Outcomes and cost analysis of patients with dementia in the intensive care unit: a population-based cohort study.

BACKGROUND: Dementia is a neurological syndrome affecting the growing elderly population. While patients with dementia are known to require significant hospital resources, little is known regarding the outcomes and costs of patients admitted to the intensive care unit (ICU) with dementia. METHODS: We conducted a population-based retrospective cohort study of patients with dementia admitted to the ICU in Ontario, Canada from 2016 to 2019. We described the characteristics and outcomes of these patients alongside those with dementia admitted to non-ICU hospital settings. The primary outcome was hospital mortality but we also assessed length of stay (LOS), discharge disposition, and costs. RESULTS: Among 114,844 patients with dementia, 11,341 (9.9%) were admitted to the ICU. ICU patients were younger, more comorbid, and had less cognitive impairment (81.8 years, 22.8% had ≥ 3 comorbidities, 47.5% with moderate-severe dementia), compared to those in non-ICU settings (84.2 years, 15.0% had ≥ 3 comorbidities, 54.1% with moderate-severe dementia). Total mean LOS for patients in the ICU group was nearly 20 days, compared to nearly 14 days for the acute care group. Mortality in hospital was nearly three-fold greater in the ICU group compared to non-ICU group (22.2% vs. 8.8%). Total healthcare costs were increased for patients admitted to ICU vs. those in the non-ICU group ($67,201 vs. $54,080). CONCLUSIONS: We find that patients with dementia admitted to the ICU have longer length of stay, higher in-hospital mortality, and higher total healthcare costs. As our study is primarily descriptive, future studies should investigate comprehensive goals of care planning, severity of illness, preventable costs, and optimizing quality of life in this high risk and vulnerable population.

Generation of the iPSC line FINi002-A from a male Parkinson's disease patient carrying compound heterozygous mutations in the PRKN gene.

The most common cause of autosomal recessive familial Parkinson's disease (PD) are mutations in the PRKN/PARK2 gene encoding an E3 ubiquitin protein-ligase PARKIN. We report the generation of an iPSC cell line from the fibroblasts of a male PD patient carrying a common missense variant in exon 7 (p.Arg275Trp), and a 133 kb deletion encompassing exon 8, using transiently-present Sendai virus. The established line displays typical human primed iPSC morphology and expression of pluripotency-associated markers, normal karyotype without SNP array-detectable copy number variations and can give rise to derivatives of all three embryonic germ layers. We envisage the usefulness of this iPSC line, carrying a common and well-studied missense mutation in the RING1 domain of the PARKIN protein, for the elucidation of PARKIN-dependent mechanisms of PD using in vitro and in vivo models.

Hydrogel oxygen reservoirs increase functional integration of neural stem cell grafts by meeting metabolic demands.

Injectable biomimetic hydrogels have great potential for use in regenerative medicine as cellular delivery vectors. However, they can suffer from issues relating to hypoxia, including poor cell survival, differentiation, and functional integration owing to the lack of an established vascular network. Here we engineer a hybrid myoglobin:peptide hydrogel that can concomitantly deliver stem cells and oxygen to the brain to support engraftment until vascularisation can occur naturally. We show that this hybrid hydrogel can modulate cell fate specification within progenitor cell grafts, resulting in a significant increase in neuronal differentiation. We find that the addition of myoglobin to the hydrogel results in more extensive innervation within the host tissue from the grafted cells, which is essential for neuronal replacement strategies to ensure functional synaptic connectivity. This approach could result in greater functional integration of stem cell-derived grafts for the treatment of neural injuries and diseases affecting the central and peripheral nervous systems.

Identifying the optimal developmental age of human pluripotent stem cell-derived midbrain dopaminergic progenitors for transplantation in a rodent model of Parkinson's disease.

Donor cell age can have a significant impact on transplantation outcomes. Despite the rapid advancement of human pluripotent stem cell (hPSC)-derived dopaminergic (DA) progenitors to the clinic for transplantation into Parkinson's Disease (PD), surprisingly limited data exists regarding the influence of cellular age on neural graft survival, composition, and integration. Here we examined the impact of transplanting ventral midbrain (VM) progenitors at varying days of differentiation (from day 13-30) into a rodent PD model, comparing two hPSC lines (an embryonic and an induced pluripotent cell line, hESC and hiPSC, respectively). Both hPSC lines expressed GFP under the promoter PITX3 enabling specific tracking of graft-derived DA neurons. Post-mortem analysis at 6 months revealed larger grafts from Day19 (D19), D22 and D25 progenitors, yet contained a higher proportion of non-DA and poorly specified (FOXA2-) cells. While D13 and D30 progenitors yielded smaller grafts. D13-derived grafts had the highest DA neuron proportion and proportionally more GIRK2+ DA neurons, the subpopulation critical for motor function. These younger progenitor grafts maintained their capacity to innervate developmentally relevant DA targets, with increased innervation capacity per DA neuron, collectively resulting in restoration of motor deficits with equal or greater proficiency than older donor cells. While donor age effects were reproducible for a given hPSC line and trends were similar between the two hPSC lines, grafts of D13 hiPSC-derived progenitors showed a 6-fold greater density of DA neurons compared to D13 hESC-derived grafts, highlighting between-line variability. These findings show that hPSC-derived VM donor age has a direct impact on graft survival, composition and maturation, and that careful assessment, on a line-to-line basis is required prior to translation.

Homophilic binding of the neural cell adhesion molecule CHL1 regulates development of ventral midbrain dopaminergic pathways.

Abnormal development of ventral midbrain (VM) dopaminergic (DA) pathways, essential for motor and cognitive function, may underpin a number of neurological disorders and thereby highlight the importance of understanding the birth and connectivity of the associated neurons. While a number of regulators of VM DA neurogenesis are known, processes involved in later developmental events, including terminal differentiation and axon morphogenesis, are less well understood. Recent transcriptional analysis studies of the developing VM identified genes expressed during these stages, including the cell adhesion molecule with homology to L1 (Chl1). Here, we map the temporal and spatial expression of CHL1 and assess functional roles of substrate-bound and soluble-forms of the protein during VM DA development. Results showed early CHL1 in the VM, corresponding with roles in DA progenitor migration and differentiation. Subsequently, we demonstrated roles for CHL1 in both axonal extension and repulsion, selectively of DA neurons, suggestive of a role in guidance towards forebrain targets and away from hindbrain nuclei. In part, CHL1 mediates these roles through homophilic CHL1-CHL1 interactions. Collectively, these findings enhance our knowledge of VM DA pathways development, and may provide new insights into understanding DA developmental conditions such as autism spectrum disorders.

Emergency department and 'Google flu trends' data as syndromic surveillance indicators for seasonal influenza.

We evaluated syndromic indicators of influenza disease activity developed using emergency department (ED) data - total ED visits attributed to influenza-like illness (ILI) ('ED ILI volume') and percentage of visits attributed to ILI ('ED ILI percent') - and Google flu trends (GFT) data (ILI cases/100 000 physician visits). Congruity and correlation among these indicators and between these indicators and weekly count of laboratory-confirmed influenza in Manitoba was assessed graphically using linear regression models. Both ED and GFT data performed well as syndromic indicators of influenza activity, and were highly correlated with each other in real time. The strongest correlations between virological data and ED ILI volume and ED ILI percent, respectively, were 0·77 and 0·71. The strongest correlation of GFT was 0·74. Seasonal influenza activity may be effectively monitored using ED and GFT data.

Medical ward admissions among HIV-positive patients in Winnipeg, Canada, 2003-10.

Canadian data regarding the characteristics of HIV-positive patients admitted to hospital as well as the causes and patterns of admissions remain limited. Chart reviews were performed to ascertain admission diagnosis, co-morbidities and CD4 counts among this sub-population, which had an over-representation of Aboriginal persons. Infectious diseases, particularly pneumonia, represent the most common admission diagnosis for HIV-positive persons in Winnipeg. Further, individuals presenting to hospital often have very low CD4 counts, representing significant immune suppression. Earlier HIV diagnosis and treatment in an effort to delay the onset of advanced disease and hospitalization is needed.

FANCG promotes formation of a newly identified protein complex containing BRCA2, FANCD2 and XRCC3.

Fanconi anemia (FA) is a human disorder characterized by cancer susceptibility and cellular sensitivity to DNA crosslinks and other damages. Thirteen complementation groups and genes are identified, including BRCA2, which is defective in the FA-D1 group. Eight of the FA proteins, including FANCG, participate in a nuclear core complex that is required for the monoubiquitylation of FANCD2 and FANCI. FANCD2, like FANCD1/BRCA2, is not part of the core complex, and we previously showed direct BRCA2-FANCD2 interaction using yeast two-hybrid analysis. We now show in human and hamster cells that expression of FANCG protein, but not the other core complex proteins, is required for co-precipitation of BRCA2 and FANCD2. We also show that phosphorylation of FANCG serine 7 is required for its co-precipitation with BRCA2, XRCC3 and FANCD2, as well as the direct interaction of BRCA2-FANCD2. These results argue that FANCG has a role independent of the FA core complex, and we propose that phosphorylation of serine 7 is the signalling event required for forming a discrete complex comprising FANCD1/BRCA2-FANCD2-FANCG-XRCC3 (D1-D2-G-X3). Cells that fail to express either phospho-Ser7-FANCG, or full length BRCA2 protein, lack the interactions amongst the four component proteins. A role for D1-D2-G-X3 in homologous recombination repair (HRR) is supported by our finding that FANCG and the RAD51-paralog XRCC3 are epistatic for sensitivity to DNA crosslinking compounds in DT40 chicken cells. Our findings further define the intricate interface between FANC and HRR proteins in maintaining chromosome stability.

Identification of ICR170-induced XPD mutations in UV-sensitive CHO cells.

We highlight selected contributions of Dr. Richard Setlow that contributed to our earlier understanding of excision repair processes and set the stage for dissecting nucleotide excision repair (NER) in mammalian cells through molecular genetics. More than 20 years ago, large-scale screens for UV-sensitive mutants of hamster CHO cells isolated approximately 200 mutants, many of which were assigned to the XPD/ERCC2 complementation group, but the nature of the mutations was not determined. The XPD protein performs not only an essential viability function as a structural component of transcription initiation factor TFIIH, but also an NER function as a 5' to 3' DNA helicase within TFIIH that unwinds DNA on the 3' side of bulky lesions. Alterations in these XPD functions are responsible for three UV-sensitivity genetic disorders that have distinguishable clinical features. In this study, we sequenced six UV-sensitive ICR170-induced Chinese hamster ovary (CHO) cell mutants that previously were assigned to the XPD complementation group to determine whether they carry frameshift mutations. All six mutants show 3- to 5-fold increased hypersensitivity to UV irradiation, similar to the XPD mutant prototype UV5. Even though ICR170 is a strong frameshift mutagen, all six cell lines contain base substitution mutations, five of which are unique among all mutations identified so far in human and rodent cells. The sixth mutation was identical to the R75W mutation previously found in CHO UVL-1. The results presented here contribute to a mutation database that should prove useful in structure-function studies of this unique DNA-structure-specific helicase and its complex mutant phenotypes.

Restoration of nucleotide excision repair in a helicase-deficient XPD mutant from intragenic suppression by a trichothiodystrophy mutation.

The UV-sensitive V-H1 cell line has a T46I substitution mutation in the Walker A box in both alleles of XPD and lacks DNA helicase activity. We characterized three partial revertants that curiously display intermediate UV cytotoxicity (2- to 2.5-fold) but normal levels of UV-induced hprt mutations. In revertant RH1-26, the efficient removal of pyrimidine (6-4) pyrimidone photoproducts from both strands of hprt suggests that global-genomic nucleotide excision repair is normal, but the pattern of cyclobutane pyrimidine dimer removal suggests that transcription-coupled repair (TCR) is impaired. To explain the intermediate UV survival and lack of RNA synthesis recovery in RH1-26 after 10 J of UV/m(2), we propose a defect in repair-transcription coupling, i.e., the inability of the cells to resume or reinitiate transcription after the first TCR event within a transcript. All three revertants carry an R658H suppressor mutation, in one allele of revertants RH1-26 and RH1-53 and in both alleles of revertant RH1-3. Remarkably, the R658H mutation produces the clinical phenotype of trichothiodystrophy (TTD) in several patients who display intermediate UV sensitivity. The XPD(R658H) TTD protein, like XPD(T46I/R658H), is codominant when overexpressed in V-H1 cells and partially complements their UV sensitivity. Thus, the suppressing R658H substitution must restore helicase activity to the inactive XPD(T46I) protein. Based on current knowledge of helicase structure, the intragenic reversion mutation may partially compensate for the T46I mutation by perturbing the XPD structure in a way that counteracts the effect of this mutation. These findings have implications for understanding the differences between xeroderma pigmentosum and TTD and illustrate the value of suppressor genetics for studying helicase structure-function relationships.

Rad52 partially substitutes for the Rad51 paralog XRCC3 in maintaining chromosomal integrity in vertebrate cells.

Yeast Rad52 DNA-repair mutants exhibit pronounced radiation sensitivity and a defect in homologous re combination (HR), whereas vertebrate cells lacking Rad52 exhibit a nearly normal phenotype. Bio chemical studies show that both yeast Rad52 and Rad55-57 (Rad51 paralogs) stimulate DNA-strand exchange mediated by Rad51. These findings raise the possibility that Rad51 paralogs may compensate for lack of Rad52 in vertebrate cells, explaining the absence of prominent phenotypes for Rad52-deficient cells. To test this hypothesis, using chicken DT40 cells, we generated conditional mutants deficient in both RAD52 and XRCC3, which is one of the five vertebrate RAD51 paralogs. Surprisingly, the rad52 xrcc3 double-mutant cells were non-viable and exhibited extensive chromosomal breaks, whereas rad52 and xrcc3 single mutants grew well. Our data reveal an overlapping (but non-reciprocal) role for Rad52 and XRCC3 in repairing DNA double-strand breaks. The present study shows that Rad52 can play an important role in HR repair by partially substituting for a Rad51 paralog.

Homologous recombinational repair of DNA ensures mammalian chromosome stability.

The process of homologous recombinational repair (HRR) is a major DNA repair pathway that acts on double-strand breaks and interstrand crosslinks, and probably to a lesser extent on other kinds of DNA damage. HRR provides a mechanism for the error-free removal of damage present in DNA that has replicated (S and G2 phases). Thus, HRR acts in a critical way, in coordination with the S and G2 checkpoint machinery, to eliminate chromosomal breaks before the cell division occurs. Many of the human HRR genes, including five Rad51 paralogs, have been identified, and knockout mutants for most of these genes are available in chicken DT40 cells. In the mouse, most of the knockout mutations cause embryonic lethality. The Brca1 and Brca2 breast cancer susceptibility genes appear to be intimately involved in HRR, but the mechanistic basis is unknown. Biochemical studies with purified proteins and cell extracts, combined with cytological studies of nuclear foci, have begun to establish an outline of the steps in mammalian HRR. This pathway is subject to complex regulatory controls from the checkpoint machinery and other processes, and there is increasing evidence that loss of HRR gene function can contribute to tumor development. This review article is meant to be an update of our previous review [Biochimie 81 (1999) 87].

Chromosome instability and defective recombinational repair in knockout mutants of the five Rad51 paralogs.

The Rad51 protein, a eukaryotic homologue of Escherichia coli RecA, plays a central role in both mitotic and meiotic homologous DNA recombination (HR) in Saccharomyces cerevisiae and is essential for the proliferation of vertebrate cells. Five vertebrate genes, RAD51B, -C, and -D and XRCC2 and -3, are implicated in HR on the basis of their sequence similarity to Rad51 (Rad51 paralogs). We generated mutants deficient in each of these proteins in the chicken B-lymphocyte DT40 cell line and report here the comparison of four new mutants and their complemented derivatives with our previously reported rad51b mutant. The Rad51 paralog mutations all impair HR, as measured by targeted integration and sister chromatid exchange. Remarkably, the mutant cell lines all exhibit very similar phenotypes: spontaneous chromosomal aberrations, high sensitivity to killing by cross-linking agents (mitomycin C and cisplatin), mild sensitivity to gamma rays, and significantly attenuated Rad51 focus formation during recombinational repair after exposure to gamma rays. Moreover, all mutants show partial correction of resistance to DNA damage by overexpression of human Rad51. We conclude that the Rad51 paralogs participate in repair as a functional unit that facilitates the action of Rad51 in HR.

Codominance associated with overexpression of certain XPD mutations.

Mutations in the XPD gene are associated with three complex clinical phenotypes, namely xeroderma pigmentosum (XP), XP in combination with Cockayne syndrome (XP-CS), and trichothiodystrophy (TTD). XP is caused by a deficiency in nucleotide excision repair (NER) that results in a high risk of skin cancer. TTD is characterized by severe developmental and neurological defects, with hallmark features of brittle hair and scaly skin, and sometimes has defective NER. We used CHO cells as a system to study how specific mutations alter the dominant/recessive behavior of XPD protein. Previously we identified the T46I and R75W mutations in two highly UV-sensitive hamster cell lines that were reported to have paradoxically high levels of unscheduled DNA synthesis. Here we report that these mutants have greatly reduced XPD helicase activity and fully defective NER in a cell-extract excision assay. We conclude that the unscheduled DNA synthesis seen in these mutants is caused by abortive "repair" that does not contribute to cell survival. These mutations, as well as the K48R canonical helicase-domain mutation, each produced codominant negative phenotypes when overexpressed in wild-type CHO cells. The common XP-specific R683W mutation also behaved in a codominant manner when overexpressed, which is consistent with the idea that this mutation may affect primarily the enzymatic activity of the protein rather than impairing protein interactions, which may underlie TTD. A C-terminal mutation uniquely found in TTD (R722W) was overexpressed but not to levels sufficiently high to rigorously test for a codominant phenotype. Overexpression of mutant XPD alleles may provide a simple means of producing NER deficiency in other cell lines.

Efficient repair of 8-oxo-7,8-dihydrodeoxyguanosine in human and hamster xeroderma pigmentosum D cells.

The repair of the endogenous lesion 8-oxo-7,8-dihydrodeoxyguanosine (8-oxodG) was investigated in the nucleotide excision repair mutant xeroderma pigmentosum D (XPD), using human normal or transformed XPD fibroblasts and the Chinese hamster XPD cell line UV5. In vivo repair of 8-oxodG induced by hydrogen peroxide treatment and analyzed by high-performance liquid chromatography/electrochemical detection was normal in the XPD mutant fibroblasts XP15PV and GM434, as compared to normal human fibroblasts GM970, GM5757, and GM6114. Similar results were obtained with the human SV40-transformed XPD mutant cell line GM8207 in comparison to the control cell line GM637. Repair of 8-oxodG was even slightly (2-3-fold) but reproducibly increased in Chinese hamster XPD mutant UV5 cells, as compared to parental AA8 cells. This unexpected effect was reversed by transfection in UV5 cells of a wild-type XPD cDNA and confirmed in in vitro experiments in which a plasmid substrate containing a single 8-oxoG was repaired by UV5 cell extracts. The data show that repair of 8-oxodG is normal in XPD cells, thus indicating that the neurological complications of XPD patients may not be linked to in vivo accumulation of this lesion.

The Rad51 paralog Rad51B promotes homologous recombinational repair.

The highly conserved Saccharomyces cerevisiae Rad51 protein plays a central role in both mitotic and meiotic homologous DNA recombination. Seven members of the Rad51 family have been identified in vertebrate cells, including Rad51, Dmc1, and five Rad51-related proteins referred to as Rad51 paralogs, which share 20 to 30% sequence identity with Rad51. In chicken B lymphocyte DT40 cells, we generated a mutant with RAD51B/RAD51L1, a member of the Rad51 family, knocked out. RAD51B(-/-) cells are viable, although spontaneous chromosomal aberrations kill about 20% of the cells in each cell cycle. Rad51B deficiency impairs homologous recombinational repair (HRR), as measured by targeted integration, sister chromatid exchange, and intragenic recombination at the immunoglobulin locus. RAD51B(-/-) cells are quite sensitive to the cross-linking agents cisplatin and mitomycin C and mildly sensitive to gamma-rays. The formation of damage-induced Rad51 nuclear foci is much reduced in RAD51B(-/-) cells, suggesting that Rad51B promotes the assembly of Rad51 nucleoprotein filaments during HRR. These findings show that Rad51B is important for repairing various types of DNA lesions and maintaining chromosome integrity.

XRCC3 promotes homology-directed repair of DNA damage in mammalian cells.

Homology-directed repair of DNA damage has recently emerged as a major mechanism for the maintenance of genomic integrity in mammalian cells. The highly conserved strand transferase, Rad51, is expected to be critical for this process. XRCC3 possesses a limited sequence similarity to Rad51 and interacts with it. Using a novel fluorescence-based assay, we demonstrate here that error-free homology-directed repair of DNA double-strand breaks is decreased 25-fold in an XRCC3-deficient hamster cell line and can be restored to wild-type levels through XRCC3 expression. These results establish that XRCC3-mediated homologous recombination can reverse DNA damage that would otherwise be mutagenic or lethal.

A summary of mutations in the UV-sensitive disorders: xeroderma pigmentosum, Cockayne syndrome, and trichothiodystrophy.

The human diseases xeroderma pigmentosum, Cockayne syndrome, and trichothiodystrophy are caused by mutations in a set of interacting gene products, which carry out the process of nucleotide excision repair. The majority of the genes have now been cloned and many mutations in the genes identified. The relationships between the distribution of mutations in the genes and the clinical presentations can be used for diagnosis and for understanding the functions and the modes of interaction among the gene products. The summary presented here represents currently known mutations that can be used as the basis for future studies of the structure, function, and biochemical properties of the proteins involved in this set of complex disorders, and may allow determination of the critical sites for mutations leading to different clinical manifestations. The summary indicates where more data are needed for some complementation groups that have few reported mutations, and for the groups for which the gene(s) are not yet cloned. These include the Xeroderma pigmentosum (XP) variant, the trichothiodystrophy group A (TTDA), and ultraviolet sensitive syndrome (UVs) groups. We also recommend that the XP-group E should be defined explicitly through molecular terms, because assignment by complementation in culture has been difficult. XP-E by this definition contains only those cell lines and patients that have mutations in the small subunit, DDB2, of a damage-specific DNA binding protein.