ObjectiveProtein–energy malnutrition and the subsequent muscle wasting (sarcopenia) are common ageing complications. It is knowing to be also associated with dementia. Our programme will test the ...cytoprotective functions of vitamin E combined with the cortisol-lowering effect of chocolate polyphenols (PP), in combination with muscle anabolic effect of adequate dietary protein intake and physical exercise to prevent the age-dependent decline of muscle mass and its key underpinning mechanisms including mitochondrial function, and nutrient metabolism in muscle in the elderly.Methods and analysisIn 2020, a 6-month double-blind randomised controlled trial in 75 predementia older people was launched to prevent muscle mass loss, in respond to the ‘Joint Programming Initiative A healthy diet for a healthy life’. In the run-in phase, participants will be stabilised on a protein-rich diet (0.9–1.0 g protein/kg ideal body weight/day) and physical exercise programme (high-intensity interval training specifically developed for these subjects). Subsequently, they will be randomised into three groups (1:1:1). The study arms will have a similar isocaloric diet and follow a similar physical exercise programme. Control group (n=25) will maintain the baseline diet; intervention groups will consume either 30 g/day of dark chocolate containing 500 mg total PP (corresponding to 60 mg epicatechin) and 100 mg vitamin E (as RRR-alpha-tocopherol) (n=25); or the high polyphenol chocolate without additional vitamin E (n=25). Muscle mass will be the primary endpoint. Other outcomes are neurocognitive status and previously identified biomolecular indices of frailty in predementia patients. Muscle biopsies will be collected to assess myocyte contraction and mitochondrial metabolism. Blood and plasma samples will be analysed for laboratory endpoints including nutrition metabolism and omics.Ethics and disseminationAll the ethical and regulatory approvals have been obtained by the ethical committees of the Azienda Ospedaliera Universitaria Integrata of Verona with respect to scientific content and compliance with applicable research and human subjects’ regulation. Given the broader interest of the society toward undernutrition in the elderly, we identify four main target audiences for our research activity: national and local health systems, both internal and external to the project; targeted population (the elderly); general public; and academia. These activities include scientific workshops, public health awareness campaigns, project dedicated website and publication is scientific peer-review journals.Trial registration numberNCT05343611.
There has been rapid scale-up of malaria vector control in the last ten years. Both of the primary control strategies, long-lasting pyrethroid treated nets and indoor residual spraying, rely on the ...use of a limited number of insecticides. Insecticide resistance, as measured by bioassay, has rapidly increased in prevalence and has come to the forefront as an issue that needs to be addressed to maintain the sustainability of malaria control and the drive to elimination. Zambia's programme reported high levels of resistance to the insecticides it used in 2010, and, as a result, increased its investment in resistance monitoring to support informed resistance management decisions.
A country-wide survey on insecticide resistance in Zambian malaria vectors was performed using WHO bioassays to detect resistant phenotypes. Molecular techniques were used to detect target-site mutations and microarray to detect metabolic resistance mechanisms. Anopheles gambiae s.s. was resistant to pyrethroids, DDT and carbamates, with potential organophosphate resistance in one population. The resistant phenotypes were conferred by both target-site and metabolic mechanisms. Anopheles funestus s.s. was largely resistant to pyrethroids and carbamates, with potential resistance to DDT in two locations. The resistant phenotypes were conferred by elevated levels of cytochrome p450s.
Currently, the Zambia National Malaria Control Centre is using these results to inform their vector control strategy. The methods employed here can serve as a template to all malaria-endemic countries striving to create a sustainable insecticide resistance management plan.
Extensive use of pyrethroids for malaria control in Africa has led to widespread pyrethroid resistance in the two major African vectors of malaria An. gambiae and An. funestus. This is often ...associated with constitutively elevated levels of cytochrome P450s involved with pyrethroid metabolism and detoxification. P450s have the capacity to metabolise diverse substrates, which raises concerns about their potential to cause cross-resistance. A bank of seven recombinant P450s from An. gambiae (CYPs 6M2, 6P2, 6P3, 6P4, 6P5, 9J5) and An. funestus (CYP6P9a) commonly associated with pyrethroid resistance were screened against twelve insecticides representing the five major classes of insecticides recommended by WHO for malaria control; permethrin, etofenprox and bifenthrin (type I pyrethroids), deltamethrin, lambda cyhalothrin and cypermethrin (type II pyrethroids), DDT (organochlorine), bendiocarb (carbamate), malathion, pirimiphos methyl and fenitrothion (organophosphates) and pyriproxyfen (juvenile hormone analogue). DDT was not metabolised by the P450 panel, while bendiocarb was only metabolised by CYP6P3. Pyrethroids and pyriproxyfen were largely susceptible to metabolism by the P450 panel, as were organophosphates, which are activated by P450s. Primiphos-methyl is increasingly used for malaria control. Examination of the pirimiphos-methyl metabolites generated by CYP6P3 revealed both the active pirimiphos-methyl-oxon form and the inactive oxidative cleavage product 2-diethylamino-6-hydroxy-4-methylpyrimidine. The inhibition profile of CYPs 6M2, 6P2, 6P3, 6P9a and 9J5 was also examined using diethoxyfluorescein (DEF) as the probe substrate. Bendiocarb was the weakest inhibitor with IC50 > 100 μM across the P450 panel, while CYP6M2 showed strongest inhibition by malathion (IC50 0.7 μM). The results suggest that P450s present at elevated levels in two major Anopheline vectors of malaria in Africa have the capacity to metabolise a diverse range of pyrethroid and organophosphate insecticides as well as pyriproxyfen that could impact vector control.
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•P450s associated with pyrethroid resistance metabolise a wide range of insecticides recommended for malaria control.•Organophosphates including pirimiphos-methyl are susceptible to metabolism by pyrethroid metabolic resistance markers.•Pirimiphos-methyl may be either activated or inactivated by CYP6P3 metabolism.•In vitro profiling of insecticides is recommended to flag potential metabolic resistance issues.
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Muscle mass and function are reduced with age in mammals and this is associated with disrupted neuromuscular interactions. Skeletal muscle mounts a robust stress response to ...contractions by an increased expression of Heat Shock Proteins (HSPs) that facilitate muscle remodeling, promote protein folding and clearance of damaged proteins. In contrast, other cell types including neuronal cells, are unable to mount a stress response. It has been proposed that HSPs may be transferred from one cell type to another in exosomes to maintain proteostasis in the recipient cells. Thus, speculation that the robust HSP response by muscle provides support to peripheral neuronal cells via exosomal transfer of HSPs is tempting. However, HSP generation by muscle following contractions is attenuated in old mice and humans. We hypothesise that inability of muscles of old mice to produce HSPs in response to contraction results in altered exosomal HSP transfer, and therefore a failure to maintain proteostasis in motor neurons, resulting in neuronal and muscle degeneration
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Muscle fibres were isolated from flexor digitorum bravis (FDB) muscles of adult (6–8 month) and old (24–26 month) mice, and initiated to contract using a non‐damaging electrical stimulation protocol
(2)
. Media was collected from quiescent fibres, and fibres immediately following contraction, and Extracellular Vesicles (EVs) purified using a Total Exosome Isolation kit (Thermo). NanoSight analysis was used to characterise the size and number of the EVs. Total protein content was determined and HSP levels were analysed by western blotting.
Data demonstrated no significant difference in the total number of EVs released from quiescent fibres from adult and old mice, along with a similar increase in EVs released by muscle fibres of adult (2.7 fold) and old mice (3.1 fold) immediately following contraction compared with quiescent fibres. In contrast, the size distribution of the EVs from fibres of old mice was altered compared with EVs from adult fibres, and the total protein content of EVs was significantly less (40% decrease) in those released by quiescent old fibres compared with EVs of quiescent adult fibres. The lower protein content for EVs from old compared with adult fibres was maintained following stimulation.
A 2.5 fold increase in HSP25 and a substantial increase in HSP60 content (from undetectable levels) was seen in EVs from fibres of adult mice immediately following contraction. In contrast, preliminary data suggested that the HSP60 content of EVs produced by quiescent muscle fibres from old mice is already elevated, with little further increase following contraction in a similar manner to muscles in vivo
(3)
. Additional data will examine the uptake of EVs by neuronal cells and the effect of age of donor on their uptake ability.
Support or Funding Information
With thanks to National Institute of Ageing (AG051442) and the University of Liverpool for their generous support.
Pyrethroid resistance is widespread in the malaria vector Anopheles gambiae leading to concerns about the future efficacy of bednets with pyrethroids as the sole active ingredient. The incorporation ...of pyriproxyfen (PPF), a juvenile hormone analogue, into pyrethroid treated bednets is being trialed in Africa. Pyrethroid resistance is commonly associated with elevated levels of P450 expression including CYPs 6M2, 6P2, 6P3, 6P4, 6P5, 6Z2 and 9J5. Having expressed these P450s in E. coli we find all are capable of metabolizing PPF. Inhibition of these P450s by permethrin, deltamethrin and PPF was also examined. Deltamethrin and permethrin were moderate inhibitors (IC50 1–10 μM) of diethoxyfluorescein (DEF) activity for all P450s apart from CYP6Z2 (IC50 > 10 μM), while PPF displayed weaker inhibition of all P450s (IC50 > 10 μM) except CYP's 6Z2 and 6P2 (IC50 1–10 μM). We found evidence of low levels of cross resistance between PPF and other insecticide classes by comparing the efficacy of PPF in inhibiting metamorphosis and inducing female sterility in an insecticide susceptible strain of An. gambiae and a multiple resistant strain from Cote d’Ivoire.
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•Pyriproxyfen is metabolized by P450s associated with pyrethroid resistance.•Pyrethroids may synergise pyriproxyfen activity.•Pyriproxyfen efficacy is compromised in a multiple resistant An. gambiae strain.•Resistance monitoring for pyriproxyfen chemistry is recommended.
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