Impact of extreme temperatures on ambulance dispatches in London, UK.

BACKGROUND: Associations between extreme temperatures and health outcomes, such as mortality and morbidity, are often observed. However, relatively little research has investigated the role of extreme temperatures upon ambulance dispatches. METHODS: A time series analysis using London Ambulance Service (LAS) incident data (2010-2014), consisting of 5,252,375 dispatches was conducted. A generalized linear model (GLM) with a quasi-likelihood Poisson regression was applied to analyse the associations between ambulance dispatches and temperature. The 99th (22.8°C) and 1st (0.0°C) percentiles of temperature were defined as extreme high and low temperature. Fourteen categories of ambulance dispatches were investigated, grouped into 'respiratory' (asthma, dyspnoea, respiratory chest infection, respiratory arrest and chronic obstructive pulmonary disease), 'cardiovascular' (cardiac arrest, chest pain, cardiac chest pain RCI, cardiac arrhythmia and other cardiac problems) and 'other' non-cardiorespiratory (dizzy, alcohol related, vomiting and 'generally unwell') categories. The effects of long-term trends, seasonality, day of the week, public holidays and air pollution were controlled for in the GLM. The lag effect of temperature was also investigated. The threshold temperatures for each category were identified and a distributed lag non-linear model (DLNM) was reported using relative risk (RR) values at 95% confidence intervals. RESULTS: Many dispatch categories show significant associations with extreme temperature. Total calls from 999 dispatches and 'generally unwell' dispatch category show significant RRs at both low and high temperatures. Most respiratory categories (asthma, dyspnoea and RCI) have significant RRs at low temperatures represented by with estimated RRs ranging from 1.392 (95%CI: 1.161-1.699) for asthma to 2.075 (95%CI: 1.673-2.574) for RCI. The RRs for all other non-cardiorespiratory dispatches were often significant for high temperatures ranging from 1.280 (95% CI: 1.128-1.454) for 'generally unwell' to 1.985 (95%CI: 1.422-2.773) for alcohol-related. For the cardiovascular group, only chest pain dispatches reported a significant RR at high temperatures. CONCLUSIONS: Ambulance dispatches can be associated with extreme temperatures, dependent on the dispatch category. It is recommended that meteorological factors are factored into ambulance forecast models and warning systems, allowing for improvements in ambulance and general health service efficiency.

A-type and B-type lamins initiate layer assembly at distinct areas of the nuclear envelope in living cells.

To investigate nuclear lamina re-assembly in vivo, Drosophila A-type and B-type lamins were artificially expressed in Drosophila lamin Dm(0)null mutant brain cells. Both exogenous lamin C (A-type) and Dm(0) (B-type) formed sub-layers at the nuclear periphery, and efficiently reverted the abnormal clustering of the NPC. Lamin C initially appeared where NPCs were clustered, and subsequently extended along the nuclear periphery accompanied by the recovery of the regular distribution of NPCs. In contrast, lamin Dm(0) did not show association with the clustered NPCs during lamina formation and NPC spacing recovered only after completion of a closed lamin Dm(0) layer. Further, when lamin Dm(0) and C were both expressed, they did not co-polymerize, initiating layer formation in separate regions. Thus, A and B-type lamins reveal differing properties during lamina assembly, with A-type having the primary role in organizing NPC distribution. This previously unknown complexity in the assembly of the nuclear lamina could be the basis for intricate nuclear envelope functions.

The Drosophila mus 308 gene product, implicated in tolerance of DNA interstrand crosslinks, is a nuclear protein found in both ovaries and embryos.

mus 308 designates one of over 30 mutagen sensitivity loci found in Drosophila. It is predicted to code for a 229-kDa polypeptide. Published sequence analyses of others indicate that this polypeptide would have helicase motifs near its N-terminus, and similarities to bacterial DNA polymerase I-like enzymes near its C-terminus. In our studies, two different and highly specific antibodies were prepared and used for identification as well as characterization of the mus 308 gene product. Western blot analyses reveal a single reactive polypeptide in both ovaries and embryos as well as in two Drosophila embryo tissue culture cell lines; it is nearly absent in homozygous mus 308 mutants. This polypeptide is about 229 kDa in size, and indirect immunofluorescence shows that the mus 308 gene product localizes throughout nuclei in wild-type cells but appears to be absent in a mus 308 mutant. Immunoblot analyses throughout development suggest greatest abundance at the end of embryogenesis, immediately before hatching of first instar larvae. They also showed a smaller ( approximately 100 kDa) antigenically and genetically related polypeptide found only in adult males. Immunoprecipitation, a highly effective method of specific purification, suggests that the mus 308 protein has DNA polymerase activity that is NEM-sensitive but largely aphidicolin-resistant. In addition, the immunoprecipitated material has DNA-dependent ATPase but lacks detectable helicase.

Site-specific mutagenesis of Drosophila proliferating cell nuclear antigen enhances its effects on calf thymus DNA polymerase delta.

BACKGROUND: We and others have shown four distinct and presumably related effects of mammalian proliferating cell nuclear antigen (PCNA) on DNA synthesis catalyzed by mammalian DNA polymerase delta(pol delta). In the presence of homologous PCNA, pol delta exhibits 1) increased absolute activity; 2) increased processivity of DNA synthesis; 3) stable binding of synthetic oligonucleotide template-primers (t1/2 of the pol deltaPCNAtemplate-primer complex >/=2.5 h); and 4) enhanced synthesis of DNA opposite and beyond template base lesions. This last effect is potentially mutagenic in vivo. Biochemical studies performed in parallel with in vivo genetic analyses, would represent an extremely powerful approach to investigate further, both DNA replication and repair in eukaryotes. RESULTS: Drosophila PCNA, although highly similar in structure to mammalian PCNA (e.g., it is >70% identical to human PCNA in amino acid sequence), can only substitute poorly for either calf thymus or human PCNA (approximately 10% as well) in affecting calf thymus pol delta. However, by mutating one or only a few amino acids in the region of Drosophila PCNA thought to interact with pol delta, all four effects can be enhanced dramatically. CONCLUSIONS: Our results therefore suggest that all four above effects depend at least in part on the PCNA-pol delta interaction. Moreover unlike mammals, Drosophila offers the potential for immediate in vivo genetic analyses. Although it has proven difficult to obtain sufficient amounts of homologous pol delta for parallel in vitro biochemical studies, by altering Drosophila PCNA using site-directed mutagenesis as suggested by our results, in vitro biochemical studies may now be performed using human and/or calf thymus pol delta preparations.

Null mutants of Drosophila B-type lamin Dm(0) show aberrant tissue differentiation rather than obvious nuclear shape distortion or specific defects during cell proliferation.

To elucidate the function of metazoan B-type lamins during development, new null mutations of the Drosophila B-type lamin gene, lamDm(0), were analyzed in parallel with the misg(sz18) mutation, a lamDm(0) allele reported previously. Although in all these mutants, lamin Dm(0) protein was undetectable in neuroblasts and imaginal disc cells from the second instar larval stage onward, cells continued to proliferate. In contrast to the embryonic lethality of another Drosophila lamDm(0) allele, lam(PM15), reported previously, lethality did not occur until late pupal stages. Chromosomal structure and the overall nuclear shape remained normal even at these late pupal stages, although obviously abnormal nuclear pore complex distribution was observed concomitant with the loss of lamin Dm(0) protein. Compensating expression of lamin C was not induced in the absence of lamin Dm(0). Thus, no lamin-containing nuclear structures were found in proliferating larval neuroblasts. We did find that developmental abnormalities appeared in specific organs during the late pupal stage, preceding lethality. Surprisingly, coordinated size increase (hypertrophy) of the ventriculus was observed accompanied by cell division and muscle layer formation. Hypertrophy of the ventriculus correlated with a decrease in ecdysteroid hormone receptor B1 (EcRB1) protein, and furthermore could be suppressed by a heat-inducible EcRB1 transgene. In contrast, both gonadal and CNS tissues exhibited underdevelopment.

In vivo phosphorylation of Drosophila melanogaster nuclear lamins during both interphase and mitosis.

To study phosphorylation of D. melanogaster nuclear lamins in vivo, we used Kc tissue culture cells. Kc cells contain products of both lamin genes, the lamin Dm0 gene encoding constitutive polypeptides expressed in almost all cell types and the developmentally regulated lamin C gene. We grew Kc cells in low phosphate medium and labelled them with (32P(H3PO4. To obtain mitotic cells we used vinblastine to arrest cells in metaphase. Cells were collected, washed, lysed and resultant extracts fractionated in the presence of protein phosphatase inhibitors. D. melanogaster proteins were then denatured by boiling in SDS plus DTT, followed by immunoaffinity chromatography and SDS-PAGE purification. As anticipated, we found that a CNBr fragment derived from the N-terminal part of lamin Dm0-derivatives (amino acid residues 2-158; fragment A) was phosphorylated during both interphase and mitosis. Interphase but not mitotic phosphorylation was found on an internal CNBr fragment (derived from the end of the central rod domain and the first part of the C-terminal lamin tail; amino acid residues 385-548; fragment D). Interphase only phosphorylation was also detected on another CNBr fragment derived from the extreme C-terminal portion of lamin Dm0-derivatives (amino acid residues 549-622; fragment E). To supplement these data, we used 2-D tryptic peptide mapping followed by phosphorImager analysis. We routinely detected at least seven 'spots' derived from interphase lamins but only a single mitotic lamin phosphopeptide.

A single amino acid change (E85K) in human PCNA that leads, relative to wild type, to enhanced DNA synthesis by DNA polymerase delta past nucleotide base lesions (TLS) as well as on unmodified templates.

Human proliferating cell nuclear antigen (hPCNA) containing a single amino acid substitution at position 85, that of lysine for glutamate (E85K), was compared to wild-type (wt) hPCNA for its ability to promote DNA synthesis by purified DNA polymerase delta (pol delta) both on unmodified templates and past chemically defined template base lesions (translesion synthesis; TLS). Significant enhancement (up to 4-5-fold or greater) was seen but depended both on the exact PCNA/pol delta ratio tested and on the specific nature of the template (e.g., unmodified versus lesion-containing; chemical nature of the template base lesion). These results suggest that human PCNA, either mutated to contain lysine (K) at position 85 or bearing similar primary mutations, would promote more secondary mutagenesis in cells and/or tissues where PCNA is normally expressed at low levels relative to pol delta. Over an entire lifetime, such secondary mutagenesis could be biomedically significant.

Barrier-to-autointegration factor plays crucial roles in cell cycle progression and nuclear organization in Drosophila.

Barrier-to-autointegration factor (BAF) is potentially a DNA-bridging protein, which directly associates with inner nuclear membrane proteins carrying LEM domains. These features point to a key role in regulation of nuclear function and organization, dependent on interactions between the nuclear envelope and chromatin. To understand the functions of BAF in vivo, Drosophila baf null mutants generated by P-element-mediated imprecise excision were analyzed. Homozygous null mutants showed a typical mitotic mutant phenotype: lethality at the larval-pupal transition with small brains and missing imaginal discs. Mitotic figures were decreased but a defined anaphase defect as reported for C. elegans RNAi experiments was not observed in these small brains, suggesting a different phase or phases of cell cycle arrest. Specific abnormalities in interphase nuclear structure were frequently found upon electron microscopic examination of baf null mutants, with partial clumping of chromatin and convolution of nuclear shape. At the light microscopic level, grossly aberrant nuclear lamina structure and B-type lamin distribution correlated well with the loss of detectable amounts of BAF protein from nuclei. Together, these data represent evidence of BAF's anticipated function in mediating interactions between the nuclear envelope and interphase chromosomes. We thus conclude that BAF plays essential roles in nuclear organization and that these BAF functions are required in both M phase and interphase of the cell cycle.