C9orf72 poly(PR) mediated neurodegeneration is associated with nucleolar stress.

The ALS/FTD-linked intronic hexanucleotide repeat expansion in the C9orf72 gene is aberrantly translated in the sense and antisense directions into dipeptide repeat proteins, among which poly proline-arginine (PR) displays the most aggressive neurotoxicity in-vitro and in-vivo. PR partitions to the nucleus when heterologously expressed in neurons and other cell types. We show that by lessening the nuclear accumulation of PR, we can drastically reduce its neurotoxicity. PR strongly accumulates in the nucleolus, a nuclear structure critical in regulating the cell stress response. We determined that, in neurons, PR caused nucleolar stress and increased levels of the transcription factor p53. Downregulating p53 levels also prevented PR-mediated neurotoxicity both in in-vitro and in-vivo models. We investigated if PR could induce the senescence phenotype in neurons. However, we did not observe any indications of such an effect. Instead, we found evidence for the induction of programmed cell death via caspase-3 activation.

Gene-nutrient interactions that impact magnesium homeostasis increase risk for neural tube defects in mice exposed to dolutegravir.

In 2018, data from a surveillance study in Botswana evaluating adverse birth outcomes raised concerns that women on antiretroviral therapy (ART) containing dolutegravir (DTG) may be at increased risk for neural tube defects (NTDs). The mechanism of action for DTG involves chelation of Mg2+ ions in the active site of the viral integrase. Plasma Mg2+ homeostasis is maintained primarily through dietary intake and reabsorption in the kidneys. Inadequate dietary Mg2+ intake over several months results in slow depletion of plasma Mg2+ and chronic latent hypomagnesemia, a condition prevalent in women of reproductive age worldwide. Mg2+ is critical for normal embryonic development and neural tube closure. We hypothesized that DTG therapy might slowly deplete plasma Mg2+ and reduce the amount available to the embryo, and that mice with pre-existing hypomagnesemia due to genetic variation and/or dietary Mg2+ insufficiency at the time of conception and initiation of DTG treatment would be at increased risk for NTDs. We used two different approaches to test our hypothesis: 1) we selected mouse strains that had inherently different basal plasma Mg2+ levels and 2) placed mice on diets with different concentrations of Mg2+. Plasma and urine Mg2+ were determined prior to timed mating. Pregnant mice were treated daily with vehicle or DTG beginning on the day of conception and embryos examined for NTDs on gestational day 9.5. Plasma DTG was measured for pharmacokinetic analysis. Our results demonstrate that hypomagnesemia prior to conception, due to genetic variation and/or insufficient dietary Mg2+ intake, increases the risk for NTDs in mice exposed to DTG. We also analyzed whole-exome sequencing data from inbred mouse strains and identified 9 predicted deleterious missense variants in Fam111a that were unique to the LM/Bc strain. Human FAM111A variants are associated with hypomagnesemia and renal Mg2+ wasting. The LM/Bc strain exhibits this same phenotype and was the strain most susceptible to DTG-NTDs. Our results suggest that monitoring plasma Mg2+ levels in patients on ART regimens that include DTG, identifying other risk factors that impact Mg2+ homeostasis, and correcting deficiencies in this micronutrient might provide an effective strategy for mitigating NTD risk.

C9orf72 poly(PR) mediated neurodegeneration is associated with nucleolar stress.

The ALS/FTD-linked intronic hexanucleotide repeat expansion in the C9orf72 gene is translated into dipeptide repeat proteins, among which poly-proline-arginine (PR) displays the most aggressive neurotoxicity in-vitro and in-vivo . PR partitions to the nucleus when expressed in neurons and other cell types. Using drosophila and primary rat cortical neurons as model systems, we show that by lessening the nuclear accumulation of PR, we can drastically reduce its neurotoxicity. PR accumulates in the nucleolus, a site of ribosome biogenesis that regulates the cell stress response. We examined the effect of nucleolar PR accumulation and its impact on nucleolar function and determined that PR caused nucleolar stress and increased levels of the transcription factor p53. Downregulating p53 levels, either genetically or by increasing its degradation, also prevented PR-mediated neurotoxic phenotypes both in in-vitro and in-vivo models. We also investigated whether PR could cause the senescence phenotype in neurons but observed none. Instead, we found induction of apoptosis via caspase-3 activation. In summary, we uncovered the central role of nucleolar dysfunction upon PR expression in the context of C9-ALS/FTD.

The overall and sex- and age-group specific incidence rates of cancer in people with schizophrenia: a population-based cohort study.

AIMS: Decades of research show that people with schizophrenia have an increased risk of death from cancer; however, the relationship between schizophrenia and cancer incidence remains less clear. This population-based study investigates the incidence of seven common types of cancer among people with a hospital diagnosis of schizophrenia and accounting for the effects of age, sex and calendar time. METHODS: This population-based study used 1990-2013 data from three nationwide Swedish registries to calculate the incidence (in total, by age group and by sex) of any cancer and of lung, oesophageal, pancreatic, stomach, colon, (in men) prostate and (in women) breast cancer in 111 306 people with a hospital diagnosis of schizophrenia. The incidence in people with diagnosed schizophrenia was compared with the incidence in the general population. Risk estimates accounted for the effects of calendar time. RESULTS: In 1 424 829 person-years of follow-up, schizophrenia did not confer an overall higher cancer risk (IRR 1.02, 95% CI 0.91-1.13) but was associated with a higher risk for female breast (IRR 1.19, 95% CI 1.12-1.26), lung (IRR 1.42, 95% CI 1.28-1.58), oesophageal (IRR 1.25, 95% CI 1.07-1.46) and pancreatic (IRR 1.10, 95% CI 1.01-1.21) and a lower risk of prostate (IRR 0.66, 95% CI 0.55-0.79) cancer. Some age- and sex-specific differences in risk were observed. CONCLUSIONS: People with schizophrenia do not have a higher overall incidence of cancer than people in the general population. However, there are significant differences in the risk of specific cancer types overall and by sex calling for efforts to develop disease-specific prevention programmes. In people with schizophrenia, higher risk generally occurs in those <75 years.

Increased cardiovascular mortality in people with schizophrenia: a 24-year national register study.

AimsPeople who have schizophrenia die earlier from somatic diseases than do people in the general population, but information about cardiovascular deaths in people who have schizophrenia is limited. We analysed mortality in all age groups of people with schizophrenia by specific cardiovascular diseases (CVDs), focusing on five CVD diagnoses: coronary heart disease, acute myocardial infarction, cerebrovascular disease, heart failure and cardiac arrhythmias. We also compared hospital admissions for CVDs in people who had schizophrenia with hospital admissions for CVDs in the general population. METHODS: This national register study of 10 631 817 people in Sweden included 46 911 people who were admitted to the hospital for schizophrenia between 1 January 1987 and 31 December 2010. Information from national registers was used to identify people who had schizophrenia and obtain data about mortality, causes of death, medical diagnoses and hospitalisations. RESULTS: CVDs were the leading cause of death in people who had schizophrenia (5245 deaths), and CVDs caused more excess deaths than suicide. The mean age of CVD death was 10 years lower for people who had schizophrenia (70.5 years) than the general population (80.7 years). The mortality rate ratio (MRR) for CVDs in all people who had schizophrenia was 2.80 (95% confidence interval (CI) 2.73-2.88). In people aged 15-59 years who had schizophrenia, the MRR for CVDs was 6.16 (95% CI 5.79-6.54). In all people who had schizophrenia, the MRR for coronary heart disease was 2.83 (95% CI 2.73-2.94); acute myocardial infarction, 2.62 (95% CI 2.49-2.75); cerebrovascular disease, 2.4 (95% CI 2.25-2.55); heart failure, 3.25 (95% CI 2.94-3.6); and cardiac arrhythmias, 2.06 (95% CI 1.75-2.43). Hospital admissions for coronary heart disease were less frequent in people who had schizophrenia than in the general population (admission rate ratio, 0.88 (95% CI 0.83-0.94). In all age groups, survival after hospital admission for CVD was lower in people who had schizophrenia than in the general population. CONCLUSIONS: People who had schizophrenia died 10 years earlier from CVDs than did people in the general population. For all five CVD diagnoses, mortality risk was higher for those with schizophrenia than those in the general population. Survival after hospitalisation for CVDs in people who had schizophrenia was comparable with that of people in the general population who were several decades older.

Mortality and life expectancy of people with alcohol use disorder in Denmark, Finland and Sweden.

OBJECTIVE: To analyse mortality and life expectancy in people with alcohol use disorder in Denmark, Finland and Sweden. METHOD: A population-based register study including all patients admitted to hospital diagnosed with alcohol use disorder (1,158,486 person-years) from 1987 to 2006 in Denmark, Finland and Sweden. RESULTS: Life expectancy was 24-28 years shorter in people with alcohol use disorder than in the general population. From 1987 to 2006, the difference in life expectancy between patients with alcohol use disorder and the general population increased in men (Denmark, 1.8 years; Finland, 2.6 years; Sweden, 1.0 years); in women, the difference in life expectancy increased in Denmark (0.3 years) but decreased in Finland (-0.8 years) and Sweden (-1.8 years). People with alcohol use disorder had higher mortality from all causes of death (mortality rate ratio, 3.0-5.2), all diseases and medical conditions (2.3-4.8), and suicide (9.3-35.9). CONCLUSION: People hospitalized with alcohol use disorder have an average life expectancy of 47-53 years (men) and 50-58 years (women) and die 24-28 years earlier than people in the general population.

Committed mast cell progenitors in mouse blood differ in maturity between Th1 and Th2 strains.

Mast cell progenitors (MCp) leave the bone marrow and migrate to peripheral tissues where they mature. Although the existence of committed MCp in adult mouse and human blood has been postulated, they have never been found. We have isolated a rare population of cells in adult mouse blood, committed to the mast cell lineage. These were identified as lineage- c-kit(hi) ST2+ integrin ß7(hi) CD16/32(hi) cells. Moreover, a major difference in maturity of these cells based on FcεRI expression was observed between the Th2-prone BALB/c strain and the Th1-prone C57BL/6 strain (66% vs. 25% FcεRI+, respectively). Therefore, the choice of mouse strain is critical when studying disease models such as experimental asthma where mast cells and their progenitors are involved.

Structural requirements and mechanism for heparin-induced activation of a recombinant mouse mast cell tryptase, mouse mast cell protease-6: formation of active tryptase monomers in the presence of low molecular weight heparin.

Mast cell tryptase is stored as an active tetramer in complex with heparin in mast cell secretory granules. Previously, we demonstrated the dependence on heparin for the activation/tetramer formation of a recombinant tryptase. Here we have investigated the structural requirements for this activation process. The ability of heparin-related saccharides to activate a recombinant murine tryptase, mouse mast cell protease-6 (mMCP-6), was strongly dependent on anionic charge density and size. The dose-response curve for heparin-induced mMCP-6 activation displayed a bell-shaped appearance, indicating that heparin acts by binding to more than one tryptase monomer simultaneously. The minimal heparin oligosaccharide required for binding to mMCP-6 was 8-10 saccharide units. Gel filtration analyses showed that such short oligosaccharides were unable to generate tryptase tetramers, but instead gave rise to active mMCP-6 monomers. The active monomers were inhibited by bovine pancreatic trypsin inhibitor, whereas the tetramers were resistant. Furthermore, monomeric (but not tetrameric) mMCP-6 degraded fibronectin. Our results suggest a model for tryptase tetramer formation that involves bridging of tryptase monomers by heparin or other highly sulfated polysaccharides of sufficient chain length. Moreover, our results raise the possibility that some of the reported activities of tryptase may be related to active tryptase monomers that may be formed according to the mechanism described here.

Heparin antagonists are potent inhibitors of mast cell tryptase.

Tryptase may be a key mediator in mast cell-mediated inflammatory reactions. When mast cells are activated, they release large amounts of these tetrameric trypsin-like serine proteases. Tryptase is present in a macromolecular complex with heparin proteoglycan where the interaction with heparin is known to be essential for maintaining enzymatic activity. Recent investigations have shown that tryptase has potent proinflammatory activity, and inhibitors of tryptase have been shown to modulate allergic reactions in vivo. Many of the tryptase inhibitors investigated previously are directed against the active site. In the present study we have investigated an alternative approach for tryptase regulation. We show that the heparin antagonists Polybrene and protamine are potent inhibitors of both human lung tryptase and of recombinant mouse tryptase (mouse mast cell protease 6). Protamine inhibited tryptase in a competitive manner whereas Polybrene showed noncompetitive inhibition kinetics. Treatment of tetrameric, active tryptase with Polybrene caused dissociation into monomers, accompanied by complete loss of enzymatic activity. The present report thus suggests that heparin antagonists potentially may be used in treatment of mast cell-mediated diseases such as asthma.

Mechanism for activation of mouse mast cell tryptase: dependence on heparin and acidic pH for formation of active tetramers of mouse mast cell protease 6.

Tryptase, a serine protease with trypsin-like substrate cleavage properties, is one of the key effector molecules during allergic inflammation. It is stored in large quantities in the mast cell secretory granules in complex with heparin proteoglycan, and these complexes are released during mast cell degranulation. In the present paper, we have studied the mechanism for tryptase activation. Recombinant mouse tryptase, mouse mast cell protease 6 (mMCP-6), was produced in a mammalian expression system. The mMCP-6 fusion protein contained an N-terminal 6 x His tag followed by an enterokinase (EK) site replacing the native activation peptide (6xHis-EK-mMCP-6). In the absence of heparin, barely detectable enzyme activity was obtained after enterokinase cleavage of 6xHis-EK-mMCP-6 over a pH range of 5.5-7.5. However, when heparin was present, 6xHis-EK-mMCP-6 yielded active enzyme when enterokinase cleavage was performed at pH 5.5-6.0 but not at neutral pH. Affinity chromatography analysis showed that mMCP-6 bound strongly to heparin-Sepharose at pH 6.0 but not at neutral pH. After enterokinase cleavage of the sample at pH 6.0, mMCP-6 occurred in inactive monomeric form as shown by FPLC analysis on a Superdex 200 column. When heparin was added at pH 6.0, enzymatically active higher molecular weight complexes were formed, e.g., a dominant approximately 200 kDa complex that may correspond to tryptase tetramers. No formation of active tetramers was observed at neutral pH. When injected intraperitoneally, mMCP-6 together with heparin caused neutrophil influx, but no signs of inflammation were seen in the absence of heparin. The present paper thus indicates a crucial role for heparin in the formation of active mast cell tryptase.

The chlB gene encoding a subunit of light-independent protochlorophyllide reductase is edited in chloroplasts of conifers.

ChlB is one of three chloroplast genes shown so far to be required for light-independent chlorophyll synthesis. It occurs in some algae, lower plants, and gymnosperms, but not in angiosperms. We have demonstrated, for the first time in conifer chloroplasts, the presence of two internal C to U editing sites in this transcript. In the chlB transcript of Pinus sylvestris, the editing of the second position in a CCG codon leads to an amino-acid substitution from proline to leucine. Editing of a nearby CGG codon, resulting in an arginine to tryptophan substitution, has also been observed. The nucleotide sequence of this region has been compared with other species of gymnosperms. Out of seven species analysed, editing at both sites has only been detected in spruce, while in Larix only the editing which results in the Arg to Trp substitution was found. In other cases, both leucine and tryptophan are encoded by cpDNA, suggesting that conservation of these amino acids, through encoding by DNA or by editing of the RNA, is critical for the protein function. Transcripts are partially edited at the CGG codon and the relative abundance of cDNA molecules with the edited C is species-specific. The possible involvement of RNA editing in the regulation of gene expression in different organs of pine seedlings is discussed.

Closing the clinical laboratory testing loop with information technology.

Information technology can enhance the effectiveness of the part of the laboratory testing loop that occurs outside of the clinical laboratory. That external component involves the presentation of laboratory results and related information to physicians and the reception of physician requests for follow-up testing. Improvements made in the clinician-computer interface can conceivably enhance the quality of information transfer of this part of the testing loop and thereby improve the effectiveness of the entire loop. Our experience has been that results presentation is easier to address than order reception and that the economic benefits from improved result presentation can be substantial. Improving order reception requires more finesse, especially with the limitations of today's health systems and their information systems in mind. For instance, effective computer-based management of prospectively developed orders (eg, clinical protocols) not only can have a substantial positive impact on test use, but is actually welcomed by clinicians. Application of information technology at the clinician-computer interface has good potential to foster more appropriate use of clinical laboratory resources.

Increased mucosal synthesis of rheumatoid factor (RF) in coeliac disease.

Increased serum levels of RF have been reported in patients with gluten sensitivity. The objective of this study was to investigate the in vivo secretion of different isotypes of RF in the small bowel in coeliac disease. Nineteen patients were investigated by perfusion of a defined jejunal segment, and the jejunal perfusion fluid was analysed for the presence of IgA and IgM anti-Fc (IgG). Five of the patients studied had serum IgA deficiency. Patients with partial/subtotal villous atrophy but no IgA deficiency (n = 7) had a four-fold increase of IgM-RF (P < 0.001) and a three-fold increase of IgA-RF (P < 0.001) compared with healthy controls (n = 29). Patients with normal jejunal mucosa but no IgA deficiency (n = 7) had similar IgA-RF and IgM-RF concentrations to healthy controls. Patients with serum IgA deficiency had no IgA-RF detectable in jejunal fluid but the highest IgM-RF concentrations, in particular in active disease. The coeliac patients had serum levels of IgA-RF and IgM-RF within the reference ranges. Jejunal fluid levels of IgA-RF and IgA-anti-gliadin antibodies were significantly correlated (P < 0.001). The data indicate that enhanced jejunal mucosal production of RF occurs above all in active coeliac disease. The findings suggest that the immune response to gluten induces a mucosal RF synthesis.

Seasonal variation in photochemical activity and hardiness in clones of Norway spruce (Picea abies).

We investigated changes in photochemical activity and cold hardiness of detached needles of three clones of Picea abies (L.) Karst. by measuring variable chlorophyll fluorescence (F(v)/F(m)), before and after artificial freezing, from September to June. Photochemical activity varied considerably during the study, but only minor differences in photochemical activity among the clones were observed before freezing. Photochemical activity was high during early fall and then declined from November until April. Photochemical activity was at a minimum in April and then increased quickly to high values in May. During the period from late September to October, and also during the winter, differences in F(v)/F(m) ratios after artificial freezing to below -10 degrees C were observed among clones, indicating clonal differences in cold hardiness and hardiness development. The clone having an average height of 2.3 m after 11 years showed consistently lower cold hardiness than clones that had reached average heights of 4.0 and 5.0 m. There were also differences in the temperature requirement for bud flushing among clones.

The genes encoding subunit 3 of NADH dehydrogenase and ribosomal protein S12 are co-transcribed and edited in Pinus sylvestris (L.) mitochondria.

The nucleotide sequence of the region encoding NADH dehydrogenase subunit 3 and ribosomal protein S12 from Pinus sylvestris (L.) mitochondrial DNA (mtDNA) has been determined. A sequence comparison of this region with six individual cDNA clones prepared by RT-PCR revealed 35 C-to-T differences, showing the occurrence of RNA editing. All but one of these alterations in mRNA sequence change codon identities to specify amino acid better conserved in evolution. Most of these modifications take place within the nad3 gene changing 20% of the amino-acid sequence, which is much more than in angiosperms. Of six cDNA clones investigated, four clones of nad3 were differentially edited, but the editing of the rps12 sequences was identical. As in angiosperms, the two genes are separated by a short sequence of 52 bp, which is not edited. Two transcripts of about 0.9 kb and 1.2 kb, each encoding both proteins, have been detected by Northern hybridisation. The hybridisation of nad3 and rps12 probes with pine mtDNA digested with different restriction enzymes indicates that both genes are present in a single copy in pine mtDNA. The analysis of PCR amplification products with gene-specific primers shows a conserved order of these genes in a wide range of gymnosperms.

The deployment of a World Wide Web (W3) based medical information system.

The development of Web technologies has revolutionized information dissemination on the Internet. The University of Minnesota Hospital and Clinic's Web Clinical Information System (CIS) demonstrates the use of the Web as an infrastructure for deploying a medical information system at a fraction of the developmental cost of more traditional client server systems. This Web CIS has been deployed since December 1994. It makes available laboratory results, including a radically improved clinical microbiology reporting system, ad hoc laboratory order entry, and an embedded expert system protocol laboratory ordering system. It provides these services to any physician or patient care area with TCP (or SLIP/PPP) connection to our hospital network backbone, whether the client computer is running MS Windows, the Macintosh OS, or X-Windows. A formal evaluation of one of this systems subcomponents, the display of clinical microbiology information, demonstrated a significant savings in clinician time (43% p < .001) and substantial reduction in interpretive errors (0 vs 15 p < .01).