necessity for complex higher order macromolecular architectures. To elucidate the regulatory mechanism for the integration and excision reactions, we have identified a Bxb1-encoded recombination directionality factor (RDF), the product of gene 47. Bxb1 gp47 is an unusual RDF in that it is relatively large (∼28 kDa), unrelated to all other RDFs, and presumably performs dual functions since it is well conserved in mycobacteriophages that utilize unrelated integration systems. Furthermore, unlike other RDFs, Bxb1 gp47 does not bind DNA and functions solely through direct interaction with integrase-DNA complexes. The nature and consequences of this interaction depend on the specific DNA substrate to which integrase is bound, generating electrophoretically stable tertiary complexes with either attB or attP that are unable to undergo integrative recombination, and weakly bound, electrophoretically unstable complexes with either attL or attR that gain full potential for excisive recombination. [ABSTRACT FROM AUTHOR]

Recombination by site-specific recombinases is a highly concerted process that requires synapsis of the correct pair of DNA substrates. Phage-encoded serine-integrases are unusual among the serine-recombinase family, which includes transposon resolvases and DNA invertases, in that they utilize two simple but different DNA substrates (attB and attP) and do not require accessory sites, additional proteins, or DNA supercoiling. Synapsis must therefore be directed solely by integrase–DNA interactions. We show here that the Bxb1 serine-integrase binds as a dimer to its two DNA substrates (attB, attP) and recombinant products (attL, attR) with similar affinities. However, synapsis occurs only between attP and attB, and not between any of the other nine possible site combinations. The Bxb1 integrase domain structure, the unusual DNA-binding properties of the integrase, and the characterization of a mutant protein with altered site-discrimination, are consistent with synaptic selectivity being derived from DNA sequence-induced changes in the conformations of integrase–DNA complexes. [Copyright &y& Elsevier]

Integration of the mycobacteriophage Bxb1 genome into its host chromosome is catalyzed by a serine-integrase, a member of the transposon-resolvase family of site-specific recombinases. These enzymes use a concerted mechanism of strand exchange involving double-stranded cleavages with two-base extensions, and covalent protein-DNA linkages via phosphoserine bonds. In contrast to the resolvase/invertase recombination systems—where there are strict requirements for a specific synaptic complex within which the catalytic potential of the enzyme is activated—synapsis of attP and attB by Bxb1 integrase is completely promiscuous, aligning the sites with equal proclivity in parallel and antiparallel alignments. Moreover, the catalytic potential of Bxb1 integrase is fully active in either alignment. As a consequence, the nonpalindomic central dinucleotide (5′-GT) at the center of attP and attB is the sole determinant of Bxb1 prophage orientation, and a single base pair substitution in the two sites is sufficient to eliminate orientation control. [Copyright &y& Elsevier]

Mycobacteriophage Bxb1 is a temperate phage of Mycobacterium smegmatis and forms stable lysogens in which the Bxb1 genome is integrated into the host chromosome. Bxb1 encodes an integrase of the large serine recombinase family that catalyses integration and excision of the Bxb1 genome. We show here that Bxb1 integrates into a chromosomal attB site located within the 3′ end of the groEL1 gene such that integration results in alteration of the C-terminal 21 amino acid residues. An integration-proficient plasmid vector containing the Bxb1 integrase gene and flanking DNA sequences efficiently transforms M. smegmatis via integration at attB. Bxb1-integrated recombinants are stable and fully compatible with L5 integration vectors. Strand exchange occurs within an 8 bp common core sequence present in attB and within an attP site situated immediately upstream of the phage integrase gene. Establishment of a defined in vitro system for Bxb1 integration shows that recombination occurs efficiently without requirement for high-energy cofactors, divalent metals, DNA supercoiling or additional proteins. [ABSTRACT FROM AUTHOR]

Omega (ω), consisting of 91 amino acids, is the smallest of all the Escherichia coli RNA polymerase subunits and is organized into an N-terminal domain of 53 amino acids followed by an unstructured tail in the C-terminal region. Our earlier experiments have shown a chaperone-like function of ω in which it helps to maintain β′ in a correct conformation and recruit it to the α2β subassembly to form a functional core enzyme (α2ββ′ω). The X-ray structure analysis of Thermus aquaticus core RNA polymerase suggests that two regions of ω latch onto the N-terminal and C-terminal ends of the β′-subunit. In the present study we have monitored the conformational changes in β′ as the denatured protein is refolded in the presence and absence of ω using tryptophan fluorescence emission of β′ as well as acrylamide quenching of Trp fluorescence. Results indicate that the presence of stoichiometric amounts of ω is helpful in β′ refolding. We have also monitored the behavior of the C-terminal tail of ω by engineering three cysteine residues at three different sites in ω and subsequently labeling them with a sulphydryl-specific fluorescent probe. Fluorescence anisotropy measurements of the labeled protein indicate that the C-terminal domain of ω is mobile in the free protein and gets restrained in the presence of β′. Calculations on side-chain interactions show that out of the three mutated positions, two have near neighbourhood interactions only with side-chains in the β′ subunit whereas the end of the C-terminal of ω, although it is restrained in the presence of β′, has no interacting partner within a 4-A˚ radius. [Copyright &y& Elsevier]

The ω subunit of Escherichia coli RNA polymerase, consisting of 90 amino acids, is present in stoichiometric amounts per molecule of core RNA polymerase (α2ββ′). The presence of ω is necessary to restore denatured RNA polymerase in vitro to its fully functional form, and, in an ω-less strain of E. coli, GroEL appears to substitute for ω in the maturation of RNA polymerase. The X-ray structure of Thermus aquaticus core RNA polymerase suggests that two regions of ω latch on to β′ at its N-terminus and C-terminus. We show here that ω binds only the intact β′ subunit and not the β′ N-terminal domain or β′ C-terminal domain, implying that ω binding requires both these regions of β′. We further show that ω can prevent the aggregation of β′ during its renaturation in vitro and that a V8-protease-resistant 52-amino-acid-long N-terminal domain of ω is sufficient for binding and renaturation of β′. CD and functional assays show that this N-terminal fragment retains the structure of native ω and is able to enhance the reconstitution of core RNA polymerase. Reconstitution of core RNA polymerase from its individual subunits proceeds according to the steps α + α → α2 + β → α2β + β′ → α2ββ′. It is shown here that ω participates during the last stage of enzyme assembly when β′ associates with the α2β subassembly. [ABSTRACT FROM AUTHOR]

Objectives: There is evidence of substantial declines in pediatric emergency department (ED) utilization in the United States in the first several months of the coronavirus disease 2019 (COVID-19) pandemic. Less is known about whether utilization changed differentially for socioeconomically disadvantaged children. This study examined how changes in pediatric ED visits during the initial months of the COVID-19 pandemic differed by two markers of socioeconomic disadvantage: minoritized race (MR) (compared with non-Hispanic White [NHW]), and publicly insured (compared with privately insured). Methods : This study used electronic medical records from a large pediatric ED for the period January to June 2020. Three time periods in 2020 were compared with corresponding time periods in 2019. Changes in overall visits, visits for MR versus NHW children, and Medicaid-enrolled versus privately insured children were considered, and changes in the acuity mix of ED visits and share of visits resulting in inpatient admits were inspected. Results : Compared with 2019, total ED visits declined in time period (TP) 1 and TP2 of 2020 (54.3%, 48.9%). Declines were larger for MR children (57.3%, 57.8%) compared with NHW children (50.5%, 39.3%), and Medicaid enrollees (56.5%, 52.0%) compared with privately insured (48.3%, 39.0%). The MR children group experienced steeper percentage declines in high-acuity visits and visits, resulting in inpatient admissions compared with NHW children. In contrast, there was little evidence of difference between TP0s of 2019 and 2020. Conclusions : The role of socioeconomic disadvantage and the potential effects on pediatric ED visits during COVID-19 is understudied. Because disadvantaged children sometimes lack access to a usual source of health care, this raises concerns about unmet health needs and worsening health disparities. [ABSTRACT FROM AUTHOR]

Mycobacterium abscessus is intrinsically resistant to antibiotics effective against other pathogenic mycobacteria largely due to the drug-induced expression of genes that confer resistance. WhiB7 is a major hub controlling the induction of resistance to ribosome-targeting antibiotics. It activates the expression of >100 genes, 7 of which are known determinants of drug resistance; the function of most genes within the regulon is however unknown, but some conceivably encode additional mechanisms of resistance. Furthermore, the hierarchy of gene expression within the regulon, if any, is poorly understood. In the present work we have identified 56 WhiB7 binding sites using chromatin immunoprecipitation sequencing (CHIP-Seq) which accounts for the WhiB7-dependent upregulation of 72 genes, and find that M. abscessus WhiB7 functions exclusively as a transcriptional activator at promoters recognized by σA/σB. We have investigated the role of 18 WhiB7 regulated genes in drug resistance. Our results suggest that while some genes within the regulon (eg. erm41, hflX, eis2 and the ABCFs) play a major role in resistance, others make smaller contributions (eg. MAB_4324c and MAB_1409c) and the observed hypersensitivity ΔMabwhiB7 is a cumulative effect of these individual contributions. Moreover, our CHIP-Seq data implicate additional roles of WhiB7 induced genes beyond antibiotic resistance. Finally, we identify a σH-dependent network in aminoglycoside and tigecycline resistance which is induced upon drug exposure and is further activated by WhiB7 demonstrating the existence of a crosstalk between components of the WhiB7-dependent and -independent circuits. Author summary: The induction of multiple genes that confer resistance to structurally diverse ribosome-targeting antibiotics is funneled through the induction of a single transcriptional activator, WhiB7, by antibiotic- stalled ribosomes. This poses a severe restriction in M. abscessus therapy as treatment with one ribosome-targeting antibiotic confers resistance to all other ribosome-targeting antibiotics. Here we uncover the intricacies of the WhiB7 regulatory circuit, identify three previously unknown determinants of aminoglycoside resistance and unveil a communication between WhiB7 dependent and independent components. This not only expands our understanding of the antibiotic resistance potential of M. abscessus but can also inform the development of much needed therapeutic options. [ABSTRACT FROM AUTHOR]

Pediatric Emergency Department (ED) utilization in the U.S. saw large declines during the COVID19 pandemic. What is relatively unexplored is whether the extent of declines differed by race and insurance status. An observational study was conducted using electronic medical record (EMR) data from the largest pediatric ED in Alabama for 2020 and 2019. The four subgroups of interest were African-American (AA), Non-Hispanic White (NHW), privately insured (PRIVATE), and publicly insured or self-insured (PUBLIC-SELF). Percentage changes in the 7-day moving average between dates in 2020 and 2019 were computed for total and high-severity ED visits by subgroup. Trends in percentage changes were plotted. T-tests were used to compare mean changes between subgroups. Large percentage declines in total ED visits and somewhat smaller percentage declines in high-severity visits were observed from March 2020. Declines were consistently larger for AA than NHW and for PUBLIC-SELF than PRIVATE. T-test results indicated mean date-specific percentage declines were significantly larger for AA than NHW for total visits (-38.92% [95% CI: -41.1, -36.8] versus -29.11% [95% CI: -30.8, -27.4]; p<0.001) and high-severity visits (-24.31% [95% CI: -26.2, -22.4] versus -19.49% [95% CI:-21.2, -17.8]; p<0.001), and larger for PUBLIC-SELF than PRIVATE for total visits (-36.32% [95% CI:-38.4, -34.3] versus 27.63% [95% CI:-29.2, -26.0]; p<0.001) and high-severity visits (-21.72% [95% CI: -23.5, -19.9] versus -20.01% [95% CI: -21.7, -18.3]; p = 0.04). In conclusion, significant differences by race and insurance status were observed in the decline in ED visits during the COVID19 pandemic, including high-severity visits. Minority-race and publicly insured or self-insured children often depend on the ED for health needs, lacking a usual source of care. Thus, these findings have worrisome implications regarding unmet healthcare needs and future exacerbations in health disparities. [ABSTRACT FROM AUTHOR]

Abstract: Mycobacteriophage Bxb1 encodes a serine-integrase that catalyzes both integrative and excisive site-specific recombination. However, excision requires a second phage-encoded protein, gp47, which serves as a recombination directionality factor (RDF). The viability of a Bxb1 mutant containing an S153A substitution in gp47 that eliminates the RDF activity of Bxb1 gp47 shows that excision is not required for Bxb1 lytic growth. However, the inability to construct a Δ47 deletion mutant of Bxb1 suggests that gp47 provides a second function that is required for lytic growth, although the possibility of an essential cis-acting site cannot be excluded. Characterization of a mutant prophage of mycobacteriophage L5 in which gene 54 – a homologue of Bxb1 gene 47 – is deleted shows that it also is defective in induced lytic growth, and exhibits a strong defect in DNA replication. Bxb1 gp47 and its relatives are also unusual in containing conserved motifs associated with a phosphoesterase function, although we have not been able to show robust phosphoesterase activity of the proteins, and amino acid substitutions with the conserved motifs do not interfere with RDF activity. We therefore propose that Bxb1 gp47 and its relatives provide an important function in phage DNA replication that has been co-opted by the integration machinery of the serine-integrases to control the directionality of recombination. [Copyright &y& Elsevier]

Structural and topological data suggest that serine site-specific DNA recombinases exchange duplex DNAs by rigid-body relative rotation of the two halves of the synapse, mediated by a flat protein-protein interaction surface. We present evidence for this rotational motion for a simple serine recombinase, the Bxb1 phage integrase, from a single-DNA-based supercoil-release assay that allows us to follow crossover site cleavage, rotation, religation, and product release in real time. We have also used a two-DNA braiding-relaxation experiment to observe the effect of synapse rotation in reactions on two long molecules. Relaxation and unbraiding are rapid (averaging 54 and 70 turns/s, respectively) and complete, with no discernible pauses. Nevertheless, the molecular friction associated with rotation is larger than that of type-I topoisomerases in a similar assay. Surprisingly we find that the synapse can stay rotationally "open" for many minutes. [ABSTRACT FROM AUTHOR]

Antibiotic resistance in bacteria is typically conferred by proteins that function as efflux pumps or enzymes that modify either the drug or the antibiotic target. Here we report an unusual mechanism of resistance to macrolide-lincosamide antibiotics mediated by mycobacterial HflX, a conserved ribosome-associated GTPase. We show that deletion of the hflX gene in the pathogenic Mycobacterium abscessus, as well as the nonpathogenic Mycobacterium smegmatis, results in hypersensitivity to the macrolide-lincosamide class of antibiotics. Importantly, the level of resistance provided by Mab_hflX is equivalent to that conferred by erm41, implying that hflX constitutes a significant resistance determinant in M. abscessus. We demonstrate that mycobacterial HflX associates with the 50S ribosomal subunits in vivo and can dissociate purified 70S ribosomes in vitro, independent of GTP hydrolysis. The absence of HflX in a ΔMs_hflX strain also results in a significant accumulation of 70S ribosomes upon erythromycin exposure. Finally, a deletion of either the N-terminal or the C-terminal domain of HflX abrogates ribosome splitting and concomitantly abolishes the ability of mutant proteins to mediate antibiotic tolerance. Together, our results suggest a mechanism of macrolide-lincosamide resistance in which the mycobacterial HflX dissociates antibiotic-stalled ribosomes and rescues the bound mRNA. Given the widespread presence of hflX genes, we anticipate this as a generalized mechanism of macrolide resistance used by several bacteria. [ABSTRACT FROM AUTHOR]

Rifampicin (RIF), the frontline drug against M. tuberculosis , is completely ineffective against M. abscessus , partially due to the presence of an ADP-ribosyltransferase (Arr) that inactivates RIF. Using RNA-seq, we show that exposure of M. abscessus to sublethal doses of RIF and Rifabutin (RBT), a close analog of RIF, results in an ∼25-fold upregulation of Mab_helR in laboratory and clinical isolates. An isogenic deletion in Mab_helR results in RIF/RBT hypersensitivity, and overexpression of Mab_helR confers RIF tolerance in M. tuberculosis. We demonstrate an increased HelR-RNAP association in RIF-exposed bacteria and a MabHelR-mediated dissociation of RNAP from stalled initiation complexes in vitro. Finally, we show that the tip of the PCh-loop of Mab_helR , present in proximity to RIF, is critical for conferring RIF resistance but dispensable for dissociation of stalled RNAP complexes, suggesting that HelR-mediated RIF resistance requires a step in addition to displacement of RIF-stalled RNAP. [Display omitted] • HelR is a significant effector of rifampicin/rifabutin resistance, independent of Arr • Interaction of HelR with RNA polymerase dissociates stalled initiation complexes • Displacement of RNAP from substrate DNA is insufficient for rifamycin resistance • PCh-loop domain and ATPase activity likely displace RIF for rifamycin resistance Mycobacterium abscessus is highly resistant to rifampicin. Hurst-Hess et al. identify a rifampicin- and rifabutin-inducible gene, helR , deletion of which results in hypersensitivity to both rifamycins, and demonstrate a critical role of the HelR PCh-loop, which binds in close proximity to rifampicin within RNA polymerase, in resistance. [ABSTRACT FROM AUTHOR]

Enzyme rates are usually considered to be dependent on local properties of the molecules involved in reactions. However, for large molecules, distant constraints might affect reaction rates by affecting dynamics leading to transition states. In single-molecule experiments we have found that enzymes that relax DNA torsional stress display rates that depend strongly on how the distant ends of the molecule are constrained; experiments with different-sized particles tethered to the end of 10-kb DNAs reveal enzyme rates inversely correlated with particle drag coefficients. This effect can be understood in terms of the coupling between molecule extension and local molecular stresses: The rate of bead thermal motion controls the rate at which transition states are visited in the middle of a long DNA. Importantly, we have also observed this effect for reactions on unsupercoiled DNA; other enzymes show rates unaffected by bead size. Our results reveal a unique mechanism through which enzyme rates can be controlled by constraints on macromolecular or supramolecular substrates. [ABSTRACT FROM AUTHOR]

Here we report an efficient, site-specific system of genetic integration into Plasmodium falciparum malaria parasite chromosomes. This is mediated by mycobacteriophage Bxb1 integrase, which catalyzes recombination between an incoming attP and a chromosomal attB site. We developed P. falciparum lines with the attB site integrated into the glutaredoxin-like cg6 gene. Transfection of these attB+ lines with a dual-plasmid system, expressing a transgene on an attP-containing plasmid together with a drug resistance gene and the integrase on a separate plasmid, produced recombinant parasites within 2 to 4 weeks that were genetically uniform for single-copy plasmid integration. Integrase-mediated recombination resulted in proper targeting of parasite proteins to intra-erythrocytic compartments, including the apicoplast, a plastid-like organelle. Recombinant attB × attP parasites were genetically stable in the absence of drug and were phenotypically homogeneous. This system can be exploited for rapid genetic integration and complementation analyses at any stage of the P. falciparum life cycle, and it illustrates the utility of Bxb1-based integrative recombination for genetic studies of intracellular eukaryotic organisms. [ABSTRACT FROM AUTHOR]

Corrections to the article "Efficient site-specific integration in Plasmodium falciparum chromosomes mediated by mycobacteriophage Bxb1 integrase," that was published in the previous issue of "Nature Methods" vol. 3, 2006 are presented.