Tackling physical inactivity and inequalities: implementing a whole systems approach to transform community provision for disabled people and people with long-term health conditions.

BACKGROUND: Physical inactivity is a global public health priority. There are known health and well-being consequences of being inactive, and the benefits of being physically active are well established. However, there are persistent inequalities when it comes to how physically active people are, with disabled people, people living with long-term health conditions, and people residing in areas of socio-economic deprivation being particularly affected. Methods such as whole system approaches (WSAs), which are dynamic, multifaceted, and engage all relevant stakeholders, have gained momentum as an approach to address such complex public health problems. However, evidence relating to the implementation of WSAs to address physical inactivity is lacking. The aim of the Prevention and Enablement Model (PEM) was to take a whole system approach in Essex to encourage and support disabled people and/or individuals living with long-term health conditions to be more active, happier, and to live more independently. METHODS: The aim of this study was to explore the enablers, challenges, and reflections associated with the process of designing and implementing the PEM. Semi-structured interviews (n = 12) were used to collect data from people involved in the PEM's design, implementation and/or delivery. Data was analysed using Braun and Clarke's reflexive thematic analysis. RESULTS: Four themes were identified: (1) Working collaboratively: Specific enablers of time and space were identified as important in the planning and implementation of a WSA (2) Leadership and planning: Distributed and flexible leadership was identified as central to successful implementation (3) Re-orientating practice: Highlighted the transformative potential of a whole system approach and how it contrasts with conventional work practices, and (4) Reflection and learning: Informing ongoing refinements and further implementation of successful system change. CONCLUSIONS: These findings highlight the challenge and complexity of implementing a WSA that involves diverse stakeholders from across adult social care, the NHS, and the third sector. Several important enablers are identified, such as leadership and planning, and the challenges and discomfort that can arise whilst changing systems. Ongoing efforts are required to ensure that different elements of the system collaborate effectively to address inequalities in physical activity participation, through the implementation of a WSA.

Characterization of Multisubstituted Corticotropin Releasing Factor (CRF) Peptide Antagonists (Astressins).

CRF mediates numerous stress-related endocrine, autonomic, metabolic, and behavioral responses. We present the synthesis and chemical and biological properties of astressin B analogues {cyclo(30-33)[D-Phe(12),Nle(21,38),C(α)MeLeu(27,40),Glu(30),Lys(33)]-acetyl-h/r-CRF(9-41)}. Out of 37 novel peptides, 17 (2, 4, 6-8, 10, 11, 16, 17, 27, 29, 30, 32-36) and 16 (3, 5, 9, 12-15, 18, 19, 22-26, 28, 31) had k(i) to CRF receptors in the high picomolar and low nanomole ranges, respectively. Peptides 1, 2, and 11 inhibited h/rCRF and urocortin 1-induced cAMP release from AtT20 and A7r5 cells. When Astressin C 2 was administered to adrenalectomized rats at 1.0 mg subcutaneously, it inhibited ACTH release for >7 d. Additional rat data based on the inhibitory effect of (2) on h/rCRF-induced stimulation of colonic secretory motor activity and urocortin 2-induced delayed gastric emptying also indicate a safe and long-lasting antagonistic effect. The overall properties of selected analogues may fulfill the criteria expected from clinical candidates.

A combination therapy for KRAS-driven lung adenocarcinomas using lipophilic bisphosphonates and rapamycin.

Lung cancer is the most common human malignancy and leads to about one-third of all cancer-related deaths. Lung adenocarcinomas harboring KRAS mutations, in contrast to those with EGFR and EML4-ALK mutations, have not been successfully targeted. We describe a combination therapy for treating these malignancies with two agents: a lipophilic bisphosphonate and rapamycin. This drug combination is much more effective than either agent acting alone in the KRAS G12D-induced mouse lung model. Lipophilic bisphosphonates inhibit both farnesyl and geranylgeranyldiphosphate synthases, effectively blocking prenylation of KRAS and other small G proteins (heterotrimeric GTP-binding protein, heterotrimeric guanine nucleotide-binding proteins) critical for tumor growth and cell survival. Bisphosphonate treatment of cells initiated autophagy but was ultimately unsuccessful and led to p62 accumulation and concomitant nuclear factor κB (NF-κB) activation, resulting in dampened efficacy in vivo. However, we found that rapamycin, in addition to inhibiting the mammalian target of rapamycin (mTOR) pathway, facilitated autophagy and prevented p62 accumulation-induced NF-κB activation and tumor cell proliferation. Overall, these results suggest that using lipophilic bisphosphonates in combination with rapamycin may provide an effective strategy for targeting lung adenocarcinomas harboring KRAS mutations.

Neural cells secrete a unique repertoire of proteins.

Proteins that are released from cells consist of those in the extracellular matrix, as well as extracellular signaling and adhesion molecules. The majority of these extracellular proteins are, however, unknown. To determine their identity, we have used a proteomics approach to define proteins released from neurons, astrocytes and neural precursor cells. Using two-dimensional gels and liquid chromatography/mass spectrometry technology, it is shown that while astrocytes release a relatively small number of proteins, neurons and neuronal precursor cells release a larger number of proteins with more functional diversity. Although there is overlap between the different cell types, the exact composition of the extracellular protein pool is unique for each cell population. The various subsets of extracellular neural proteins include those involved in cellular Redox regulation and chaperones. In addition, many proteolytic enzymes are found outside of the cell. These data show that the extracellular space within the nervous system has a more diverse protein composition than previously thought.

Somatostatin receptor 1 selective analogues: 2. N(alpha)-Methylated scan.

Des-AA(1,2,5)-[d-Trp(8)/d-Nal(8),IAmp(9)]SRIF (AA = amino acid, Nal = 3-(2-naphthyl)-alanine, IAmp = 4-(N-isopropyl)-aminomethylphenylalanine, SRIF = somatostatin), with or without a tyrosine or monoiodotyrosine, were scanned with the introduction of a backbone N-methyl group and tested for binding affinity at the five human somatostatin receptors (sst(1)(-)(5)). N(alpha)-Methylation resulted in loss of sst affinity (2- to >5-fold) when introduced at residues Lys(4) (6), Phe(6) (7), Phe(7) (8), Thr(10) (11), and Phe(11) (12) of the parent compound Des-AA(1,2,5)-[d-Nal(8),IAmp(9)]SRIF (4). N(alpha)-Methylation was tolerated at residues Cys(3) (5), d-Nal(8) (9), Thr(12) (13), and Cys(14) (15) with retention of binding sst affinity and selectivity and resulted in an increase in sst binding affinity at positions IAmp(9) (10) and Ser(13) (14). In these series, the d-Trp(8) substitution versus d-Nal(8) is clearly superior. C-Terminally lysine-extended analogues (21-25) retained sst(1) selectivity and binding affinity when compared to their d-Nal(8)- (4) or d-Trp(8)- (3) containing parent. Des-AA(1,2,5)-[d-Trp(8), (N(alpha)Me)IAmp(9)]SRIF (17), Des-AA(1,2,5)-[d-Trp(8),IAmp(9),(N(alpha)Me)Ser(13)]SRIF (19), Des-AA(1,2,5)-[d-Trp(8),IAmp(9),(N(alpha)Me)Cys(14)]SRIF (20), Des-AA(1,2,5)-[d-Trp(8),(N(alpha)Me)IAmp(9),Tyr(11)]SRIF (34), and Des-AA(1,2,5)-[d-Agl(8)(N(beta)Me,2-naphthoyl),IAmp(9),Tyr(11)]SRIF (42) (Agl = aminoglycine) are sst(1) agonists in their ability to inhibit forskolin-induced cAMP production.

Total chemical synthesis and NMR characterization of the glycopeptide tx5a, a heavily post-translationally modified conotoxin, reveals that the glycan structure is alpha-D-Gal-(1-->3)-alpha-D-GalNAc.

The 13-amino acid glycopeptide tx5a (Gla-Cys-Cys-Gla-Asp-Gly-Trp*-Cys-Cys-Thr*-Ala-Ala-Hyp-OH, where Trp* = 6-bromotryptophan and Thr* = Gal-GalNAc-threonine), isolated from Conus textile, causes hyperactivity and spasticity when injected intracerebral ventricularly into mice. It contains nine post-translationally modified residues: four cysteine residues, two gamma-carboxyglutamic acid residues, and one residue each of 6-bromotryptophan, 4-trans-hydroxyproline and glycosylated threonine. The chemical nature of each of these has been determined with the exception of the glycan linkage pattern on threonine and the stereochemistry of the 6-bromotryptophan residue. Previous investigations have demonstrated that tx5a contains a disaccharide composed of N-acetylgalactosamine (GalNAc) and galactose (Gal), but the interresidue linkage was not characterized. We hypothesized that tx5a contained the T-antigen, beta-D-Gal-(1-->3)-alpha-D-GalNAc, one of the most common O-linked glycan structures, identified previously in another Conus glycopeptide, contalukin-G. We therefore utilized the peracetylated form of this glycan attached to Fmoc-threonine in an attempted synthesis. While the result-ing synthetic peptide (Gla-Cys-Cys-Gla-Asp-Gly-Trp*-Cys-Cys-Thr*-Ala-Ala-Hyp-OH, where Trp* =6-bromotryptophan and Thr* = beta-D-Gal-(1-->3)-alpha-D-GalNAc-threonine) and the native peptide had almost identical mass spectra, a comparison of their RP-HPLC chromatograms suggested that the two forms were not identical. Two-dimensional 1H homonuclear and 13C-1H heteronuclear NMR spectroscopy of native tx5a isolated from Conus textile was then used to determine that the glycan present on tx5a indeed is not the aforementioned T-antigen, but rather alpha-D-Gal-(1-->3)-alpha-D-GalNAc.

MALDI-MS analysis of peptides modified with photolabile arylazido groups.

The ability of MALDI-MS to analyze photolabile arylazido peptide derivatives was investigated. Peptides containing UV-labile p-azidobenzoyl groups were subjected to MALDI-MS analysis in a variety of matrices. As standard MALDI-MS employs a UV laser (337 nm), we investigated conditions that would allow detection of the intact molecule ions for these light-sensitive peptides. When using alpha-cyano-4-hydroxycinnamic acid (ACHC) or 2,5 dihydroxybenzoic acid (DHB) as the matrix, photoinduced degradation products were prevalent. In contrast, when employing the matrix sinapinic acid, the intact molecule ion corresponding with the azido peptide was the predominant signal. The protection of photolabile azido derivatives correlates with the UV absorbance properties of the matrix employed, i.e., sinapinic acid, which exhibits a strong absorbance near 337 nm, most efficiently protects the azido derivative from photodegradation.

Novel sst(4)-selective somatostatin (SRIF) agonists. 1. Lead identification using a betide scan.

Hypothesizing that structural constraints in somatostatin (SRIF) analogues may result in receptor selectivity, and aiming to characterize the bioactive conformation of somatostatin at each of its five receptors, we carried out an N(beta)-methylated aminoglycine (Agl) scan of the octapeptide H-c[Cys(3)-Phe(6)-Phe(7)-dTrp(8)-Lys(9)-Thr(10)-Phe(11)-Cys(14)]-OH (SRIF numbering) (ODT-8) that is potent at all SRIF receptor subtypes (sst's) but sst(1). We found that H-c[Cys-LAgl(N(beta)Me,benzoyl)-Phe-DTrp-Lys-Thr-Phe-Cys]-OH (4), H-c[Cys-Phe-LAgl(N(beta)Me,benzoyl)-Trp-Lys-Thr-Phe-Cys]-OH (6), H-c[Cys-Phe-LAgl(N(beta)Me,benzoyl)-dTrp-Lys-Thr-Phe-Cys]-OH (8), and H-c[DCys-Phe-LAgl(N(beta)Me,benzoyl)-DTrp-Lys-Thr-Phe-Cys]-OH (10) had high affinity (IC(50) = 14.3, 5.4, 5.2, and 3.4 nM, respectively) and selectivity for sst(4) (>50-fold over the other receptors). The l-configuration at positions 7 and 8 (l(7), l(8)) yields greater sst(4) selectivity than the l(7), d(8) configuration (6 versus 8). Peptides with the d(7), l(8) (7) and d(7), d(8) (9) configurations are significantly less potent at all receptors. H-c[Cys-Phe-Phe-DTrp-LAgl(betaAla)-Thr-Phe-Cys]-OH (16), H-c[Cys-Phe-Phe-DTrp-DAgl(betaAla)-Thr-Phe-Cys]-OH (17), and their N(beta)Me derivatives at position 9 (18, 19) were essentially inactive. Potent but less sst(4)-selective were members of the Agl-scan at positions 10, H-c[Cys-Phe-Phe-dTrp-Lys-lAgl(N(beta)Me,HO-Ac)-Phe-Cys]-OH (20, IC(50) = 6.5 nM), and 11, H-c[Cys-Phe-Phe-DTrp-Lys-Thr-LAgl(N(beta)Me,benzoyl)-Cys]-OH (22, IC(50) = 6.9 nM), while the d-configuration at positions 10 (21) and 11 (23) led to reduced affinity. One of our best analogues, 8, is an agonist when tested for its ability to inhibit forskolin-stimulated cAMP accumulation in sst(4)-transfected CCL39 cells (EC(50) = 1.01 nM). All Agl-containing analogues were first synthesized using unresolved Fmoc-Agl(N(beta)Me,Boc)-OH, and the diastereomers were separated using HPLC. Chiral assignment at the Agl-containing residue was subsequently done using enzymatic degradation and by de novo synthesis in the cases of H-c[Cys-Phe-DAgl(N(beta)Me,benzoyl)-DTrp-Lys-Thr-Phe-Cys]-OH (9) and H-c[DCys-Phe-DAgl(N(beta)Me,benzoyl)-DTrp-Lys-Thr-Phe-Cys]-OH (11), starting with the papain-resolved Fmoc-DAgl(Boc). These results suggested that the orientation of side chains at position 6, 7, or 11 with respect to the side chains of residues 8 and 9 may be independently responsible for sst(4) selectivity.