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A Drosophila third chromosome Minute locus encodes a ribosomal protein.

Minutes (M) are a group of over 50 phenotypically similar Drosophila mutations widely believed to affect ribosomal protein genes. This report describes the characterization of the P element-induced M(3)95A(Plac92) mutation [allelic to M(3)95A]. This mutation can be reversed by the mobilization of the P element, demonstrating that the mutation is caused by insertion of this transposable element. The gene interrupted by insertion of the P element was cloned by use of inverse polymerase chain reaction. Nucleotide sequence analysis revealed a 70-75% identity to the human and rat ribosomal protein S3 genes, and to the Xenopus ribosomal protein S1a gene. At the amino acid level, the overall identity is approximately 78% for all three species. This is only the second time that a Minute has been demonstrated to encode a ribosomal protein.

Amino Acid Sequence↗

Ribosome dynamics at the conserved PGP motif governs 2A peptide-bond-skipping efficiency.

Viral 2A oligopeptides drive an unusual ribosome recoding event in which peptide-bond formation fails at a conserved PG↓P motif, producing two discrete proteins without canonical termination. Despite decades of study, the molecular basis of 2A-mediated peptide-bond skipping remains poorly understood. Here, we combine quantitative 2A reporters with high-resolution ribosome profiling to interrogate ribosome dynamics at the core 2A sequences. We identify a pausing event at the terminal proline codon of the PGP motif that functions as a kinetic decision point: ribosome dwell time at this site inversely correlates with skipping efficiency. Increasing nascent chain flexibility by inserting linkers immediately upstream of the 2A sequence reduces ribosome occupancy at the terminal proline codon and enhances peptide-bond skipping. Strikingly, amino acid repeats positioned distally upstream also modulate 2A activity, indicating long-range coupling between nascent chain properties outside of the ribosome and the peptidyl transferase center inside the ribosome. In particular, hydrophobic residues potently suppress skipping, an effect that can be rescued by extending flexible segments within the peptide exit tunnel. Together, our findings support a model in which nascent chain features-beyond the core 2A motif-dynamically tune ribosomal recoding efficiency through co-translational feedback into the catalytic center.

Ribosomes↗

Pedicle screw pullout strength. Correlation with insertional torque.

This study was designed to correlate several parameters regarding pedicle screw bone/metal interface strength. The insertional torque measured during tapping and placement of pedicle screws was correlated with the bone mineral density of the vertebral body, the dimensions of the pedicle, the method of preparation of the pedicle, and the amount of load and number of cycles to failure of the bone/metal interface. Thirty human cadaveric lumbar vertebrae were instrumented with 6.5-mm pedicle screws. The maximum torque achieved during insertion was digitally recorded. A cyclic pedicle screw pullout test was performed. A linear correlation existed between both the insertional torque when tapping or when inserting a screw and the number of cycles to ultimate pedicle screw pullout. An inverse linear relationship was found between the pedicle width and cycles to failure. There was no linear correlation found when comparing the number of cycles to failure to bone mineral density. These findings suggest that insertional torque is a good predictor of bone-metal interface failure. Bone mineral density of the vertebral body was less effective as a predictor of failure. Smaller pedicle width correlated with increased insertional torque and cycles to failure. This may explain why patients with osteoporosis on radiography may still obtain stable fixation with pedicle screws. Other factors, such as pedicle dimensions and shape, affect screw purchase as much as vertebral body bone density. Insertional torque less than 4.0 inch-pounds led to early pedicle screw pullout. This study forms the basis for the authors' clinical use of an instrumented torque screwdriver to measure insertional torque in the operating room.

Aged↗

Poxvirus genomes: a phylogenetic analysis.

The evolutionary relationships of 26 sequenced members of the poxvirus family have been investigated by comparing their genome organization and gene content and by using DNA and protein sequences for phylogenetic analyses. The central region of the genome of chordopoxviruses (ChPVs) is highly conserved in gene content and arrangement, except for some gene inversions in Fowlpox virus (FPV) and species-specific gene insertions in FPV and Molluscum contagiosum virus (MCV). In the central region 90 genes are conserved in all ChPVs, but no gene from near the termini is conserved throughout the subfamily. Inclusion of two entomopoxvirus (EnPV) sequences reduces the number of conserved genes to 49. The EnPVs are divergent from ChPVs and between themselves. Relationships between ChPV genera were evaluated by comparing the genome size, number of unique genes, gene arrangement and phylogenetic analyses of protein sequences. Overall, genus Avipoxvirus is the most divergent. The next most divergent ChPV genus is Molluscipoxvirus, whose sole member, MCV, infects only man. The Suipoxvirus, Capripoxvirus, Leporipoxvirus and Yatapoxvirus genera cluster together, with Suipoxvirus and Capripoxvirus sharing a common ancestor, and are distinct from the genus Orthopoxvirus (OPV). Within the OPV genus, Monkeypox virus, Ectromelia virus and Cowpox virus strain Brighton Red (BR) do not group closely with any other OPV, Variola virus and Camelpox virus form a subgroup, and Vaccinia virus is most closely related to CPV-GRI-90. This suggests that CPV-BR and GRI-90 should be separate species.

Amino Acid Sequence↗

Construction of a mini-Tn5-luxCDABE mutant library in Pseudomonas aeruginosa PAO1: a tool for identifying differentially regulated genes.

Pseudomonas aeruginosa is a major cause of nosocomial (hospital-derived) infections, is the predominant pathogen in chronic cystic fibrosis lung infections, and remains difficult to treat due to its high intrinsic antibiotic resistance. The completion of the P. aeruginosa PAO1 genome sequence provides the opportunity for genome-wide studies to increase our understanding of the pathogenesis and biology of this important pathogen. In this report, we describe the construction of a mini-Tn5-luxCDABE mutant library and a high-throughput inverse PCR method to amplify DNA flanking the site of insertion for sequencing and insertion site mapping. In addition to producing polar knockout mutations in nonessential genes, the promoterless luxCDABE reporter present in the transposon serves as a real-time reporter of gene expression for the inactivated gene. A total of 2519 transposon insertion sites were mapped, 77% of which were nonredundant insertions. Of the insertions within an ORF, -55% of total and unique insertion sites were transcriptional luxCDABE fusions. A bias toward low insertion-site density in the genome region that surrounds the predicted terminus of replication was observed. To demonstrate the utility of chromosomal lux fusions, we performed extensive regulatory screens to identify genes that were differentially regulated under magnesium or phosphate limitation. This approach led to the discovery of many known and novel genes necessary for these environmental adaptations, including genes involved in resistance to cationic antimicrobial peptides. This dual-purpose mutant library allows for functional and regulation studies and will serve as a resource for the research community to further our understanding of P. aeruginosa biology.

Antimicrobial Cationic Peptides↗

Comparative genomics of two Leptospira interrogans serovars reveals novel insights into physiology and pathogenesis.

Leptospira species colonize a significant proportion of rodent populations worldwide and produce life-threatening infections in accidental hosts, including humans. Complete genome sequencing of Leptospira interrogans serovar Copenhageni and comparative analysis with the available Leptospira interrogans serovar Lai genome reveal that despite overall genetic similarity there are significant structural differences, including a large chromosomal inversion and extensive variation in the number and distribution of insertion sequence elements. Genome sequence analysis elucidates many of the novel aspects of leptospiral physiology relating to energy metabolism, oxygen tolerance, two-component signal transduction systems, and mechanisms of pathogenesis. A broad array of transcriptional regulation proteins and two new families of afimbrial adhesins which contribute to host tissue colonization in the early steps of infection were identified. Differences in genes involved in the biosynthesis of lipopolysaccharide O side chains between the Copenhageni and Lai serovars were identified, offering an important starting point for the elucidation of the organism's complex polysaccharide surface antigens. Differences in adhesins and in lipopolysaccharide might be associated with the adaptation of serovars Copenhageni and Lai to different animal hosts. Hundreds of genes encoding surface-exposed lipoproteins and transmembrane outer membrane proteins were identified as candidates for development of vaccines for the prevention of leptospirosis.

Animals↗

Four of the seven zinc fingers of the Evi-1 myeloid-transforming gene are required for sequence-specific binding to GA(C/T)AAGA(T/C)AAGATAA.

Expression of the Evi-1 gene is activated in murine myeloid leukemias by retroviral insertions and in human acute myelogenous leukemia by translocations and inversions involving chromosome band 3q26 where the gene resides. Aberrant expression of the Evi-1 gene has been shown to interfere with myeloid differentiation, which is proposed to be the basis for its role in leukemias. The Evi-1 gene encodes a 145-kDa DNA-binding protein containing two domains of seven and three Cys2-His2 zinc fingers. Previous studies identified a portion of the consensus DNA-binding sequence for the first domain of zinc fingers. The experiments presented here extend these studies and demonstrate that the first domain recognizes a consensus of 15 nucleotides consisting of GA(C/T)AAGA(T/C)AAGATAA. The first three fingers of the first domain do not detectably bind DNA but contribute to the binding by conferring a relative specificity for GACAA verses GATAA in the first position. The first three fingers also contribute to optimal binding of the 15-nucleotide consensus sequence.

Amino Acid Sequence↗

Gene verification and discovery by Walking Tree Method.

The Walking Tree Method [3, 4, 5, 18] is an approximate string alignment method that can handle insertions, deletions, substitutions, translocations, and more than one level of inversions all together. Moreover, it tends to highlight gene locations, and helps discover unknown genes. Its recent improvements in runtime and space use extends its capability in exploring large strings. We will briefly describe the Walking Tree Method with its recent improvements [18], and demonstrate its speed and ability to align real complete genomes such as Borrelia burgdorferi (910724 base pairs of its single chromosome) and Chlamydia trachomatis (1042519 base pairs) in reasonable time, and to locate and verify genes.

Animals↗

Rapid identification and mapping of insertion sequences in Escherichia coli genomes using vectorette PCR.

BACKGROUND: Insertion sequences (IS) are small DNA segments capable of transposing within and between prokaryotic genomes, often causing insertional mutations and chromosomal rearrangements. Although several methods are available for locating ISs in microbial genomes, they are either labor-intensive or inefficient. Here, we use vectorette PCR to identify and map the genomic positions of the eight insertion sequences (IS1, 2, 3, 4, 5, 30, 150, and 186) found in E. coli strain CGSC6300, a close relative of MG1655 whose genome has been sequenced. RESULTS: Genomic DNA from strain CGSC6300 was digested with a four-base cutter Rsa I and the resulting restriction fragments ligated onto vectorette units. Using IS-specific primers directed outward from the extreme ends of each IS and a vectorette primer, flanking DNA fragments were amplified from all but one of the 37 IS elements identified in the genomic sequence of MG1655. Purification and sequencing of the PCR products confirmed that they are IS-associated flanking DNA fragments corresponding to the known IS locations in the MG1655 genome. Seven additional insertions were found in strain CGSC6300 indicating that very closely related isolates of the same laboratory strain (the K12 isolate) may differ in their IS complement. Two other E. coli K12 derivatives, TD2 and TD10, were also analyzed by vectorette PCR. They share 36 of the MG1655 IS sites as well as having 16 and 18 additional insertions, respectively. CONCLUSION: This study shows that vectorette PCR is a swift, efficient, reliable method for typing microbial strains and identifying and mapping IS insertion sites present in microbial genomes. Unlike Southern hybridization and inverse PCR, our approach involves only one genomic digest and one ligation step. Vectorette PCR is then used to simultaneously amplify all IS elements of a given type, making it a rapid and sensitive means to survey IS elements in genomes. The ability to rapidly identify the IS complements of microbial genomes should facilitate subtyping closely related pathogens during disease outbreaks.

Bacterial Typing Techniques↗

[Age-related changes in crossing over in Drosophila resemble the picture of interchromosomal effect of chromosome rearrangement on crossing over].

Crossing over in the left arm of chromosome 2 (2L) was studied in successive broods of Drosophila melanogaster females carrying intact chromosomes (+/+), inversion Muller-5 in the X chromosome (M-5/+), and insertion of the Y-chromosome material into region 34A (Is(2L)/+). The regions net-dp, dp-b, b-pr and pr-cn were examined in 14 two-day-old broods of females +/+ and M-5/+ and in 10 broods of females Is(2L)/+. In all lines, the highest level of crossing over was in the first three broods (eggs laid during the first 6 days of oviposition) and the lowest level in the broods 7-8 (eggs laid at days 14-16). A high rate of crossing over in the first broods of females +/+ and M-5/+ was due to an increment of exchanges in the proximal euchromatin regions (b-pr and pr-cn) and to an increase in the number of tetrads with double exchanges. These changes are similar to a pattern of the interchromosomal effect on crossing over (IEC) in structurally normal chromosomes. In Is(2L)/+ females, a high level of crossing over was due to extensive exchanges in the interstitial regions net-dp and dp and an increase in the number of tetrads with single exchanges. These changes resembled the IEC in rearranged chromosomes (in this case, in chromosomes bearing an insertion). Thus, the age changes of crossing over are similar to the consequences of the presence or absence of IEC. Age changes in crossing over in a chromosome depended both on the local rearrangements in this chromosome (the local effect on crossing over, LEC) and on rearrangements in nonhomologous chromosomes (IEC). In the first broods, both LEC and IEC decreased with an increase in the level of crossing over. In subsequent broods, the reduced level of crossing over was accompanied by an increase in both LEC and IEC. This suggests that the mechanisms responsible for the age changes in crossing over and IEC may have common steps. The contact model of crossing over may explain the similarity between the age changes in crossing-over and IEC. It is suggested that both phenomena result from delayed determination of crossing over in a meiotic cell. This may occur due to the retarded formation of the local contacts in one of the homologous chromosome pairs or because a higher number of local contacts is required to trigger crossing over in a meiotic cell (of early age).

Aging↗

A new approach to fetal echocardiography: digital casts of the fetal cardiac chambers and great vessels for detection of congenital heart disease.

OBJECTIVE: The purpose of this study was to describe a method of 4-dimensional (4D) reconstruction of the cardiac chambers and outflow tracts using a combination of spatiotemporal image correlation, "inversion mode," and "B-flow" imaging. METHODS: Spatiotemporal image correlation and the inversion mode were used in the examination of the volume data sets of 23 fetuses with congenital heart anomalies. A subset was also examined with B-flow imaging using the gradient light algorithm. Digital reconstructions from abnormal hearts were compared with a library obtained from fetuses without abnormalities. RESULTS: Rendered images of the 4-chamber view using the inversion mode were characterized by: (1) echogenic chambers; (2) sharp delineation of chamber contours when compared with 2-dimensional (2D) images; and (3) distinct display of the myocardium, interventricular septum, interatrial septum, and mitral and tricuspid valves as anechoic structures. Ventricular septal defects, abnormal differential insertion of the atrioventricular valves, and valve atresia were well visualized with the inversion mode. The application of inversion mode or B-flow imaging to 4D rendering of the outflow tracts resulted in "digital casts" displaying the spatial relationships between the outflow tracts as well as the connections between the great arteries and ventricular chambers. The spatial relationships and communications among cardiac structures cannot be visualized with conventional 2D ultrasonography. CONCLUSIONS: The application of spatiotemporal image correlation, inversion mode, and B-flow imaging generates information about the anatomy and pathologic characteristics of the fetal heart (digital casts) that cannot be obtained with 2D fetal echocardiography. We propose that these modalities enhance the information provided by ultrasonographic interrogation of the fetal heart and will improve prenatal diagnosis.

Echocardiography, Four-Dimensional↗

Intrauterine devices and pelvic inflammatory disease: an international perspective.

The risk of pelvic inflammatory disease (PID) associated with use of an intrauterine device (IUD) has been an important concern that has dominated decisions on its use throughout the world, especially in the USA. Early research that suggested such an association led to both a dramatic decline in use of the method and its withdrawal from the US market by two manufacturers. However, factors other than use of an IUD are now thought to be major determinants of PID risk. To address these concerns, we have reviewed the World Health Organisation's IUD clinical trial data to explore the incidence and patterns of PID risk with use of an IUD. The overall rate of PID among 22,908 IUD insertions and during 51,399 woman-years of follow-up was 1.6 cases per 1000 woman-years of use. After adjustment for confounding factors, PID risk was more than six times higher during the 20 days after insertion than during later times (unadjusted rates, 9.7 vs 1.4 per 1000 woman-years, respectively); the risk was low and constant for up to eight years of follow-up. Rates varied according to geographical area (highest in Africa and lowest in China) and were inversely associated with age. PID rates were lower among women who had IUDs inserted more recently. Our findings indicate that PID among IUD users is most strongly related to the insertion process and to background risk of sexually transmissible disease. PID is an infrequent event beyond the first 20 days after insertion. Because of this increased risk with insertion, IUDs should be left in place up to their maximum lifespan and should not routinely be replaced earlier, provided there are no contraindications to continued use and the woman wishes to continue with the device.

Adolescent↗

Large-scale identification of virulence genes from Streptococcus pneumoniae.

Streptococcus pneumoniae is the major cause of bacterial pneumonia, and it is also responsible for otitis media and meningitis in children. Apart from the capsule, the virulence factors of this pathogen are not completely understood. Recent technical advances in the field of bacterial pathogenesis (in vivo expression technology and signature-tagged mutagenesis [STM]) have allowed a large-scale identification of virulence genes. We have adapted to S. pneumoniae the STM technique, originally used for the discovery of Salmonella genes involved in pathogenicity. A library of pneumococcal chromosomal fragments (400 to 600 bp) was constructed in a suicide plasmid vector carrying unique DNA sequence tags and a chloramphenicol resistance marker. The recent clinical isolate G54 was transformed with this library. Chloramphenicol-resistant mutants were obtained by homologous recombination, resulting in genes inactivated by insertion of the suicide vector carrying a unique tag. In a mouse pneumonia model, 1.250 candidate clones were screened; 200 of these were not recovered from the lungs were therefore considered virulence-attenuated mutants. The regions flanking the chloramphenicol gene of the attenuated mutants were amplified by inverse PCR and sequenced. The sequence analysis showed that the 200 mutants had insertions in 126 different genes that could be grouped in six classes: (i) known pneumococcal virulence genes; (ii) genes involved in metabolic pathways; (iii) genes encoding proteases; (iv) genes coding for ATP binding cassette transporters; (v) genes encoding proteins involved in DNA recombination/repair; and (vi) DNA sequences that showed similarity to hypothetical genes with unknown function. To evaluate the virulence attenuation for each mutant, all 126 clones were individually analyzed in a mouse septicemia model. Not all mutants selected in the pneumonia model were confirmed in septicemia, thus indicating the existence of virulence factors specific for pneumonia.

Animals↗

Integrated light-scattering spectroscopy, a sensitive probe for peptide-vesicle binding: application to the membrane-bound colicin E1 channel peptide.

Integrated light-scattering (ILS) spectroscopy was used to monitor the binding of the colicin E1 channel peptide to POPC:POPG large unilamellar vesicles (LUV; 60:40, mol:mol) at acidic pH (3.5). Binding conditions were chosen such that nearly all of the channel peptide was bound to the vesicles with little free peptide remaining in solution. The increase in vesicle size upon the insertion of the channel peptide was measured by performing a discrete inversion technique on data obtained from an ILS spectrometer. Vesicle size number distributions were determined for five different systems having peptide/vesicle ratios of approximately 0, 77, 154, 206, and 257. The experiment was repeated four times (twice at two different vesicle concentrations) to determine reproducibility. The relative changes in vesicle radius upon peptide binding to the membrane vesicles was remarkably reproducible even though these changes represented only a few nanometers. A comparison of vesicle size number distributions in the absence of bound peptide was made between ILS and dynamic light scattering (DLS) data and showed similar results. However, DLS was incapable of detecting the small changes due to peptide-induced vesicle swelling. The membrane-bound volume of the colicin E1 channel peptide was approximately 177 +/- 22 nm3. These data indicate that in the absence of a membrane potential (closed channel state) the colicin E1 channel peptide inserts into the membrane resulting in a significant displacement of the lipid bilayer as evidenced from the dose-dependent increase in the vesicle radius. These results indicate that ILS spectroscopy is a sensitive sizing technique that is capable of detecting relatively small changes in membrane vesicles and may have a wide application in the determination of peptide binding to membrane vesicles.

Biophysical Phenomena↗

Fine-mapping of cytogenetically undetectable EWS/ERG fusions on DNA fibers of Ewing tumors.

In contrast to the EWS/FLI1 fusion which is represented by a t(11;22)(q24;q12), EWS/ERG fusions are frequently cytogenetically not detectable. Three Ewing tumors (ET), two with apparently normal chromosomes 21 and 22, and one ET with a t(2;22)(p25;q12), were studied by FISH on interphase nuclei, metaphase chromosomes and on DNA fibers. EWS/ERG transcripts were detected by RT-PCR in all cases. FISH, using cosmids located proximally (F10, G9) and distally (F7) to the EWS breakpoint region, revealed no detectable separation of these probes in two cases. In contrast, co-hybridization of probe PT1526 containing the ERG breakpoint region with G9 revealed the juxtaposition of two signals per interphase nucleus in all three cases indicating the EWS/ERG fusions. Chromosome preparations displayed the juxtaposed signals on the der(22), and hybridization signals of the probes PT1526 and G9 on the non-rearranged chromosomes 21 and 22 in all cases, respectively. The PT1526 signal on the der(21) was seen only in cases 1 and 2. These results were confirmed by triple-target FISH on tumor DNA fibers. In all three cases, the hybridization pattern F10 - G9 - PT1526 indicates a centromere to telomere orientation. This finding suggests that EWS/ERG fusions in ETs may be generated by an inversion of the ERG gene or a part thereof followed by an insertion into the EWS gene on the der(22). Double-target FISH on interphase nuclei using probes flanking the EWS breakpoint region and probe PT1526 enables the detection of virtually all 22q12 rearrangements in ETs, thus providing a reliable diagnostic assay.

Cation Transport Proteins↗

[Associated changes in recombination length, number of exchanges, and interference in Drosophila chromosome 2].

A set of eight strains differing in recombinational length of the left arm of chromosome 2(2L) was obtained by means of inserting genetic material of the Y chromosome into the 34A region and two inversions, In(1)dl-49 + BM1 and In(3LR)D, demonstrating interchromosomal effects on crossing over. The length of 2L ranged from 15.0 to 64.9 cM. Interchromosomal effects on crossing over differed in extent and in distribution over 2L between females carrying wild-type chromosome 2 and the chromosome with inserts. Tetrad analysis showed that the 2L recombinational length was increased up to the normal level (females +/+; +/+, +/+) or more due to increased proportions of single- or double-exchange tetrads, respectively. A model of pairing and crossing over proposed earlier was used to interpret crossing-over patterns. The model implies that initial pairing of homologous chromosomes involves the formation of local contacts between them. The number of exchanges in a bivalent is determined by the number of local contacts at the onset of an event termed the determination of crossing-over sites (DCS). Interference is determined by the mechanism of contact formation. The extent of interference varies both temporally and spatially (within a chromosome), decreasing from the periphery to a contact site as the time from contact formation increases.

Animals↗

Rapid amplification and cloning of Tn5 flanking fragments by inverse PCR.

A simple approach is described to efficiently amplify DNA sequences flanking transposon Tn5 insertions. The method involves: (i) digestion with a restriction enzyme that cuts within Tn5; (ii) self-ligation under conditions favouring the production of monomeric circles; (iii) four parallel PCR reactions using primers designed to amplify left or right flanking sequences, and to distinguish target amplicons from non-specific products. This reveals the number of Tn5 insertions and the size of flanking genomic restriction fragments, without Southern blot analysis. The amplified product contains restriction sites that facilitate cohesive-end cloning. This rapid method is demonstrated using Tn5 and Tn5-Mob tagged DNA sequences involved in albicidin biosynthesis in Xanthomonas albilineans. It is generally applicable for efficient recovery of DNA sequences flanking transposon Tn5 derivatives in insertional mutagenesis studies.

Anti-Bacterial Agents↗

[Abdominal reposition of puerperally inverted uterus (author's transl)].

Inversion of uterus in puerperium is an extremely rare case and always accompanied by severe shock. To the obstetrician such event is almost always unexpected and calls for immediate decision and action. Dry manual reposition usually is impossible for tough inversion pressure, while severe disadvantages are implied in vaginal surgery with incision of the anterior and posterior walls of the uterus. Therefore, an abdominal method is recommended by the author, leaning on Huntington's technique. First, the shock is brought under control. Then the inverted uterus is levered out of the inversion funnel to complete reversion, step by step, with a pair of bullet forceps inserted after each step, four altogether. The uterus of two cases was completely reversed by means of that simple method in 30 minutes only. Even the most careful and vigilant obstetrician may find himself trapped in such rare situation of spontaneous uterus inversion due to the constitution of a particular patient. The procedure suggested in this paper, therefore, may be an approach practicable even in smaller and less equipped hospitals, when it comes to the need for immediate remedial action to cope with that most dangerous complication.

Female↗