Biopsychosocial risk factors for subjective cognitive decline among older adults during the COVID-19 pandemic: a population-based study.

OBJECTIVES: There have been concerns that the COVID-19 pandemic and the measures used to contain it impacted the cognitive health of older adults. We therefore examined the prevalence of subjective cognitive decline, and its associated risk factors and health consequencs, among dementia-free older adults 2 years into the pandemic in Switzerland. STUDY DESIGN: Population-based cohort study. METHODS: Prevalence of SCD was estimated using the cognitive complaint questionnaire administered to adults aged ≥65 years in June-September 2022 (Specchio-COVID19 cohort, N = 1414), and compared to prepandemic values from 2014 to 2018 (CoLaus|PsyCoLaus cohort, N = 1181). Associated risk factors and health consequences were assessed using logistic and/or linear regression. RESULTS: Prevalence of SCD in 2022 (18.9% [95% CI, 16.2-21.9]) was comparable to prepandemic levels in 2014-2018 (19.5% [17.2-22.1]). Risk factors included established risks for dementia-namely health issues, health behaviours, and depressive symptoms. Self-reported post-COVID, perceived worsening of mental health since the start of the pandemic, less frequent social club attendance, and increased loneliness were also risk factors for SCD. In turn, SCD was associated with poorer objective cognitive performance, difficulty performing instrumental activities of daily living, greater risk of falls, and lower well-being at one-year follow-up. CONCLUSIONS: While the overall prevalence of SCD in 2022 was comparable to prepandemic levels, we identified several pandemic-related risk factors for SCD, including perceived worsening of mental health and increased isolation since the start of the pandemic. These findings highlight the importance of mental health promotion strategies in reducing cognitive complaints and preventing cognitive decline.

Characterization of the EP receptor subtype that mediates the inhibitory effects of prostaglandin E2 on IgE-dependent secretion from human lung mast cells.

BACKGROUND: Prostaglandin E2 (PGE2 ) has been shown to inhibit IgE-dependent histamine release from human lung mast cells. This effect of PGE2 is believed to be mediated by EP2 receptors. However, definitive evidence that this is the case has been lacking in the absence of EP2 -selective antagonists. Moreover, recent evidence has suggested that PGE2 activates EP4 receptors to inhibit respiratory cell function. OBJECTIVE: The aim of this study was to determine the receptor by which PGE2 inhibits human lung mast cell responses by using recently developed potent and selective EP2 and EP4 receptor antagonists alongside other established EP receptor ligands. METHODS: The effects of non-selective (PGE2 , misoprostol), EP2 -selective (ONO-AE1-259, AH13205, butaprost-free acid) and EP4 -selective (L-902,688, TCS251) agonists on IgE-dependent histamine release and cyclic-AMP generation in mast cells were determined. The effects of EP2 -selective (PF-04418948, PF-04852946) and EP4 -selective (CJ-042794, L-161,982) antagonists on PGE2 responses of mast cells were studied. The expression of EP receptor subtypes was determined by RT-PCR. RESULTS: Prostaglandin E2 , EP2 agonists and EP4 agonists inhibited IgE-dependent histamine release from mast cells. PGE2 and EP2 agonists, but not EP4 agonists, increased cyclic-AMP levels in mast cells. EP4 -selective antagonists did not affect the PGE2 inhibition of histamine release, whereas EP2 -selective antagonists caused rightward shifts in the PGE2 concentration-response curves. RT-PCR studies indicated that mast cells expressed EP2 and EP4 receptors. CONCLUSIONS AND CLINICAL RELEVANCE: Although human lung mast cells may express both EP2 and EP4 receptors, the principal mechanism by which PGE2 inhibits mediator release in mast cells is by activating EP2 receptors.

An inhibitor of thrombin activated fibrinolysis inhibitor (TAFI) can reduce extracellular matrix accumulation in an in vitro model of glucose induced ECM expansion.

Chronic kidney disease (CKD) is characterised by the pathological accumulation of extracellular matrix (ECM) proteins leading to progressive kidney scarring via glomerular and tubular basement membrane expansion. Increased ECM synthesis and deposition, coupled with reduced ECM breakdown contribute to the elevated ECM level in CKD. Previous pre-clinical studies have demonstrated that increased plasmin activity has a beneficial effect in the protein overload model of CKD. As plasmin activation is downregulated by the action of the thrombin activated fibrinolytic inhibitor (TAFI), we tested the hypothesis that inhibition of TAFI might increase plasmin activity and reduce ECM accumulation in an in vitro model of glucose induced ECM expansion. Treatment of NRK52E tubular epithelial cells with increasing concentrations of glucose resulted in a 40% increase in TAFI activity, a 38% reduction in plasmin activity and a subsequent increase in ECM accumulation. In this model system, application of the previously reported TAFI inhibitor UK-396082 [(2S)-5-amino-2-[(1-n-propyl-1H-imidazol-4-yl)methyl]pentanoic acid] caused a reduction in TAFI activity, increased plasmin activity and induced a parallel decrease in ECM levels. In contrast, RNAi knockdown of plasmin resulted in an increase in ECM levels. The data presented here indicate that high glucose induces TAFI activity, inhibiting plasmin activation which results in elevated ECM levels in tubular epithelial cells. The results support the hypothesis that UK-396082 is able to reduce TAFI activity, normalising plasmin activity and preventing excess ECM accumulation suggesting that TAFI inhibition may have potential as an anti-scarring strategy in CKD.

Cellular exchange in an endometriosis-adhesion model using GFP transgenic mice.

BACKGROUND: Endometriosis is a debilitating disease that affects women of reproductive age and may lead to impaired fertility. Cell attachment, invasion of the underlying tissue, and vascular ingrowth are important processes in endometrial lesion development. However, the degree of cellular exchange between host peritoneum and endometrial tissue is unclear. METHODS: An experimental endometriosis model was employed whereby uterine horn fragments from wild-type mice were implanted into genetically identical eGFP (enhanced green fluorescent protein) host mice and vice versa. Hormone sensitivity of the ectopic lesions was assessed and cellular exchange determined histologically. RESULTS: White cyst-like lesions developed from implanted fibrin-rich fragments by day 7. Lesions consisted of a well-developed stroma with glandular and luminal epithelium. Both ovariectomy and treatment with a GnRH agonist, leuprorelin, resulted in the suppression of ectopic lesion growth, whereas estradiol treatment increased the size of the ectopic lesion (4 mice per group on day 14). Ingrowth and outgrowth of blood vessels was apparent as well as the exchange of cells between host peritoneum and lesion. CONCLUSION: These findings support the proposal that there is a close cellular interplay between host peritoneum and ectopic tissue and the suitability of this mouse model to study these interactions.

In vitro and in vivo characterization of PF-04418948, a novel, potent and selective prostaglandin EP2 receptor antagonist.

BACKGROUND AND PURPOSE: Studies of the role of the prostaglandin EP(2) receptor) have been limited by the availability of potent and selective antagonist tools. Here we describe the in vitro/in vivo pharmacological characterization of a novel EP(2) receptor antagonist, PF-04418948 (1-(4-fluorobenzoyl)-3-{[(6-methoxy-2-naphthyl)oxy]methyl} azetidine-3-carboxylic acid). EXPERIMENTAL APPROACH: Functional antagonist potency was assessed in cell-based systems expressing human EP(2) receptors and native tissue preparations from human, dog and mouse. The selectivity of PF-04418948 was assessed against related receptors and a panel of GPCRs, ion channels and enzymes. The ability of PF-04418948 to pharmacologically block EP(2) receptor function in vivo was tested in rats. KEY RESULTS: PF-04418948 inhibited prostaglandin E(2)(PGE(2))-induced increase in cAMP in cells expressing EP(2) receptors with a functional K(B) value of 1.8 nM. In human myometrium, PF-04418948 produced a parallel, rightward shift of the butaprost-induced inhibition of the contractions induced by electrical field stimulation with an apparent K(B) of 5.4 nM. In dog bronchiole and mouse trachea, PF-04418948 produced parallel rightward shifts of the PGE(2)-induced relaxation curve with a K(B) of 2.5 nM and an apparent K(B) of 1.3 nM respectively. Reversal of the PGE(2)-induced relaxation in the mouse trachea by PF-04418948 produced an IC(50) value of 2.7 nM. Given orally, PF-04418948 attenuated the butaprost-induced cutaneous blood flow response in rats. PF-04418948 was selective for EP(2) receptors over homologous and unrelated receptors, enzymes and channels. CONCLUSIONS AND IMPLICATIONS: PF-04418948 is an orally active, potent and selective surmountable EP(2) receptor antagonist that should aid further elaboration of EP(2) receptor function.

Pharmacological characterization of PF-00547659, an anti-human MAdCAM monoclonal antibody.

BACKGROUND AND PURPOSE: The adhesion molecule mucosal addressin cell adhesion molecule (MAdCAM) plays an essential role in the recruitment of lymphocytes to specialized high endothelial venules of the gastrointestinal tract and in their excessive tissue extravasation observed in inflammatory conditions, such as Crohn's disease. We have characterized the in vitro pharmacological properties of two monoclonal antibodies blocking MAdCAM, MECA-367 and PF-00547659, and determined their pharmacokinetic/pharmacodynamic profiles in vivo. EXPERIMENTAL APPROACH: Functional adhesion assays and surface plasmon resonance were used to characterize, in vitro, the pharmacological properties of MECA-367 and PF-00547659. The in vivo effects of MECA-367 and PF-00547659 on restriction of beta(7) (+) memory T lymphocytes were determined in mice and macaques, respectively, over the pharmacological dose range to confirm pharmacokinetic/pharmacodynamic relationships. KEY RESULTS: MECA-367 and PF-00547659 bound with high affinity to mouse and human MAdCAM with K(d) values of 5.1 and 16.1 pmol.L(-1) respectively and blocked the adhesion of alpha(4)beta(7) (+) leukocytes to MAdCAM with similar potency. MECA-367 and PF-00547659 induced a similar, dose-dependent two- to threefold increase in circulating populations of beta(7) (+) memory T-cells in the mouse and macaque; without affecting the beta(7) (-) populations. CONCLUSIONS AND IMPLICATIONS: PF-00547659 has potential utility in the treatment of inflammatory conditions by blocking tissue homing of activated alpha(4)beta(7) (+) leukocytes. The characterization of a rodent cross-reacting antibody as a surrogate for PF-00547659 in the search for potential pharmacological biomarkers and the determination of efficacious doses was effective in addressing the restricted orthologous cross-reactivity of PF-00547659 and the challenges this poses with respect to efficacy and safety testing.

The working practices of the police in relation to mentally disordered offenders and diversion services.

A research study was undertaken to examine the working practices of four bridewells in Hampshire in relation to mentally disordered offenders (MDOs) and diversion services. A consecutive sample of individuals detained in cells and not identified by the police as having a mental disorder were screened for the presence of mental disorder and their suitability for diversion. Custody and detention staff were observed and interviewed to elicit their views and working practices in relation to MDOs. The findings revealed that in bridewells with diversion schemes an average of about 7% of detained individuals had mental disorders who were suitable for diversion but were not detected by the police. In the bridewell without a diversion scheme the figure was 14%. Conversely, many individuals without a formal mental disorder were inappropriately referred to diversion schemes. The effectiveness of screening processes by custody staff was variable. Facilities in the bridewells were not suitable for containing disturbed individuals. Delays in obtaining mental health assessments caused considerable concern for police officers and prolonged the discomfort of vulnerable individuals. Further preparation and training of custody staff is needed to improve screening procedures. Reception and detention facilities for mentally disordered individuals should be reviewed and response times for approved social workers (ASWs) and psychiatrists would benefit from improvement.

Effects of oncogenic ErbB2 on G1 cell cycle regulators in breast tumour cells.

The ErbB2 receptor tyrosine kinase is overexpressed in a variety of human tumours. In order to understand the mechanism by which ErbB2 mediates tumour proliferation we have functionally inactivated the receptor using an intracellularly expressed, ER-targeted single-chain antibody (scFV-5R). Inducible expression of scFv-5R in the ErbB2-overexpressing SKBr3 breast tumour cell line leads to loss of plasma membrane localized ErbB2. Simultaneously, the activity of ErbB3, MAP kinase and PKB/Akt decreased dramatically, suggesting that active ErbB2/ErbB3 dimers are necessary for sustained activity of these kinases. Loss of functional ErbB2 caused the SKBr3 tumour cells to accumulate in the G1 phase of the cell cycle. This was a result of reduction in CDK2 activity, which was mediated by a re-distribution of p27Kip1 from sequestering complexes to cyclin E/CDK2 complexes. The level of c-Myc and D-cyclins, proteins involved in p27KiP1 sequestration, decreased in the absence of functional ErbB2. Ectopic expression of c-Myc led to an increase in D cyclin levels, CDK2 activity and resulted in a partial G1 rescue. We propose that c-Myc is a primary effector of ErbB2-mediated oncogenicity and functions to prevent normal p27Kip1 control of cyclinE/CDK2.

Manipulating mammalian genome by gene targeting.

The development of strategies which allow the inactivation of specific murine genes by homologous recombination in embryonic cells has revolutionized biological science in the last 10 years. A large number of mice carrying genetic lesions, generated by gene targeting technology, has tremendously increased our knowledge in many areas of biology, culminating in the identification of mouse models for human genetic disorders. These findings have been recently complemented by "conditional" gene targeting technology, allowing gene inactivation in a defined tissue and at a specific time point during development or adulthood, thereby extending the sophistication and potential of this technology.

Phosphorylation sites in the autoinhibitory domain participate in p70(s6k) activation loop phosphorylation.

Here we have employed p70(s6k) truncation and point mutants to elucidate the role played by the carboxyl-terminal autoinhibitory domain S/TP phosphorylation sites in kinase activation. Earlier studies showed that truncation of the p70(s6k) amino terminus severely impaired kinase activation but that this effect was reversed by deleting the carboxyl terminus, which in parallel led to deregulation of Thr229 phosphorylation in the activation loop (Dennis, P. B., Pullen, N., Kozma, S. C., and Thomas, G. (1996) Mol. Cell. Biol. 16, 6242-6251). In this study, substitution of acidic residues for the four autoinhibitory domain S/TP sites mimics the carboxyl-terminal deletion largely by rescuing kinase activation caused by the amino-terminal truncation. However, these mutations do not deregulate Thr229 phosphorylation, suggesting the involvement of another regulatory element in the intact kinase. This element appears to be Thr389 phosphorylation, because substitution of an acidic residue at this position in the p70(s6k) variant containing the S/TP mutations leads to a large increase in basal Thr229 phosphorylation and kinase activity. In contrast, an alanine substitution at Thr389 blocks both responses. Consistent with these data, we show that a mutant harboring the acidic S/TP and Thr389 substitutions is an excellent in vitro substrate for the newly identified Thr229 kinase, phosphoinositide-dependent kinase-1 (Pullen, N., Dennis, P. B., Andjelkovic, M., Dufner, A., Kozma, S., Hemmings, B. A., and Thomas, G. (1998) Science 279, 707-710), whereas phosphoinositide-dependent kinase-1 poorly utilizes the two p70(s6k) variants that have only one set of mutations. These findings indicate that phosphorylation of the S/TP sites, in cooperation with Thr389 phosphorylation, controls Thr229 phosphorylation through an intrasteric mechanism.

Phosphorylation and activation of p70s6k by PDK1.

Activation of the protein p70s6k by mitogens leads to increased translation of a family of messenger RNAs that encode essential components of the protein synthetic apparatus. Activation of the kinase requires hierarchical phosphorylation at multiple sites, culminating in the phosphorylation of the threonine in position 229 (Thr229), in the catalytic domain. The homologous site in protein kinase B (PKB), Thr308, has been shown to be phosphorylated by the phosphoinositide-dependent protein kinase PDK1. A regulatory link between p70s6k and PKB was demonstrated, as PDK1 was found to selectively phosphorylate p70s6k at Thr229. More importantly, PDK1 activated p70s6k in vitro and in vivo, whereas the catalytically inactive PDK1 blocked insulin-induced activation of p70s6k.

The insulin-induced signalling pathway leading to S6 and initiation factor 4E binding protein 1 phosphorylation bifurcates at a rapamycin-sensitive point immediately upstream of p70s6k.

Employing specific inhibitors and docking-site mutants of growth factor receptors, recent studies have indicated that the insulin-induced increase in 40S ribosomal protein S6 and initiation factor 4E binding protein 1 (4E-BP1) phosphorylation is mediated by the mTOR/FRAP-p70s6k signal transduction pathway. However, it has not been resolved whether the phosphorylation of both proteins is mediated by p70s6k or whether they reside on parallel pathways which bifurcate upstream of p70s6k. Here we have used either rapamycin-resistant, kinase-dead, or wild-type p70s6k variants to distinguish between these possibilities. The rapamycin-resistant p70s6k, which has high constitutive activity, was able to signal to S6 in the absence of insulin and to prevent the rapamycin-induced block of S6 phosphorylation. This same construct did not increase the basal state of 4E-BP1 phosphorylation or protect it from the rapamycin-induced block in phosphorylation. Unexpectedly, the rapamycin-resistant p70s6k inhibited insulin-induced 4E-BP1 phosphorylation in a dose-dependent manner. This effect was mimicked by the kinase-dead and wild-type p70s6k constructs, which also blocked insulin-induced dissociation of 4E-BP1 from initiation factor 4E. Both the kinase-dead and wild-type constructs also blocked reporter p70s6k activation, although only the kinase-dead p70s6k had a dominant-interfering effect on S6 phosphorylation. Analysis of phosphopeptides from reporter 4E-BP1 and p70s6k revealed that the kinase-dead p70s6k affected the same subset of sites as rapamycin in both proteins. The results demonstrate, for the first time, that activated p70s6k mediates increased S6 phosphorylation in vivo. Furthermore, they show that increased 4E-BP1 phosphorylation is controlled by a parallel signalling pathway that bifurcates immediately upstream of p70s6k, with the two pathways sharing a common rapamycin-sensitive activator.

Dual requirement for a newly identified phosphorylation site in p70s6k.

The activation of p70s6k is associated with multiple phosphorylations at two sets of sites. The first set, S411, S418, T421, and S424, reside within the autoinhibitory domain, and each contains a hydrophobic residue at -2 and a proline at +1. The second set of sites, T229 (in the catalytic domain) and T389 and S404 (in the linker region), are rapamycin sensitive and flanked by bulky aromatic residues. Here we describe the identification and mutational analysis of three new phosphorylation sites, T367, S371, and T447, all of which have a recognition motif similar to that of the first set of sites. A mutation of T367 or T447 to either alanine or glutamic acid had no apparent effect on p70s6k activity, whereas similar mutations of S371 abolished kinase activity. Of these three sites and their surrounding motifs, only S371 is conserved in p70s6k homologs from Drosophila melanogaster, Arabidopsis thaliana, and Saccharomyces cerevisiae, as well as many members of the protein kinase C family. Serum stimulation increased S371 phosphorylation; unlike the situation for specific members of the protein kinase C family, where the homologous site is regulated by autophosphorylation, S371 phosphorylation is regulated by an external mechanism. Phosphopeptide analysis of S371 mutants further revealed that the loss of activity in these variants was paralleled by a block in serum-induced T389 phosphorylation, a phosphorylation site previously shown to be essential for kinase activity. Nevertheless, the substitution of an acidic residue at T389, which mimics phosphorylation at this site, did not rescue mutant p70s6k activity, indicating that S371 phosphorylation plays an independent role in regulating intrinsic kinase activity.

The modular phosphorylation and activation of p70s6k.

The activation of p70s6k is accompanied by a complex series of phosphorylation events. In this review we propose a model of activation which divides p70s6k into four functional modules that cooperate in leading to full enzyme activity. In the light of the model, we suggest how candidate effectors of p70s6k activation might function by directing the phosphorylation of specific sites.

The principal rapamycin-sensitive p70(s6k) phosphorylation sites, T-229 and T-389, are differentially regulated by rapamycin-insensitive kinase kinases.

Mitogen-induced activation of p70(s6k) is associated with the phosphorylation of specific sites which are negatively affected by the immunosuppressant rapamycin, the fungal metabolite wortmannin, and the methylxanthine SQ20006. Recent reports have focused on the role of the amino terminus of the p85(s6k) isoform in mediating kinase activity, with the observation that amino-terminal truncation mutants are activated in the presence of rapamycin while retaining their sensitivity to wortmannin. Here we show that the effects of previously described amino- and carboxy-terminal truncations on kinase activity are ultimately reflected in the phosphorylation state of the enzyme. Mutation of the principal rapamycin-targeted phosphorylation site, T-389, to an acidic residue generates a form of the kinase which is as resistant to wortmannin or SQ20006 as it is to rapamycin, consistent with the previous observation that T-389 was a common target of all three inhibitors. Truncation of the first 54 residues of the amino terminus blocks the serum-induced phosphorylation of three rapamycin-sensitive sites, T-229 in the activation loop and T-389 and S-404 in the linker region. This correlates with a severe reduction in the ability of the kinase to be activated by serum. However, loss of mitogen activation conferred by the removal of the amino terminus is reversed by additional truncation of the carboxy-terminal domain, with the resulting mutant demonstrating phosphorylation of the remaining two rapamycin-sensitive sites, T-229 and T-389. In this double-truncation mutant, phosphorylation of T-229 occurs in the basal state, whereas mitogen stimulation is required to induce acute upregulation of T-389 phosphorylation. The phosphorylation of both sites proceeds unimpaired in the presence of rapamycin, indicating that the kinases responsible for the phosphorylation of these sites are not inhibited by the macrolide. In contrast, activation of the double-truncation mutant is blocked in the presence of wortmannin or SQ20006, and these agents completely block the phosphorylation of T-389 while having only a marginal effect on T-229 phosphorylation. When the T-389 site is mutated to an acidic residue in the double-truncation background, the activation of the resulting mutant is insensitive to the wortmannin and SQ20006 block, but interestingly, the mutant is activated to a significantly greater level than a control in the presence of rapamycin. These data are consistent with the hypothesis that T-389 is the principal regulatory phosphorylation site, which, in combination with hyperphosphorylation of the autoinhibitory domain S/TP sites, is acutely regulated by external effectors, whereas T-229 phosphorylation is regulated primarily by internal mechanisms.

Rhodopsin kinase: studies on the sequence of and the recognition motif for multiphosphorylations.

Peptides of 10-12 amino acids in length, which overlapped with the sequence of the last 20 amino acids in the C-terminal tail of rhodopsin, were synthesized and used as substrates for rhodopsin kinase. In all cases the phosphorylation of the peptides was found to be greatly stimulated (> 20-fold) by the presence of light-activated rhodopsin (Rho*). The incorporation of 32P at seven Ser/Thr residues that are the potential sites of phosphorylation was quantified, and the results were analyzed in terms of two parameters. First, a global comparison of phosphorylation at each site was made when the propensity for the modification was found to be in the order: Ser 343 > Ser 338 > Thr 336 > Ser 334, Thr 342 > Thr 335, Thr 340. Second, the peptides were aligned on a hypothetical template with the residue to be phosphorylated occupying the P-position, and the manner in which the nature of the surrounding residues effected the phosphorylation was assessed. It was found that the optimal phosphorylation of the P-site Ser/Thr occurs if it has at least one residue on the amino side and five on the acyl side and also contains a neutral residue, preferably small (A, P, S, T) at the P+4 position.(ABSTRACT TRUNCATED AT 250 WORDS)

Cooperativity during multiple phosphorylations catalyzed by rhodopsin kinase: supporting evidence using synthetic phosphopeptides.

Rhodopsin kinase is a key component in the shutdown of visual transduction. The phosphorylation of rhodopsin's C-terminus was evaluated using synthetic peptides derived from the last 12 amino acids (337-348) as substrates and their phosphorylated counterparts as inhibitors. It was found that synthetic peptides were phosphorylated at the serine residue corresponding to Ser-343 in the primary sequence of bovine rhodopsin. The phosphopeptides were prepared by incorporating into the peptide chain a trityl-protected serine derivative at the site destined to contain the phosphoryl group. The trityl group was selectively released with 20% (v/v) dichloroacetic acid; the free hydroxyl group was then phosphitylated with di-tert-butyl N,N-diethylphosphoramidite, and the resulting phosphite derivative was oxidized with m-chloroperoxybenzoic acid. The phosphopeptides were found to have a greater affinity for the kinase compared with their nonphosphorylated counterparts; for the peptides corresponding to residues 337-348 of rhodopsin the affinity increased in the order VSKTETSQVAPA < VSKTETS[PO3H2]QVAPA < VS[PO3H2]KTETS[PO3H2]QVAPA. The results are interpreted to support the cooperativity hypotheses proposed previously [Wilden, U., & Kühn, H. (1982) Biochemistry 21, 3014-3022; Aton, B. R., Litman, B. J., & Jackson, M. L. (1984) Biochemistry 23, 1737-1741].