Sex determination in insects: variations on a common theme.

Recent studies in a representative selection of holometabolous insects suggest that, despite diversity at the instructive level, the signal-relaying part of the sex-determining pathway is remarkably well conserved. In principle, it is composed of the transformer gene (tra), which acts as a common binary switch that transduces the selected sexual fate, female when ON, male when OFF, to the downstream effector doublesex(dsx) that controls overt sexual differentiation. An interesting recurrent feature is that tra is switched ON in the early zygote by maternally provisioned tra activity. Different male-determining signals evolved, which prevent maternal activation of zygotic tra to allow for male development. In some species, where lack of maternal activation leaves tra in the OFF state, novel female-determining signals were deployed to activate zygotic tra. It appears that both the instructive end of the pathway upstream of tra as well as the executive end downstream of dsx are primary targets of evolutionary divergence, while the transduction part seems less prone to changes. We propose that this is a feature shared with many other signaling cascades that regulate developmental fates.

Public health approach to detection of non-O157 Shiga toxin-producing Escherichia coli: summary of two outbreaks and laboratory procedures.

Routine laboratory testing may not detect non-O157 Shiga toxin-producing Escherichia coli (STEC) reliably. Active clinical, epidemiological, environmental health, and laboratory collaboration probably influence successful detection and study of non-O157 STEC infection. We summarized two outbreak investigations in which such coordinated efforts identified non-O157 STEC disease and led to effective control measures. Outbreak 1 involved illness associated with consuming unpasteurized apple cider from a local orchard. Public health personnel were notified by a local hospital; stool specimens from ill persons contained O111 STEC. Outbreak 2 involved bloody diarrhoea at a correctional facility. Public health personnel were notified by the facility infection control officer; O45 STEC was the implicated agent. These reports highlight the ability of non-O157 STEC to cause outbreaks and demonstrate that a coordinated effort by clinicians, infection-control practitioners, clinical diagnostic laboratorians, and public health personnel can lead to effective identification, investigation, and prevention of non-O157 STEC disease.

Demonstration of ignition radiation temperatures in indirect-drive inertial confinement fusion hohlraums.

We demonstrate the hohlraum radiation temperature and symmetry required for ignition-scale inertial confinement fusion capsule implosions. Cryogenic gas-filled hohlraums with 2.2 mm-diameter capsules are heated with unprecedented laser energies of 1.2 MJ delivered by 192 ultraviolet laser beams on the National Ignition Facility. Laser backscatter measurements show that these hohlraums absorb 87% to 91% of the incident laser power resulting in peak radiation temperatures of T(RAD)=300 eV and a symmetric implosion to a 100 µm diameter hot core.

Salmonella Cerro isolated over the past twenty years from various sources in the US represent a single predominant pulsed-field gel electrophoresis type.

Salmonella Cerro prevalence in US dairy cattle has increased significantly during the past decade. Comparison of 237 Salmonella isolates collected from various human and animal sources between 1986 and 2009 using pulsed-field gel electrophoresis, antimicrobial resistance typing, and spvA screening, showed very limited genetic diversity, indicating clonality of this serotype. Improved subtyping methods are clearly needed to analyze the potential emergence of this serotype. Our results thus emphasize the critical importance of population-based pathogen surveillance for the detection and characterization of potentially emerging pathogens, and caution to critically evaluate the adequacy of diagnostic tests for a given study population and diagnostic application.

A test cassette for x-ray-exposure experiments at the National Ignition Facility.

We present the design and operation of a test cassette for exposure of samples to radiation environments at the National Ignition Facility. The cassette provides options for square and round samples and exposure areas; the cassette provides for multiple levels of filtration on a single sample, which allows dynamic range in experiments. The samples had normal lines of sight to the x-ray source in order to have uniform x-ray illumination. The incident x-radiation onto the samples was determined by the choice of filter thicknesses and materials. The samples were held at precise locations, accurate to within a few hundred microns, in the target chamber in order to have a known fluence incident. In the cassette, the samples were held in place in such a way that a minimal "line contact" allows them to have the maximal mechanical response to the x-ray load. We present postshot images of the debris found on films used for filters, and pre- and postexposure specimens.

The prevalence of multidrug resistance is higher among bovine than human Salmonella enterica serotype Newport, Typhimurium, and 4,5,12:i:- isolates in the United States but differs by serotype and geographic region.

Salmonella represents an important zoonotic pathogen worldwide, but the transmission dynamics between humans and animals as well as within animal populations are incompletely understood. We characterized Salmonella isolates from cattle and humans in two geographic regions of the United States, the Pacific Northwest and the Northeast, using three common subtyping methods (pulsed-field gel electrophoresis [PFGE], multilocus variable number of tandem repeat analysis [MLVA], and multilocus sequence typing [MLST]). In addition, we analyzed the distribution of antimicrobial resistance among human and cattle Salmonella isolates from the two study areas and characterized Salmonella persistence on individual dairy farms. For both Salmonella enterica subsp. enterica serotypes Newport and Typhimurium, we found multidrug resistance to be significantly associated with bovine origin of isolates, with the odds of multidrug resistance for Newport isolates from cattle approximately 18 times higher than for Newport isolates from humans. Isolates from the Northwest were significantly more likely to be multidrug resistant than those from the Northeast, and susceptible and resistant isolates appeared to represent distinct Salmonella subtypes. We detected evidence for strain diversification during Salmonella persistence on farms, which included changes in antimicrobial resistance as well as genetic changes manifested in PFGE and MLVA pattern shifts. While discriminatory power was serotype dependent, the combination of PFGE data with either MLVA or resistance typing data consistently allowed for improved subtype discrimination. Our results are consistent with the idea that cattle are an important reservoir of multidrug-resistant Salmonella infections in humans. In addition, the study provides evidence for the value of including antimicrobial resistance data in epidemiological investigations and highlights the benefits and potential problems of combining subtyping methods.

Genotypic characterization of environmental isolates of Cryptococcus gattii from Puerto Rico.

Cryptococcus neoformans and Cryptococcus gattii are found in distinct environments with some overlap around different parts of the world. However, no systematic surveys of these two pathogens have been reported from Puerto Rico, a tropical island uniquely situated between mainland USA and countries in South America. We carried out an exhaustive environmental survey in southwestern Puerto Rico for pathogenic Cryptococcus species. Twenty-two presumptive isolates of C. gattii from cacti and tree detritus were characterized in detail by physiological and molecular methods and seventeen strains were confirmed as C. gattii. Cryptococcus gattii isolates were haploid and majority of them were MATa [corrected] strains. Sixteen out of seventeen C. gattii isolates belonged to VGII/AFLP6 genotype while one isolate was a VGIV/AFLP7 genotype. The results are significant as Puerto Rico strains are distinct from VGIII/AFLP5 strains reported from Southern California, but similar to C. gattii VGII/AFLP6 molecular type implicated in recent outbreaks of cryptococcosis in Pacific Northwest and British Columbia, Canada, but different in its M13 fingerprinting, and a common genotype in South America.

Salmonella enterica serotype 4,5,12:i:-, an emerging Salmonella serotype that represents multiple distinct clones.

The prevalence, among human clinical cases, of Salmonella enterica serotype 4,5,12:i:-, a serotype antigenically similar to Salmonella enterica serotype Typhimurium but lacking second-phase flagellar antigens, has increased considerably over the last 10 years. To probe the evolution and ecology of this emerging serotype, we characterized 190 Salmonella isolates initially classified as Salmonella serotypes 4,5,12:i:- (n = 90) and Typhimurium (n = 100) and obtained from various sources in the United States and Spain. These isolates were characterized into six sequence types (determined by multilocus sequence typing [MLST]) and 79 pulsed-field gel electrophoresis types. The majority of Salmonella serotype 4,5,12:i:- and Typhimurium isolates (85 and 84 isolates, respectively) represented a single MLST type. Existing genome information revealed different genome deletions (which included genes responsible for phase 2 flagellum expression) in four Spanish Salmonella serotype 4,5,12:i:- isolates and one U.S. Salmonella serotype 4,5,12:i:- isolate. Fifty-nine isolates of both serotypes, representing different sources and geographical locations as well as different molecular subtypes, were thus screened for the presence of six genes and one specific region, all of which were previously found to show variable presence among Salmonella serotype 4,5,12:i:- and Typhimurium strains. All Salmonella serotype 4,5,12:i:- isolates lacked the phase 2 flagella genes fljA and fljB, which were present in all Salmonella serotype Typhimurium isolates. While all Spanish Salmonella serotype 4,5,12:i:- isolates carried the same deletion surrounding fljAB, all but two U.S. isolates showed a different genomic deletion; the two atypical U.S. isolates represented the "Spanish" deletion genotype and a unique deletion genotype. Salmonella serotype 4,5,12:i:- thus appears to represent at least two common clones, which cannot easily be differentiated with standard diagnostic procedures.

PulseNet USA standardized pulsed-field gel electrophoresis protocol for subtyping of Vibrio parahaemolyticus.

PulseNet is a national molecular subtyping network for foodborne disease surveillance composed of public health and food regulatory agencies. Participants employ molecular subtyping of foodborne pathogens using a standardized method of pulsed-field gel electrophoresis (PFGE) for conducting laboratory-based surveillance of foodborne pathogens. The PulseNet standardized PFGE protocols are developed through a comprehensive testing process. The reproducibility of the protocol undergoes an internal evaluation at the Centers for Disease Control and Prevention and an external evaluation in multiple PulseNet laboratories. Here we describe the development and evaluation of a rapid PFGE protocol for subtyping Vibrio parahaemolyticus for use in PulseNet activities. The protocol was derived from the existing standardized PulseNet protocols for Escherichia coli O157:H7 and Vibrio cholerae. An external evaluation of this protocol was undertaken in collaboration with three PulseNet USA participating public health laboratories. Comparative analysis of the PFGE fingerprints generated by each of these laboratories demonstrated that the protocol is both reliable and reproducible in the hands of multiple users.

Community-associated methicillin-resistant Staphylococcus aureus skin infections in a religious community.

In September 2004, an outbreak of community-associated methicillin-resistant Staphylococcus aureus (MRSA) skin and soft tissue infections (SSTI) was reported among members of a religious community. We conducted a retrospective cohort study on all 175 community members; performed a nasal carriage survey, and environmental swab testing. We identified 24 MRSA cases (attack rate 14%). In multivariate analysis, sauna use [odds ratio (OR) 19.1, 95% confidence interval (CI) 2.7-206.1] and antimicrobial use within 12 months before infection (OR 11.7, 95% CI 2.9-47.6) were risk factors for infection. MRSA nasal carriage rate was 0.6% (1/174). Nine of 10 clinical isolates and an isolate from an administrative office within the community had the pulsed-field gel electrophoresis type USA300. Targeted hygiene improvement, wound care, and environmental cleaning were implemented. We describe the first reported outbreak of MRSA SSTI in a religious community. Adherence to appropriate personal and environmental hygiene might be critical factors in controlling transmission.

Genetic transformation of the housefly Musca domestica with the lepidopteran derived transposon piggyBac.

The piggyBac transposable element was successfully used for stable genetic transformation of the housefly Musca domestica. The construct contains the EGFP marker under the control of Pax-6 binding sites, which can drive eye-specific expression in insect species as distantly related as Drosophila melanogaster and Tribolium castaneum [Berghammer, A.J., Klingler, M. and Wimmer, E.A. (1999) Nature 402: 370-371]. We obtained seven independent integration events among 41 fertile G0 Musca flies. Most of the transformed lines contained two or more chromosomal insertions of the EGFP marker which were stably inherited over more than 15 generations. piggyBac-mediated transposition was verified by identifying the characteristic TTAA duplication at the insertion sites. This first report of stable transmission of a genetic marker in Musca confirms the use of this vector-marker system for effective gene transfer in a broad range of insect species.

Merging sex and position.

The choice between male and female development arises from a simple binary decision made in early development. Studies in a few model organisms led to a detailed understanding of the regulatory mechanisms that convey male or female identity at the cellular level. We have learned little, however, of how this information translates into the actual sexual phenotype with regionally dimorphic characters. Where does positional information come from and how does it integrate with the sex-determining pathway? A recent report sheds light onto this enigma and reveals possible intersections between sex-determining and homeotic pathways in Drosophila. Such intersections may also play an important part in evolution, providing a basis for phenotypic diversity among related species. BioEssays 23:304-306, 2001.

Rapid restructuring of bicoid-dependent hunchback promoters within and between Dipteran species: implications for molecular coevolution.

Interacting genetic elements need to coevolve if their joint function is to be maintained; for example, the correct binding of transcriptional regulators to defined binding sites in gene promoters needs to be maintained during evolution to ensure proper function. As part of a wider investigation into the molecular coevolution of the Dipteran homeodomain-bearing regulator bicoid (bcd) and Bcd-dependent promoters, we present data on the functional, structural, and sequence differences between the promoters of the segmentation gene hunchback (hb), in several species of Cyclorrhaphan (higher) Diptera. The result of phenocopying hb mutations using RNA interference (RNAi) in Musca domestica shows broadly similar functions to the hb gene in Drosophila melanogaster. However, the Bcd-binding sites in the hb promoters of Drosophila, Musca, and the two blowfly species Lucilia sericata and Calliphora vicina differ in copy number, sequence, orientation, and spacing. Furthermore, all promoters are subject to rapid turnover by slippage-like processes leading to high densities of short repetitive motifs. A study of polymorphism among six strains of M. domestica reveals that turnover by slippage also occurs in the promoter, untranslated leader, and exonic coding sequences of hb, but to different extents. We discuss these results in terms of the known interspecific differences in bcdand the potential coevolution of selected compensatory mutations in trans and cis in response to continuous promoter restructuring.

Sex determination in the Drosophila germline is dictated by the sexual identity of the surrounding soma.

It has been suggested that sexual identity in the germline depends upon the combination of a nonautonomous somatic signaling pathway and an autonomous X chromosome counting system. In the studies reported here, we have examined the role of the sexual differentiation genes transformer (tra) and doublesex (dsx) in regulating the activity of the somatic signaling pathway. We asked whether ectopic somatic expression of the female products of the tra and dsx genes could feminize the germline of XY animals. We find that Tra(F) is sufficient to feminize XY germ cells, shutting off the expression of male-specific markers and activating the expression of female-specific markers. Feminization of the germline depends upon the constitutively expressed transformer-2 (tra-2) gene, but does not seem to require a functional dsx gene. However, feminization of XY germ cells by Tra(F) can be blocked by the male form of the Dsx protein (Dsx(M)). Expression of the female form of dsx, Dsx(F), in XY animals also induced germline expression of female markers. Taken together with a previous analysis of the effects of mutations in tra, tra-2, and dsx on the feminization of XX germ cells in XX animals, our findings indicate that the somatic signaling pathway is redundant at the level tra and dsx. Finally, our studies call into question the idea that a cell-autonomous X chromosome counting system plays a central role in germline sex determination.

Effect of the 30 degree lateral recumbent position on pulmonary artery and pulmonary artery wedge pressures in critically ill adult cardiac surgery patients.

BACKGROUND: Despite demonstrated benefits of lateral positioning, critically ill patients may require prolonged supine positioning to obtain reproducible hemodynamic measurements. OBJECTIVES: TO determine the effect of 30 degree right and left lateral positions on pulmonary artery and pulmonary artery wedge pressures after cardiac surgery in critically ill adult patients. METHODS: An experimental repeated-measures design was used to study 35 patients with stable hemodynamics after cardiac surgery. Subjects were randomly assigned to 1 of 2 position sequences. Pulmonary artery and pulmonary artery wedge pressures were measured in each position. RESULTS: Measurements obtained from patients in the 30 degree left lateral position differed significantly (all Ps < .05) from measurements obtained from patients in the supine position for pulmonary artery systolic, end-diastolic, and mean pressures. Pulmonary artery wedge pressures did not differ significantly; however, data were available from only 17 subjects. The largest mean difference in pressures between the 2 positions was 2.0 +/- 2.1 mm Hg for pulmonary artery systolic pressures, whereas maximum differences for end-diastolic and pulmonary artery wedge pressures were 1.4 +/- 2.7 mm Hg and 1.6 +/- 2.4 mm Hg, respectively. Clinically significant position-related changes in pressure occurred in 12 (2.1%) of 581 pressure pairs. Clinically significant changes occurred in end-diastolic pressure in 2 subjects and in pulmonary artery wedge pressure in 1 subject. CONCLUSIONS: In patients with stable hemodynamics during the first 12 to 24 hours after cardiac surgery, measurements of pulmonary artery and pulmonary artery wedge pressures obtained in the 30 degree lateral and supine positions are clinically interchangeable.

Recombination and disjunction in female germ cells of Drosophila depend on the germline activity of the gene sex-lethal.

Gametogenesis in males and females differs in many ways. An important difference in Drosophila is that recombination between homologous chromosomes occurs only in female meiosis. Here, we report that this process relies on the correct functioning of Sex-lethal (Sxl) which is primarily known as the master gene in somatic sex determination. Certain alleles of this gene (Sxl(fs)) disrupt the germline, but not the somatic function of Sxl and cause an arrest of germ cell development during cystocyte proliferation. Using dominant suppressor mutations that relieve this early block in Sxl(fs) mutant females, we discovered additional requirements of Sxl for normal meiotic differentiation of the oocyte. Females mutant for Sxl(fs) and carrying a suppressor become fertile, but pairing of homologous chromosomes and formation of chiasmata is severely perturbed, resulting in an almost complete lack of recombinants and a high incidence of non-disjunction events. Similar results were obtained when germline expression of wild-type Sxl was compromised by mutations in virilizer (vir), a positive regulator of Sxl. Ectopic expression of a Sxl transgene in premeiotic stages of male germline development, on the other hand, is not sufficient to allow recombination to take place, which suggests that Sxl does not have a discriminatory role in this female-specific process. We propose that Sxl performs at least two tasks in oogenesis: an 'early' function in formation of the egg chamber, and a 'late' function in progression of the meiotic cell cycle, suggesting that both events are coordinated by a common mechanism.

Sex-lethal, the master sex-determining gene in Drosophila, is not sex-specifically regulated in Musca domestica.

Sex-lethal (Sxl) is the master switch gene for somatic sex determination in Drosophila melanogaster. In XX animals, Sxl becomes activated and imposes female development; in X(Y) animals, Sxl remains inactive and male development ensues. A switch gene for sex determination, called F, has also been identified in the housefly, Musca domestica. An active F dictates female development, while male development ensues when F is inactive. To test if the switch functions of Sxl and F are founded on a common molecular basis, we isolated the homologous Sxl gene in the housefly. Though highly conserved in sequence, Musca-Sxl is not sex-specifically regulated: the same transcripts and protein isoforms are expressed in both male and female animals throughout development. Musca-Sxl is apparently not controlled by the primary sex-determining signal and, thus, is unlikely to correspond to the F gene. Ectopic expression of Musca-SXL protein in Drosophila does not exert any noticeable effects on the known target genes of endogenous Sxl. Instead, forced overexpression of the transgene eventually results in lethality of both XY and XX animals and in developmental abnormalities in some escaper XY animals. Similar results were obtained with the Sxl homologue of Ceratitis capitata (Saccone, G., Peluso, I., Artiaco, D. , Giodano, E., Bopp, D. and Polito, L. C. (1998) Development 125, 1495-1500) suggesting that, in these non-drosophilid species, Sxl performs a function different from that in sex determination.

The Ceratitis capitata homologue of the Drosophila sex-determining gene sex-lethal is structurally conserved, but not sex-specifically regulated.

In Drosophila, Sxl functions as a binary switch in sex determination. Under the control of the primary sex-determining signal, it produces functional protein only in XX animals to implement female development. Here we report that, in contrast to Drosophila, the Sxl homologue in the Medfly, Ceratitis capitata, expresses the same mRNAs and protein isoforms in both XX and XY animals irrespective of the primary sex-determining signal. Also, experiments with two inducible transgenes demonstrate that the corresponding Ceratitis SXL product has no significant sex-transforming effects when expressed in Drosophila. Similar results have been obtained for the Sxl homologue of Musca domestica (Meise, M., Hilfiker-Kleiner, D., Brunner, C., DLbendorfer, A., N¿thiger, R. and Bopp, D. (1998) Development 125, 1487-1494). Our findings suggest that Sxl acquired its master regulatory role in sex determination during evolution of the Acalyptratae group, most probably after phylogenetic divergence of the genus Drosophila from other genera of this group.

Sex-specific control of Sex-lethal is a conserved mechanism for sex determination in the genus Drosophila.

In D. melanogaster the binary switch gene Sex-lethal (Sxl) plays a pivotal role in somatic sex determination -- when the Sxl gene is on the female pathway is followed, while the male pathway is followed when the gene is off. In the present study we have asked whether the Sxl gene is present in other species of the genus Drosophila and whether it is subject to a similar sex-specific on-off regulation. Sxl proteins were found in all of the drosophilids examined, and they display a sex-specific pattern of expression. Furthermore, characterization of the Sxl gene in the distant drosophilan relative, D. virilis, reveals that the structure and sequence organization of the gene has been well conserved and that, like melanogaster, alternative RNA processing is responsible for its sex-specific expression. Hence, this posttranscriptional on-off regulatory mechanism probably existed before the separation of the drosophilan and sophophoran subgenera and it seems likely that Sxl functions as a sex determination switch gene in most species in the Drosophila genus. Although alternative splicing appears to be responsible for the on-off regulation of the Sxl gene in D. virilis, this species is unusual in that Sxl proteins are present not only in females but also in males. The D. virilis female and male proteins appear to be identical over most of the length except for the amino-terminal approx. 25 aa which are encoded by the differentially spliced exons. In transcriptionally active polytene chromosomes, the male and female proteins bind to the same cytogenetic loci, including the sites corresponding to the D. virilis Sxl and tra genes. Hence, though the male proteins are able to interact with appropriate target pre-mRNAs, they are apparently incapable of altering the splicing pattern of these pre-mRNAs.