A conifer genome spruces up plant phylogenomics

The Norway spruce genome provides key insights into the evolution of plant genomes, leading to testable new hypotheses about conifer, gymnosperm, and vascular plant evolution.In the past year a burst of plant genome sequences have been published, providing enhanced phylogenetic coverage of green plants (Figure ​(Figure1)1) and inclusion of new agricultural, ecological, and evolutionary models. Collectively, these sequences are revealing some extraordinary structural and evolutionary attributes in plant genomes. Perhaps most surprising is the exceptionally high frequency of whole-genome duplication (WGD): nearly every genome that has been analyzed has borne the signature of one or more WGDs, with particularly notable events having occurred in the common ancestors of seed plants, of angiosperms, and of core eudicots (the latter ''WGD'' represents two WGDs in close succession) [1,2]. Given this tendency for plant genomes to duplicate and then return to an essentially diploid genetic system (an example is the cotton genomes, which have accumulated the effects of perhaps 15 WGDs [3]), the conservation of genomes in terms of gene number, chromosomal organization, and gene content is astonishing. From the publication of the first plant genome, Arabidopsis thaliana [4], the number of inferred genes has been between 25,000 and 30,000, with many gene families shared across all land plants, although the number of members and patterns of expansion and contraction vary. Furthermore, conserved synteny has been detected across the genomes of diverse angiosperms, despite WGDs, diploidization, and millions of years of evolution.Open in a separate windowFigure 1Simplified phylogeny of land plants, showing major clades and their component lineages. Asterisks indicate species (or lineage) for which whole-genome sequence (or sequences) is (are) available. Increases and decreases in genome size are shown by arrows.Despite the proliferation of genome sequences available for angiosperms, genome-level data for both ferns (and their relatives, collectively termed monilophytes; Figure ​Figure1)1) and gymnosperms have been conspicuously lacking - until recently, with the publication of the genome sequence of the gymnosperm Norway spruce (Picea abies) [5]. The large genome sizes for both monilophytes and gymnosperms have discouraged attempts at genome sequencing and assembly, whereas the smaller genome size of angiosperms has resulted in more genome sequences being available (Table ​(Table1)1) [6]. Because of this limited phylogenetic sample, our understanding of the timing and phylogenetic positions of WGDs, the core number of plant genes, possible conserved syntenic regions, and patterns of expansion and contraction of gene families across both tracheophytes (vascular plants) and across all land plants is imperfect. This sampling problem is particularly acute in analyses of the genes and genomes of seed plants; many hundreds of genes are present in angiosperms that are not present in mosses or lycophytes, but whether these genes arose in the common ancestor of seed plants or of angiosperms cannot be determined without a gymnosperm genome sequence. The Norway spruce genome therefore offers tremendous power, not only for understanding the structure and evolution of conifer genomes, but also as a reference for interpreting gene and genome evolution in angiosperms.

Table 1

Genome sizes in land plants

Peptidoglycan Fine Structure of the Radiotolerant Bacterium Deinococcus radiodurans Sark

Peptidoglycan from Deinococcus radiodurans was analyzed by high-performance liquid chromatography and mass spectrometry. The monomeric subunit was: N-acetylglucosamine–N-acetylmuramic acid–l-Ala–d-Glu-(γ)–l-Orn-[(δ)Gly-Gly]–d-Ala–d-Ala. Cross-linkage was mediated by (Gly)₂ bridges, and glycan strands were terminated in (1→6)anhydro-muramic acid residues. Structural relations with the phylogenetically close Thermus thermophilus are discussed.The gram-positive bacterium Deinococcus radiodurans is remarkable because of its extreme resistance to ionizing radiation (14). Phylogenetically the closest relatives of Deinococcus are the extreme thermophiles of the genus Thermus (4, 11). In 16S rRNA phylogenetic trees, the genera Thermus and Deinococcus group together as one of the older branches in bacterial evolution (11). Both microorganisms have complex cell envelopes with outer membranes, S-layers, and ornithine-Gly-containing mureins (7, 12, 19, 20, 22, 23). However, Deinococcus and Thermus differ in their response to the Gram reaction, having positive and negative reactions, respectively (4, 14). The murein structure for Thermus thermophilus HB8 has been recently elucidated (19). Here we report the murein structure of Deinococcus radiodurans with similar detail.D. radiodurans Sark (23) was used in the present study. Cultures were grown in Luria-Bertani medium (13) at 30°C with aeration. Murein was purified and subjected to amino acid and high-performance liquid chromatography (HPLC) analyses as previously described (6, 9, 10, 19). For further analysis muropeptides were purified, lyophilized, and desalted as reported elsewhere (6, 19). Purified muropeptides were subjected to plasma desorption linear time-of-flight mass spectrometry (PDMS) as described previously (1, 5, 16, 19). Positive and negative ion mass spectra were obtained on a short linear ²⁵²californium time-of-flight instrument (BioIon AB, Uppsala, Sweden). The acceleration voltage was between 17 and 19 kV, and spectra were accumulated for 1 to 10 million fission events. Calibration of the mass spectra was done in the positive ion mode with H⁺ and Na⁺ ions and in the negative ion mode with H⁻ and CN⁻ ions. Calculated m/z values are based on average masses.Amino acid analysis of muramidase (Cellosyl; Hoechst, Frankfurt am Main, Germany)-digested sacculi (50 μg) revealed Glu, Orn, Ala, and Gly as the only amino acids in the muramidase-solubilized material. Less than 3% of the total Orn remained in the muramidase-insoluble fraction, indicating an essentially complete solubilization of murein.Muramidase-digested murein samples (200 μg) were analyzed by HPLC as described in reference 19. The muropeptide pattern (Fig. ​(Fig.1)1) was relatively simple, with five dominating components (DR5 and DR10 to DR13 [Fig. 1]). The muropeptides resolved by HPLC were collected, desalted, and subjected to PDMS. The results are presented in Table ​Table11 compared with the m/z values calculated for best-matching muropeptides made up of N-acetylglucosamine (GlucNAc), N-acetylmuramic acid (MurNAc), and the amino acids detected in the murein. The more likely structures are shown in Fig. ​Fig.1.1. According to the m/z values, muropeptides DR1 to DR7 and DR9 were monomers; DR8, DR10, and DR11 were dimers; and DR12 and DR13 were trimers. The best-fitting structures for DR3 to DR8, DR11, and DR13 coincided with muropeptides previously characterized in T. thermophilus HB8 (19) and had identical retention times in comparative HPLC runs. The minor muropeptide DR7 (Fig. ​(Fig.1)1) was the only one detected with a d-Ala–d-Ala dipeptide and most likely represents the basic monomeric subunit. The composition of the major cross-linked species DR11 and DR13 confirmed that cross-linking is mediated by (Gly)₂ bridges, as proposed previously (20). Open in a separate windowFIG. 1HPLC muropeptide elution patterns of murein purified from D. radiodurans. Muramidase-digested murein samples were subjected to HPLC analysis, and the A₂₀₄ of the eluate was recorded. The most likely structures for each muroeptide as deduced by PDMS are shown. The position of residues in brackets is the most likely one as deduced from the structures of other muropeptides but could not be formally demonstrated. R = GlucNac–MurNac–l-Ala–d-Glu-(γ)→.

TABLE 1

Calculated and measured m/z values for the molecular ions of the major muropeptides from D. radiodurans

Intra- and inter-generic transfer of pathogenicity island-encoded virulence genes by cos phages

Bacteriophage-mediated horizontal gene transfer is one of the primary driving forces of bacterial evolution. The pac-type phages are generally thought to facilitate most of the phage-mediated gene transfer between closely related bacteria, including that of mobile genetic elements-encoded virulence genes. In this study, we report that staphylococcal cos-type phages transferred the Staphylococcus aureus pathogenicity island SaPIbov5 to non-aureus staphylococcal species and also to different genera. Our results describe the first intra- and intergeneric transfer of a pathogenicity island by a cos phage, and highlight a gene transfer mechanism that may have important implications for pathogen evolution.Classically, transducing phages use the pac site-headful system for DNA packaging. Packaging is initiated on concatemeric post-replicative DNA by terminase cleavage at the sequence-specific pac site, a genome slightly longer than unit length is packaged, and packaging is completed by non-sequence-specific cleavage (reviewed in Rao and Feiss, 2008). Generalized transduction results from the initiation of packaging at pac site homologs in host chromosomal or plasmid DNA, and typically represents ∼1% of the total number of phage particles. In the alternative cos site mechanism packaging is also initiated on concatemeric post-replicative DNA by terminase cleavage at a sequence-specific (cos) site. Here, however, packaging is completed by terminase cleavage at the next cos site, generating a precise monomer with the cohesive termini used for subsequent circularization (Rao and Feiss, 2008). Although cos site homologs may exist in host DNA, it is exceedingly rare that two such sites would be appropriately spaced. Consequently, cos phages, of which lambda is the prototype, do not engage in generalized transduction. For this reason, cos-site phages have been preferred for possible phage therapy, since they would not introduce adventitious host DNA into target organisms.The Staphylococcus aureus pathogenicity islands (SaPIs) are the best-characterized members of the phage-inducible chromosomal island family of mobile genetic elements (MGEs; Novick et al., 2010). SaPIs are ∼15 kb mobile elements that encode virulence factors and are parasitic on specific temperate (helper) phages. Helper phage proteins are required to lift their repression (Tormo-Más et al., 2010, 2013), thereby initiating their excision, circularization and replication. Phage-induced lysis releases vast numbers of infectious SaPI particles, resulting in high frequencies of transfer. Most SaPI helper phages identified to date are pac phages, and many well-studied SaPIs are packaged by the headful mechanism (Ruzin et al., 2001; Ubeda et al., 2007). Recently, we have reported that some SaPIs, of which the prototype is SaPIbov5 (Viana et al., 2010), carry phage cos sequences in their genomes, and can be efficiently packaged and transferred by cos phages to S. aureus strains at high frequencies (Quiles-Puchalt et al., 2014). Here we show that this transfer extends to non-aureus staphylococci and to Listeria monocytogenes.Since the pac phages transfer SaPIs to non-aureus staphylococci and to the Gram-positive pathogen Listeria monocytogenes (Maiques et al., 2007; Chen and Novick, 2009), we reasoned that cos phages might also be capable of intra- and intergeneric transfer. We tested this with SaPIbov5, into which we had previously inserted a tetracycline resistance (tetM) marker to enable selection, and with lysogens of two helper cos phages, φ12 and φSLT, carrying SaPIbov5 (strains JP11010 and JP11194, respectively; Supplementary Table 1). The prophages in these strains were induced with mitomycin C, and the resulting lysates were adjusted to 1 μg ml⁻¹ DNase I and RNase A, filter sterilized (0.2 μm pore), and tested for SaPI transfer with tetracycline selection, as previously described (Ubeda et al., 2008). To test for trans-specific or trans-generic transduction, coagulase-negative staphylococci species and L. monocytogenes strains were used as recipients for SaPIbov5 transfer, respectively, as previously described (Maiques et al., 2007; Chen and Novick, 2009). As shown in Figure 1 and Supplementary Table 2). In contrast, deletion of the SaPIbov5 cos site (strains JP11229 and JP11230) did not affect SaPI replication (Supplementary Figure 1), but completely eliminated SaPIbov5 transfer (Supplementary Table 2). The TerS protein is essential for φ12 and SaPIbov5 DNA packaging, but not for phage-mediated lysis (Quiles-Puchalt et al., 2014). As expected, this mutation abolished SaPIbov5 transfer (Open in a separate windowFigure 1(a) Map of SaPIbov5. Arrows represent the localization and orientation of ORFs greater than 50 amino acids in length. Rectangles represent the position of the ori (in purple) or cos (in red) sites. Positions of different primers described in the text are shown. (b) Amplimers generated for detection of SaPIbov5 in the different recipient strains. Supplementary Table 2 lists the sequence of the different primers used. The element was detected in S. epidermidis JP829 (Se-1), S. epidermidis JP830 (Se-2), L. monocytogenes SK1351 (Lm-1), L. monocytogenes EGDe (Lm-2), S. xylosus C2a (Sx) and S. aureus JP4226 (Sa).

Table 1

Intra- and intergeneric SaPIbov5 transfer^a

Transfer of a Phage T4 Gene into Enterobacteriaceae,Determined at the Single-Cell Level

The transfer range of phage genes was investigated at the single-cell level by using an in situ DNA amplification technique. After absorption of phages, a phage T4 gene was maintained in the genomes of non-plaque-forming bacteria at frequencies of 10⁻² gene copies per cell. The gene transfer decreased the mutation frequencies in nonhost recipients.Recently, whole-genome analyses have revealed that many bacterial genomes contain foreign genes, especially phage genes (9). The phage genes in bacterial genomes include genes for virulence or fitness factors such as extracellular toxins, superantigens, lipopolysaccharide-modifying enzymes, and proteins conferring serum resistance, etc. (1). These findings suggest that the horizontal transfer of phage genes has contributed significantly to the acquisition of new genetic traits and to the genetic diversity of bacteria (1, 9, 10). To truly appreciate the mechanisms behind phage-associated evolution, it is important to understand the frequency and range of transfer of phage genes.Most phage genomes consist of many genes derived from different origins (5, 8). Some genes are similar to those of other phages with phylogenetically different hosts or are found in the genomes of bacteria that are not the phage hosts. The mosaic nature of phage genomes has been known for some time, and a body of molecular genetic studies of phages to explain the mechanisms that drive this feature have been attempted previously (1, 5). More importantly, the horizontal transfer of phage genes has emerged as a major factor in the evolution of the phage genome. Since recombination between phage and phage/prophage can occur when these elements coexist in the same cell, coinfection with multiple phage species may result in the production of hybrid phage genomes (5). The pathways by which phages exchange genetic material vary dramatically in concert with host ranges. However, conventional plaque assays have shown that the host ranges of the phages studied are narrow. We hypothesized that phage genes can be transferred to more diverse species than previously thought.In order to accurately quantify DNA movement, gene targeting that does not require cultivation or gene expression is necessary (7). In situ DNA amplification methods allow the visualization of specific DNA sequences inside bacterial cells. In this study, we employed cycling primed in situ amplification-fluorescent in situ hybridization (CPRINS-FISH) to examine the possible range and frequency of the transfer of phage genes. CPRINS uses one primer and results in linear amplification of the target DNA inside cells, and multiply labeled fluorescent probe sets are applied for detection of the amplicons to improve the specificity and sensitivity of CPRINS (3). Previously, CPRINS-FISH did clarify the movement of DNA of a specific gene among Escherichia coli cells at the single-cell level (4).Enterobacterial phages P1 and T4 infect E. coli and have been well studied. P1 can exist as circular DNA within the bacterial cell as if it were a plasmid. Phage T4 is capable of undergoing only a lytic life cycle and not the lysogenic life cycle. Conventional methods using plaque assays have shown that the host of P1 and T4 is E. coli, but orthologous phage genes have been found in bacteria other than E. coli (6, 8). In the present study, strains of Enterobacteriaceae were allowed to grow on agar medium after the phage was adsorbed, and the maintenance of the transferred phage gene in the bacterial genomes was examined at the community level by quantitative real-time PCR and at the single-cell level by CPRINS-FISH.The following bacterial strains were used for maintenance experiments: Citrobacter freundii IFO 12681, Enterobacter aerogenes BM 2688, E. coli NBRC 12713, a Proteus mirabilis clinical isolate, Salmonella enterica serovar Enteritidis IID 640, and Yersinia enterocolitica IID 981. The bacterial strains were grown in Luria-Bertani (LB) medium (1% tryptone, 0.5% yeast extract, 0.5% NaCl; Nacalai Tesque Inc., Kyoto, Japan) at 37°C overnight.Stationary-phase cultures of 500 μl were incubated with 500 μl of SM buffer (50 mmol liter⁻¹ Tris-HCl [pH 7.5], 100 mmol liter⁻¹ NaCl, 8 mmol liter⁻¹ MgSO₄, 0.01% gelatin) containing the phage P1kc NBRC 20008 (2) or T4GT7 (11) at 37°C for 10 min at a multiplicity of infection of 1:1 (ratio of PFU of the phage to CFU of the recipient bacterium). The concentration of bacterial cells was adjusted to 10⁹ cells ml⁻¹. After 10 min of incubation, the diluted cell suspension (10⁵ cells) was filtered through a polycarbonate filter (Advantec, Tokyo, Japan) with a pore size of 0.2 μm and a diameter of 25 mm. Cells trapped on the filter were cultured on LB agar medium at 37°C for 24 h. The filter was transferred into a microtube, and cells on the filter were suspended in 1 ml of sterile deionized water. The numbers of cells in the suspension and cells remaining on the filter were determined by using an epifluorescence microscope (see below) after staining of the samples with 1 μg ml⁻¹ of 4′,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich Japan, Tokyo). The level of recovery of cells from the filter into sterile deionized water was about 99%. The cultured cells were subjected to real-time PCR and CPRINS-FISH.For real-time PCR, bacterial DNA was extracted using a QIAamp DNA isolation kit (Qiagen, Tokyo, Japan). The cell suspension was mixed with 10 mg ml⁻¹ of lysozyme solution and incubated at 37°C for 1 h. DNA extraction was then performed according to the kit manufacturer''s instructions. Table ​Table11 lists the oligonucleotide primers for PCR and CPRINS and the polynucleotide probes used in the present study. Tail fiber genes from phages P1kc and T4GT7 were quantified by real-time PCR with a LightCycler system (Roche Diagnostics, Tokyo, Japan). LightCycler FastStart DNA master SYBR green I (Roche Diagnostics) was used with 5 mmol liter⁻¹ Mg²⁺ and 0.5 μmol liter⁻¹ (each) primers targeting the tail fiber genes of P1kc (P1-tail931f and P1-tail1148r) and T4GT7 (T4-tail2770f and T4-tail2983r). After a hot start for 10 min at 95°C, 40 cycles of PCR were run with denaturation at 94°C for 15 s, annealing at 60°C for 10 s, extension at 72°C for 10 s, and fluorescence detection at 83°C for 5 s. The known amounts of PCR products from the phage DNA (10¹ to 10⁷ copies per reaction) were used for the standard curves to quantify the target DNA. To confirm the specificity of the reaction after real-time PCR, the PCR mixture was collected in a glass capillary and subjected to agarose gel electrophoresis in addition to a melting-curve analysis with the LightCycler system. The maintenance frequencies determined by real-time PCR were recorded as the copy number of the phage tail fiber gene per bacterial genome detected by staining with PicoGreen (Invitrogen, Tokyo, Japan) after cultivation of cells on LB agar medium for 24 h as described above. The frequencies were determined in triplicate for each sample. The increase in the phage gene copy number was determined by comparing the copy numbers in cells on the filter before and after cultivation. The phage gene copy number in cells on the filter was determined by the following formula: (total number of cells determined by DAPI staining) × (phage tail fiber gene copy number determined by real-time PCR)/(bacterial genome copy number determined by PicoGreen staining).

TABLE 1.

Probes and primers designed in this study

Safety and feasibility of umbilical cord mesenchymal stem cells in treatment-refractory systemic lupus erythematosus nephritis: time for a double-blind placebo-controlled trial to determine efficacy

As translational clinical researchers familiar with the risk-benefit of hematopoietic stem cell transplantation in autoimmune diseases, we are intrigued by the recent report of umbilical cord mesenchymal stem cell (UC-MSC) transplantation in treatment-refractory systemic lupus erythematosus nephritis by Wang and colleagues. They report the results of an open-label single-arm multicenter phase I/II study. This stimulated us to examine whether collective data from this group provide sufficient evidence for the feasibility, safety, dose rationale, and potential efficacy of UC-MSCs to conduct a randomized controlled trial in such patients. Results, though confounded by variable baseline prednisone and immuno-suppressive treatment, appear to indicate near-term response rates of approximately 50%, which are comparable to those seen with hematopoietic stem cell transplantation but with less morbidity and mortality.Wang and colleagues have been in the forefront of evaluating the potential for mesenchymal stem cells (MSCs) to treat systemic lupus erythematosus (SLE), based on studies in murine autoimmune disease models, demonstrating immunomodulatory properties of MSCs [1]. We have reviewed the additional reports published in peer-reviewed journals from this group [2–5], together with two protocols available on ClinicalTrials.gov ({"type":"clinical-trial","attrs":{"text":"NCT00698191","term_id":"NCT00698191"}}NCT00698191 and {"type":"clinical-trial","attrs":{"text":"NCT01741857","term_id":"NCT01741857"}}NCT01741857) (Table 

Antimicrobial Activity of Simulated Solar Disinfection against Bacterial,Fungal, and Protozoan Pathogens and Its Enhancement by Riboflavin

Riboflavin significantly enhanced the efficacy of simulated solar disinfection (SODIS) at 150 watts per square meter (W m⁻²) against a variety of microorganisms, including Escherichia coli, Fusarium solani, Candida albicans, and Acanthamoeba polyphaga trophozoites (>3 to 4 log₁₀ after 2 to 6 h; P < 0.001). With A. polyphaga cysts, the kill (3.5 log₁₀ after 6 h) was obtained only in the presence of riboflavin and 250 W m⁻² irradiance.Solar disinfection (SODIS) is an established and proven technique for the generation of safer drinking water (11). Water is collected into transparent plastic polyethylene terephthalate (PET) bottles and placed in direct sunlight for 6 to 8 h prior to consumption (14). The application of SODIS has been shown to be a simple and cost-effective method for reducing the incidence of gastrointestinal infection in communities where potable water is not available (2-4). Under laboratory conditions using simulated sunlight, SODIS has been shown to inactivate pathogenic bacteria, fungi, viruses, and protozoa (6, 12, 15). Although SODIS is not fully understood, it is believed to achieve microbial killing through a combination of DNA-damaging effects of ultraviolet (UV) radiation and thermal inactivation from solar heating (21).The combination of UVA radiation and riboflavin (vitamin B₂) has recently been reported to have therapeutic application in the treatment of bacterial and fungal ocular pathogens (13, 17) and has also been proposed as a method for decontaminating donor blood products prior to transfusion (1). In the present study, we report that the addition of riboflavin significantly enhances the disinfectant efficacy of simulated SODIS against bacterial, fungal, and protozoan pathogens.Chemicals and media were obtained from Sigma (Dorset, United Kingdom), Oxoid (Basingstoke, United Kingdom), and BD (Oxford, United Kingdom). Pseudomonas aeruginosa (ATCC 9027), Staphylococcus aureus (ATCC 6538), Bacillus subtilis (ATCC 6633), Candida albicans (ATCC 10231), and Fusarium solani (ATCC 36031) were obtained from ATCC (through LGC Standards, United Kingdom). Escherichia coli (JM101) was obtained in house, and the Legionella pneumophila strain used was a recent environmental isolate.B. subtilis spores were produced from culture on a previously published defined sporulation medium (19). L. pneumophila was grown on buffered charcoal-yeast extract agar (5). All other bacteria were cultured on tryptone soy agar, and C. albicans was cultured on Sabouraud dextrose agar as described previously (9). Fusarium solani was cultured on potato dextrose agar, and conidia were prepared as reported previously (7). Acanthamoeba polyphaga (Ros) was isolated from an unpublished keratitis case at Moorfields Eye Hospital, London, United Kingdom, in 1991. Trophozoites were maintained and cysts prepared as described previously (8, 18).Assays were conducted in transparent 12-well tissue culture microtiter plates with UV-transparent lids (Helena Biosciences, United Kingdom). Test organisms (1 × 10⁶/ml) were suspended in 3 ml of one-quarter-strength Ringer''s solution or natural freshwater (as pretreated water from a reservoir in United Kingdom) with or without riboflavin (250 μM). The plates were exposed to simulated sunlight at an optical output irradiance of 150 watts per square meter (W m⁻²) delivered from an HPR125 W quartz mercury arc lamp (Philips, Guildford, United Kingdom). Optical irradiances were measured using a calibrated broadband optical power meter (Melles Griot, Netherlands). Test plates were maintained at 30°C by partial submersion in a water bath.At timed intervals for bacteria and fungi, the aliquots were plated out by using a WASP spiral plater and colonies subsequently counted by using a ProtoCOL automated colony counter (Don Whitley, West Yorkshire, United Kingdom). Acanthamoeba trophozoite and cyst viabilities were determined as described previously (6). Statistical analysis was performed using a one-way analysis of variance (ANOVA) of data from triplicate experiments via the InStat statistical software package (GraphPad, La Jolla, CA).The efficacies of simulated sunlight at an optical output irradiance of 150 W m⁻² alone (SODIS) and in the presence of 250 μM riboflavin (SODIS-R) against the test organisms are shown in Table ​Table1.1. With the exception of B. subtilis spores and A. polyphaga cysts, SODIS-R resulted in a significant increase in microbial killing compared to SODIS alone (P < 0.001). In most instances, SODIS-R achieved total inactivation by 2 h, compared to 6 h for SODIS alone (Table ​(Table1).1). For F. solani, C. albicans, ands A. polyphaga trophozoites, only SODIS-R achieved a complete organism kill after 4 to 6 h (P < 0.001). All control experiments in which the experiments were protected from the light source showed no reduction in organism viability over the time course (results not shown).

TABLE 1.

Efficacies of simulated SODIS for 6 h alone and with 250 μM riboflavin (SODIS-R)

Characterization of a Thermostable Short-Chain Alcohol Dehydrogenase from the Hyperthermophilic Archaeon Thermococcus sibiricus

Short-chain alcohol dehydrogenase, encoded by the gene Tsib_0319 from the hyperthermophilic archaeon Thermococcus sibiricus, was expressed in Escherichia coli, purified and characterized as an NADPH-dependent enantioselective oxidoreductase with broad substrate specificity. The enzyme exhibits extremely high thermophilicity, thermostability, and tolerance to organic solvents and salts.Alcohol dehydrogenases (ADHs; EC 1.1.1.1.) catalyze the interconversion of alcohols to their corresponding aldehydes or ketones by using different redox-mediating cofactors. NAD(P)-dependent ADHs, due to their broad substrate specificity and enantioselectivity, have attracted particular attention as catalysts in industrial processes (5). However, mesophilic ADHs are unstable at high temperatures, sensitive to organic solvents, and often lose activity during immobilization. In this relation, there is a considerable interest in ADHs from extremophilic microorganisms; among them, Archaea are of great interest. The representatives of all groups of NAD(P)-dependent ADHs have been detected in genomes of Archaea (11, 12); however, only a few enzymes have been characterized, and the great majority of them belong to medium-chain (3, 4, 14, 16, 19) or long-chain iron-activated ADHs (1, 8, 9). Up to now, a single short-chain archaeal ADH from Pyrococcus furiosus (10, 18) and only one archaeal aldo-keto reductase also from P. furiosus (11) have been characterized.Thermococcus sibiricus is a hyperthermophilic anaerobic archaeon isolated from a high-temperature oil reservoir capable of growth on complex organic substrates (15). The complete genome sequence of T. sibiricus has been recently determined and annotated (13). Several ADHs are encoded by the T. sibiricus genome, including three short-chain ADHs (Tsib_0319, Tsib_0703, and Tsib_1998) (13). In this report, we describe the cloning and expression of the Tsib_0319 gene from T. sibiricus and the purification and the biochemical characterization of its product, the thermostable short-chain ADH (TsAdh319).The Tsib_0319 gene encodes a protein with a size of 234 amino acids and the calculated molecular mass of 26.2 kDa. TsAdh319 has an 85% degree of sequence identity with short-chain ADH from P. furiosus (AdhA; PF_0074) (18). Besides AdhA, close homologs of TsAdh319 were found among different bacterial ADHs, but not archaeal ADHs. The gene flanked by the XhoI and BamHI sites was PCR amplified using two primers (sense primer, 5′-GTTCTCGAGATGAAGGTTGCTGTGATAACAGGG-3′, and antisense primer, 5′-GCTGGATCCTCAGTATTCTGGTCTCTGGTAGACGG-3′) and cloned into the pET-15b vector. TsAdh319 was overexpressed, with an N-terminal His₆ tag in Escherichia coli Rosetta-gami (DE3) and purified to homogeneity by metallochelating chromatography (Hi-Trap chelating HP column; GE Healthcare) followed by gel filtration on Superdex 200 10/300 GL column (GE Healthcare) equilibrated in 50 mM Tris-HCl (pH 7.5) with 200 mM NaCl. The homogeneity and the correspondence to the calculated molecular mass of 28.7 kDa were verified by SDS-PAGE (7). The molecular mass of native TsAdh319 was 56 to 60 kDa, which confirmed the dimeric structure in solution.The standard ADH activity measurement was made spectrophotometrically at the optimal pH by following either the reduction of NADP (in 50 mM Gly-NaOH buffer; pH 10.5) or the oxidation of NADPH (in 0.1 M sodium phosphate buffer; pH 7.5) at 340 nm at 60°C. The enzyme exhibited a strong preference for NADP(H) and broad substrate specificity (Table ​(Table1).1). The highest oxidation rates were found with pentoses d-arabinose (2.0 U mg⁻¹) and d-xylose (2.46 U mg⁻¹), and the highest reduction rates were found with dimethylglyoxal (5.9 U mg⁻¹) and pyruvaldehyde (2.2 U mg⁻¹). The enzyme did not reduce sugars which were good substrates for the oxidation reaction. The kinetic parameters of TsAdh319 determined for the preferred substrates are shown in Table ​Table2.2. The enantioselectivity of the enzyme was estimated by measuring the conversion rates of 2-butanol enantiomers. TsAdh319 showed an evident preference, >2-fold, for (S)-2-butanol over (RS)-2-butanol. The enzyme stereoselectivity is confirmed by the preferred oxidation of d-arabinose over l-arabinose (Table ​(Table1).1). The fact that TsAdh319 is metal independent was supported by the absence of a significant effect of TsAdh319 preincubation with 10 mM Me²⁺ for 30 min before measuring the activity in the presence of 1 mM Me²⁺ or EDTA (Table ​(Table3).3). TsAdh319 also exhibited a halophilic property, so the enzyme activity increased in the presence of NaCl and KCl and the activation was maintained even at concentration of 4 M and 3 M, respectively (Table ​(Table33).

TABLE 1.

Substrate specificity of TsAdh319

DNA microarray analysis of rheumatoid arthritis susceptibility genes identified by genome-wide association studies

In a recent interesting review, Alex Clarke and Timothy Vyse described the genetics of rheumatic disease [1]. In the past several years, genome-wide association studies (GWAS) have led to the identification of six high-risk rheumatoid arthritis (RA) susceptibility genes - namely, CD244, PADI4, SLC22A2, PTPN22, CTLA4, and STAT4 (summarized in [2]). In vitro studies using mutant alleles and cultured cells have revealed the individual upregulation of CD244, PADI4, SLC22A2, and PTPN22 [2-6]; however, studies on the expression of RA susceptibility genes in RA patients are rare. We therefore investigated the expression of the above-mentioned six RA susceptibility genes in 112 RA patients using DNA microarray analysis. This study aims to clarify whether DNA microarray analysis and GWAS produce comparable results with respect to RA susceptibility genes.Total RNA extracted from total peripheral blood cells obtained from 112 RA patients and 45 healthy individuals was used to prepare aminoallyl RNA. As a reference, mixed RNA from 45 healthy individuals was used. The aminoallyl RNA of each individual and the reference was subjected to Cy3 and Cy5 labeling, respectively, and was hybridized with an oligonucleotide-based DNA microarray. The data obtained were analyzed by nonparametric statistical group comparison. The intensities of the noprobe spots were used as the background. The median and standard deviation of the background intensity were calculated. The genes with an intensity value that was less than the median plus 2 standard deviation of the background intensity were identified as null. The Cy3/Cy5 ratios of all spots on the DNA microarray were normalized using the global ratio median. Only gene expression data that were collected from at least 80% of samples from each group were selected for further analysis. The unpaired Mann-Whitney test was used to determine statistically significant differences in the mRNA expression levels between the RA and healthy groups. Statistical significance was set at P < 0.05.The results of our DNA microarray analysis showed that the expressions of four out of the six RA susceptibility genes were significantly higher in RA patients than in healthy individuals (1.0 × 10^-16 to 2.32 × 10^-5) (Table ​(Table1).1). As described above, the upregulation of these four genes (CD244, PADI4, SLC22A2, and PTPN22) has been previously confirmed in in vitro studies. We found, however, that CTLA4 expression levels were similar between the RA and control groups, whereas STAT4 expression was significantly downregulated in the RA group (1.38 × 10^-8). We investigated the expression of other RA susceptibility genes - namely, TRF1/C5 [7], CD40 [8], and CCL21 [8] - and found that their expressions were similar in both groups. The genetic risk factors for RA were recently reported to differ between Caucasian and Asian (Korean) populations [9]. The samples used in our microarray analysis were derived from the same Asian (Japanese) cohort. The expression profiles for these three genes may therefore not be consistent with the profiles determined by GWAS.

Table 1

Candidate genes identified from rheumatoid arthritis genome-wide association studies

Introducing Titratable Water to All-Atom Molecular Dynamics at Constant?pH

Recent development of titratable coions has paved the way for realizing all-atom molecular dynamics at constant pH. To further improve physical realism, here we describe a technique in which proton titration of the solute is directly coupled to the interconversion between water and hydroxide or hydronium. We test the new method in replica-exchange continuous constant pH molecular dynamics simulations of three proteins, HP36, BBL, and HEWL. The calculated pK_a values based on 10-ns sampling per replica have the average absolute and root-mean-square errors of 0.7 and 0.9 pH units, respectively. Introducing titratable water in molecular dynamics offers a means to model proton exchange between solute and solvent, thus opening a door to gaining new insights into the intricate details of biological phenomena involving proton translocation.Solution pH is an important factor in biology. Although neutral pH in extracellular medium accounts for balanced electrostatics and proper folding of protein structures, pH gradients across cell membranes induce large conformational changes that are necessary for biological functions, such as ATP synthesis and efflux of small molecules out of the cell. To gain detailed insights into pH-dependent conformational phenomena, several constant pH molecular dynamics (pHMD) methods, based on either discrete or continuous titration coordinates, have been developed in the last decade (1–4). In the continuous pHMD (CpHMD) framework (2,4), a set of titration coordinates {λ_i} are simultaneously propagated along with the conformational degrees of freedom. Although the original CpHMD method based on the generalized Born (GB) implicit-solvent models (2,4) offers quantitative prediction of pK_a values and pH dependence of folding and conformational dynamics of proteins (5), its accuracy and applicability to highly charged systems and those with dominantly hydrophobic regions are limited due to the approximate nature of the underlying implicit-solvent models.Motivated by the above-mentioned need, three groups have made efforts to develop a CpHMD method using exclusively the explicit-solvent models (6–8). In our development, the titration of acidic and basic sites is coupled with that of coions to level the total charge of the system (8). To further improve physical realism, here we replace the coions by titratable water molecules, which not only absorb the excess charge but also enable direct modeling of solute-solvent proton exchange in classical molecular dynamics simulations.To illustrate the utility of the new methodology, we applied it to the titration simulations of three proteins that were previously used to benchmark the GB-based CpHMD. Although this work does not explore specific interactions between titratable waters and proteins, the methodology can be further tested or improved to provide a rigorous way for modeling proton transfer in molecular dynamics, which is a computationally efficient alternative to the empirical valence-bond theory-based methodologies (9,10).We define titration of water as:

• 1.Loss of a proton to give a negatively charged hydroxide,

H₂O ? OH^? + H⁺, (1)or

• 2.Gain of a proton to give a positively charged hydronium,

H₂O + H⁺ ? H₃O⁺.(2)We now couple the titration of hydroxide (Eq. 1) with that of an acidic site of the solute in the CpHMD simulation,HA+OH−⇌KaA−+H2O.(3)The use of hydronium is avoided here to prevent a potential artifact due to prolonged attraction with A⁻. Analogously, we couple the titration of hydronium (Eq. 2) with that of a basic site,BH++H2O⇌KbH3O++B.(4)Thus, effectively, a proton is transferred between the solute and solvent. However, we should note that in CpHMD simulations, titratable protons are represented by covalently attached dummies (2,4). Through varying the atomic charges and van der Waals interactions, they are seen by other atoms in the protonated state but not in the unprotonated state (see Table S1 in the Supporting Material). Furthermore, the solution proton concentration is implicitly modeled through a free energy term (2,4).In CpHMD, the reference potential of mean force (PMF) for titration is that of the model compound (blocked single amino acid in water) along λ (2,4). In the presence of cotitrating water molecules, it is necessary to add the PMF for the conversion of water to hydroxide or hydronium. One-nanosecond NPT simulations at ambient pressure and temperature were performed to calculate the average force, 〈dU/d,θ〉 at given θ-values, which are related to λ by λ = sin² θ (see Fig. S1 in the Supporting Material). Thermodynamic integration was then applied to calculate the PMF. We found that the average force can be accurately fit when assuming the PMF is quadratic in λ (Fig. 1). The same applies to the PMFs for titration of models Asp, Glu, and His. After testing on the titration of model compounds (see Table S2), we performed 10-ns all-atom CpHMD simulations with the pH replica-exchange protocol for three proteins: HP36, BBL and HEWL (see the Supporting Material for details). Most of the calculated pK_a values were converged in 10 ns per replica (see Fig. S3). Results are summarized in Fig. S4. Based on the 10-ns data, the root-mean-square (RMS) and average absolute errors are 0.9 and 0.7 pH units, respectively, while the largest absolute error is 2.5 (Glu³⁵ of HEWL). Linear regression of the calculation versus experiment gives R² of 0.8 and slope of 1.2.Open in a separate windowFigure 1Average force and potential of mean force for converting a water molecule to hydroxide (A) and hydronium. (B) (Data points) Average forces. (Dashed curves) Best fits using a linear function, 2A(λ − B). (Solid curves) Corresponding potential of mean force.

Table 1

Calculated and experimental pK_a values of three proteins

Does an Eye-Hand Coordination Test Have Added Value as Part of Talent Identification in Table Tennis? A Validity and Reproducibility Study

This study investigated the added value, i.e. discriminative and concurrent validity and reproducibility, of an eye-hand coordination test relevant to table tennis as part of talent identification. Forty-three table tennis players (7–12 years) from national (n = 13), regional (n = 11) and local training centres (n = 19) participated. During the eye-hand coordination test, children needed to throw a ball against a vertical positioned table tennis table with one hand and to catch the ball correctly with the other hand as frequently as possible in 30 seconds. Four different test versions were assessed varying the distance to the TotalNationalRegionalLocalTotal43131119Boys268810Girls17539Age (years)10.4±1.410.9±1.510.4±1.510.1±1.47 year olds1--18 year olds51139 year olds3-3-10 year olds1232711 year olds1151512 year olds11443Length (cm)149±11150±12150±12148±10Weight (kg )38±837±737±738±9Right-handed359917Left-handed8422Training (hours*week-1)6 (0–20)11 (7–20)7 (4–11)2 (0–3)Competition (points)173 (−52–430)297 (144–430)188 (72–317)36 (−52–130)Open in a separate windowData are frequencies, except for age, length and weight (years±SD), and training and competition (mean (range)).     

DdlN from Vancomycin-Producing Amycolatopsis orientalis C329.2 Is a VanA Homologue with d-Alanyl-d-Lactate Ligase Activity

Vancomycin-resistant enterococci acquire high-level resistance to glycopeptide antibiotics through the synthesis of peptidoglycan terminating in d-alanyl-d-lactate. A key enzyme in this process is a d-alanyl-d-alanine ligase homologue, VanA or VanB, which preferentially catalyzes the synthesis of the depsipeptide d-alanyl-d-lactate. We report the overexpression, purification, and enzymatic characterization of DdlN, a VanA and VanB homologue encoded by a gene of the vancomycin-producing organism Amycolatopsis orientalis C329.2. Evaluation of kinetic parameters for the synthesis of peptides and depsipeptides revealed a close relationship between VanA and DdlN in that depsipeptide formation was kinetically preferred at physiologic pH; however, the DdlN enzyme demonstrated a narrower substrate specificity and commensurately increased affinity for d-lactate in the C-terminal position over VanA. The results of these functional experiments also reinforce the results of previous studies that demonstrated that glycopeptide resistance enzymes from glycopeptide-producing bacteria are potential sources of resistance enzymes in clinically relevant bacteria.The origin of antibiotic resistance determinants is of significant interest for several reasons, including the prediction of the emergence and spread of resistance patterns, the design of new antimicrobial agents, and the identification of potential reservoirs for resistance elements. Antibiotic resistance can occur either through spontaneous mutation in the target or by the acquisition of external genetic elements such as plasmids or transposons which carry resistance genes (7). The origins of these acquired genes are varied, but it has long been recognized that potential reservoirs are antibiotic-producing organisms which naturally harbor antibiotic resistance genes to protect themselves from the actions of toxic compounds (6).High-level resistance to glycopeptide antibiotics such as vancomycin and teicoplanin in vancomycin-resistant enterococci (VRE) is conferred by the presence of three genes, vanH, vanA (or vanB), and vanX, which, along with auxiliary genes necessary for inducible gene expression, are found on transposons integrated into plasmids or the bacterial genome (1, 20). These three genes are essential to resistance and serve to change the C-terminal peptide portion of the peptidoglycan layer from d-alanyl-d-alanine (d-Ala-d-Ala) to d-alanyl-d-lactate (d-Ala-d-Lac). This change results in the loss of a critical hydrogen bond between vancomycin and the d-Ala-d-Ala terminus and in a 1,000-fold decrease in binding affinity between the antibiotic and the peptidoglycan layer, which is the basis for the bactericidal action of this class of compounds (5). The vanH gene encodes a d-lactate dehydrogenase which provides the requisite d-Lac (3, 5), while the vanX gene encodes a highly specific dd-peptidase which cleaves only d-Ala-d-Ala produced endogenously while leaving d-Ala-d-Lac intact (19, 21). The final gene, vanA or vanB, encodes an ATP-dependent d-Ala-d-Lac ligase (4, 8, 10). This enzyme has sequence homology with the chromosomal d-Ala-d-Ala ligases, which are essential for peptidoglycan synthesis but which generally lack the ability to synthesize d-Ala-d-Lac (9).We have recently cloned vanH, vanA, and vanX homologues from two glycopeptide antibiotic-synthesizing organisms: Amycolatopsis orientalis C329.2, which produces vancomycin, and Streptomyces toyocaensis NRRL 15009, which produces {"type":"entrez-nucleotide","attrs":{"text":"A47934","term_id":"2301796","term_text":"A47934"}}A47934 (14). In addition, the vanH-vanA-vanX gene cluster was identified in several other glycopeptide producers. We have also demonstrated that the VanA homologue from S. toyocaensis NRRL 15009 can synthesize d-Ala-d-Lac in vitro and in the glycopeptide-sensitive host Streptomyces lividans (15, 16). We now report the expression of the A. orientalis C329.2 VanA homologue DdlN in Escherichia coli, its purification, and its enzymatic characterization. These data reinforce the striking similarity between vancomycin resistance elements in VRE and glycopeptide-producing organisms and support the possibility of a common origin for these enzymes.

Expression, purification, and specificity of DdlN.

DdlN was overexpressed in E. coli under the control of the bacteriophage T7 promoter. The construct gave good yields of highly purified enzyme following a four-step purification procedure (Table ​(Table1;1; Fig. ​Fig.1).1). Like other dd-ligases, DdlN behaved like a dimer in solution (not shown).

TABLE 1

Purification of DdlN from E. coli BL21 (DE3)/pETDdlN

Economic Benefits of Investing in Women’s Health: A Systematic Review

Background

Globally, the status of women’s health falls short of its potential. In addition to the deleterious ethical and human rights implications of this deficit, the negative economic impact may also be consequential, but these mechanisms are poorly understood. Building on the literature that highlights health as a driver of economic growth and poverty alleviation, we aim to systematically investigate the broader economic benefits of investing in women’s health.

Methods

Using the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines, we systematically reviewed health, gender, and economic literature to identify studies that investigate the impact of women’s health on micro- and macroeconomic outcomes. We developed an extensive search algorithm and conducted searches using 10 unique databases spanning the timeframe 01/01/1970 to 01/04/2013. Articles were included if they reported on economic impacts stemming from changes in women’s health (table of outcome measures included in full review, Outcome measures   FertilityIntergenerational Health SpilloverEducationProductivitySavingsMicroeconomic level    Total fertility rateChild survivalEnrollment in schoolIncomeMoneyChange in fertilityChild wellbeing and behaviorYears of schoolingPurchasing powerAssetsAge at first birth/ teenage pregnanciesAnthropometryEarly drop outPerformanceBirth spacingImproved cognitive developmentPerformance in school Life expectancyHigher education  Adult health outcomesLiteracy  Nutrition   Intrauterine growth  Macroeconomic level    Open in a separate windowGross domestic product/gross national product, gross domestic product/gross national product growth, income per capita, labor force participation, per capita income.

Results

The existing literature indicates that healthier women and their children contribute to more productive and better-educated societies. This study documents an extensive literature confirming that women’s health is tied to long-term productivity: the development and economic performance of nations depends, in part, upon how each country protects and promotes the health of women. Providing opportunities for deliberate family planning; healthy mothers before, during, and after childbirth, and the health and productivity of subsequent generations can catalyze a cycle of positive societal development.

Conclusions

This review highlights the untapped potential of initiatives that aim to address women’s health. Societies that prioritize women’s health will likely have better population health overall, and will remain more productive for generations to come.     

Heterosexual Transmission of a Murine AIDS Virus

Heterosexual transmission of a murine leukemia virus mixture named LP-BM5 MuLV, which is known as the murine AIDS virus, was investigated. Our results indicated that the heterosexual transmission of LP-BM5 MuLV occurs in both directions with high frequency and that the frequencies of virus transmission in the cervix and penis are higher than those in other genital organs. The results suggested that infection by LP-BM5 MuLV via heterosexual transmission may initially take place at particular retrovirus-sensitive sites (cells) in the genital organs.Human immunodeficiency virus (HIV) infection is now pandemic. In many countries, HIV has been spread mainly by heterosexual transmission (3, 5). For the prevention of HIV infection, as well as for the development of vaccines against HIV, it is of a great importance to understand the mechanisms of the heterosexual transmission of retroviruses. Since it is difficult to investigate the mechanisms of heterosexual transmission of HIV in humans experimentally, an animal model with a retrovirus which induces an acquired immunodeficiency syndrome like human AIDS would be useful. A murine leukemia virus mixture called LP-BM5 MuLV induces a severe acquired immunodeficiency syndrome termed murine AIDS (MAIDS) in susceptible strains of mice (10). The mixture includes a replication-competent ecotropic virus, mink cell focus-inducing virus, and a replication-defective virus which has been considered to be involved in the pathogenesis of MAIDS (4). With many similarities to human AIDS patients, mice infected with the LP-BM5 MuLV mixture develop splenomegaly, systemic lymphadenopathy, and severe immunodeficiency (4, 11). We previously reported that maternal transmission of LP-BM5 MuLV occurs via mother’s milk with high frequency (12). In the present study, we demonstrate that the heterosexual transmission of LP-BM5 MuLV also occurs with high frequency via genital organs.C57BL/10 (B10) mice were purchased from Japan SLC Inc., Shizuoka, Japan. All mice were specific-pathogen free and were housed in an air-conditioned room. They were given autoclaved water and sterilized pelleted feed. An SC-1 clone chronically infected with LP-BM5 MuLV, the G6 cell line, was kindly supplied by H. C. Morse III, National Institutes of Health, Bethesda, Md. Virus was prepared from the supernatant of G6 cells as previously described (12). The virus preparation was stored at −70°C until use. B10 mice were inoculated by the intraperitoneal route with 0.3 ml of the LP-BM5 MuLV preparation. To increase the frequency of sexual contacts and to avoid pregnancy in the female mice, all male mice were sterilized by vasectomy under anesthesia with pentobarbital (Nembutal). The vasectomized male mice were mated with female mice at least 4 weeks postoperation, since sperm are usually kept alive for 2 to 3 weeks in spermiducts. Excised genital organs were crushed with plastic sticks in 1 ml of lysis buffer containing 10 mM Tris-HCl (pH 8.0), 100 mM NaCl, 1 mM EDTA, 0.5% sodium dodecyl sulfate, and proteinase K (0.5 mg/ml). Spleen cells were lysed after hemolysis with 0.83% NH₄Cl. Lysed samples were incubated at 50°C for 3 h. DNA was extracted three times with phenol-chloroform, precipitated with cold ethanol, treated with RNase and proteinase K, and dissolved in 0.1 ml of H₂O. LP-BM5 MuLV defective virus genome was detected by Southern blot hybridization combined with PCR as described previously (12). In brief, template DNAs (1 μg per tube) were added to a cocktail adjusted to final concentrations of 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl₂, 0.01% gelatin, 200 μM deoxynucleoside triphosphate, 100 pmol of each primer (5′-CCTCTTCCTTTATCGACACT-3′ [sense] and 5′-ATTAGGGGGGGAATAGCTCG-3′ [antisense]), and 2 U of Taq DNA polymerase (Boehringer Mannheim) in a total volume of 100 μl and were subjected to 32 cycles of amplification. In each cycle of PCR, the mixture was denatured at 95°C for 1 min (5 min for the first cycle), annealed at 55°C for 3 min, and extended at 72°C for 1 min. The PCR-amplified products were subjected to gel electrophoresis (1.5% agarose) and transferred to a Hybond N+ membrane (Amersham) by the alkaline blotting method. Hybridization was achieved with a 5′ ³²P-labeled probe (5′-TGTCAAAGGGACCAGTTAAG-3′) at 45°C overnight in 6× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate)–0.5% sodium dodecyl sulfate–100 μg of salmon sperm DNA per ml. Hybridized membranes were washed twice in 2× SSC at 37°C for 10 min and then in 0.5× SSC at 45°C for 30 min. DNA derived from uterine cervices of uninfected mice was used as a negative control. The limit of sensitivity was approximately 10 copies per tube, as assessed by Southern blot analysis with plasmid DNAs (1/10 of the PCR product).Concanavalin A (ConA) was obtained from Pharmacia Fine Chemicals, Uppsala, Sweden. Responder spleen cells (2 × 10⁵) were cultured with ConA (5 μg/ml) in 96-well flat-bottomed microculture plates in 0.2 ml of culture medium at 37°C in 7.5% CO₂. The culture medium consisted of RPMI 1640 supplemented with 10% fetal calf serum, penicillin (5,000 IU/100 ml), streptomycin (5,000 μg/100 ml), nonessential amino acids, sodium pyruvate (11.0 mg/100 ml), 2-mercaptoethanol (5 × 10⁻⁵ M), and l-glutamine (29.2 mg/100 ml). On day 2, cultures were pulsed with 1 μCi of [³H]thymidine and incubated for an additional 12 to 18 h. Incorporation of [³H]thymidine into responder spleen cells was quantitated by liquid scintillation counting. Determinations were performed in triplicate; standard errors of the means were generally <5% and therefore have not been indicated.As illustrated in Fig. ​Fig.1,1, in order to investigate the heterosexual transmission of LP-BM5 MuLV from male to female mice, normal male mice were inoculated with LP-BM5 MuLV and vasectomized 1 week later. At 5 weeks after virus inoculation, they were mated with uninfected female mice. After 8 weeks of breeding, female mice were sacrificed and their vaginae, cervices uteri, corpora uteri, inguinal lymph nodes, and spleens were removed and stored at −70°C until use. In the opposite direction, to investigate virus transmission from female to male, normal female mice were inoculated with LP-BM5 MuLV and then mated with uninfected, vasectomized male mice as described above. After 8 weeks of breeding, male mice were sacrificed and their penes, prepuces, inguinal lymph nodes and spleens were removed and stored at −70°C until use. Figure ​Figure22 shows the detection by PCR of the LP-BM5 defective virus genome in genital organs and spleens that were taken from mice mated with their virus-infected counterparts. It was demonstrated that although the defective virus genome was detected in both spleens and genital organs in some male mice (2 of 17 [see Table ​Table1]),1]), as shown in Fig. ​Fig.2,2, lanes 3 and 4, the defective virus genome was detected only in the genital organs, not the spleens (Fig. ​(Fig.2,2, lanes 5 and 6), from most of the male mice. In contrast, all of the female mice were positive for defective virus genome only in the genital organs (Fig. ​(Fig.2,2, lanes 1 and 2). None of the mice examined were positive for the virus genome only in the spleens (this issue is discussed below). It should be noted here that the efficacy of PCR amplification, which was measured by experiments using the mixture of genomic DNA and plasmid DNA containing the defective virus, did not differ among the genital organs and spleens. By using the above strategy, the heterosexual transmission of LP-BM5 MuLV was investigated according to the protocol shown in Fig. ​Fig.1.1. Open in a separate windowFIG. 1Experimental design for examination of heterosexual transmission of the MAIDS virus in B10 mice. i.p., intraperitoneal.Open in a separate windowFIG. 2Detection of the LP-BM5 MuLV defective virus genome by PCR in genital organs and spleens. The template DNAs (1 μg) derived from female or male mice which were bred with LP-BM5 MuLV-infected mice were amplified by PCR. Samples were prepared from either female (lanes 1 and 2) or male (lanes 3 to 6) mice. Lanes 1, 3, and 5, spleen; lane 2, uterine cervix; lanes 4 and 6, penis (from two representative male mice). The PCR products (5 μl) were applied to a 1.5% agarose gel and analyzed by Southern blotting with a probe for the defective virus (12).

TABLE 1

Heterosexual transmission of LP-BM5 MuLV