The incidence of vaccine preventable disease in Pakistan remains high despite a long-standing Expanded Program on Immunization (EPI). We describe vaccine completeness, timeliness and determinants of ...coverage from a remote rural cohort (2012-2014). Vaccination histories were taken from EPI records. Vaccination was complete if all doses were received according to the EPI schedule and timely if doses were not ≥3 days early or ≥ 28 days late. Three models are presented: a multivariable logistic regression of household demographic and socioeconomic factors associated with complete vaccination, a multivariable mixed effects logistic regression assessing whether or not the vaccine was administered late (versus on-time), and a mixed effects multivariable Poisson regression model analysing the interval (in days) between vaccine doses. Of 959 enrolled children with full vaccination histories, 88.2 and 65.1% were fully vaccinated following either the pentavalent or DPT/HBV schedules if measles was excluded; coverage dropped to 50.0 and 27.1% when both doses of measles were included. Sixty-four (6.7%) were unvaccinated. Coverage and timeliness declined with subsequent doses. Migrating into the village after 1995 (95%CI 1.88 to 5.17) was associated with late vaccination. Being male, having an older father, and having parents with at least some formal education reduced the likelihood of a late dose. The interval between doses was consistent at 5 weeks (compared with the 4 weeks recommended by EPI). None of the socio-demographic variables were related to the likelihood of receiving full coverage. Vaccine coverage in Oshikhandass was higher than national averages. Measles vaccine coverage and timeliness were low; special consideration should be paid to this vaccine. The local vaccination schedule differed from the EPI, but the consistency suggests good local administration.
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Activation of cannabinoid CB1 receptors (CB1R) by delta9-tetrahydrocannabinol (THC) produces a variety of negative effects with major consequences in cannabis users that constitute important ...drawbacks for the use of cannabinoids as therapeutic agents. For this reason, there is a tremendous medical interest in harnessing the beneficial effects of THC. Behavioral studies carried out in mice lacking 5-HT2A receptors (5-HT2AR) revealed a remarkable 5-HT2AR-dependent dissociation in the beneficial antinociceptive effects of THC and its detrimental amnesic properties. We found that specific effects of THC such as memory deficits, anxiolytic-like effects, and social interaction are under the control of 5-HT2AR, but its acute hypolocomotor, hypothermic, anxiogenic, and antinociceptive effects are not. In biochemical studies, we show that CB1R and 5-HT2AR form heteromers that are expressed and functionally active in specific brain regions involved in memory impairment. Remarkably, our functional data shows that costimulation of both receptors by agonists reduces cell signaling, antagonist binding to one receptor blocks signaling of the interacting receptor, and heteromer formation leads to a switch in G-protein coupling for 5-HT2AR from Gq to Gi proteins. Synthetic peptides with the sequence of transmembrane helices 5 and 6 of CB1R, fused to a cell-penetrating peptide, were able to disrupt receptor heteromerization in vivo, leading to a selective abrogation of memory impairments caused by exposure to THC. These data reveal a novel molecular mechanism for the functional interaction between CB1R and 5-HT2AR mediating cognitive impairment. CB1R-5-HT2AR heteromers are thus good targets to dissociate the cognitive deficits induced by THC from its beneficial antinociceptive properties.
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Recent advances in synthetic biology research have been underpinned by an exponential increase in available genomic information and a proliferation of advanced DNA assembly tools. The adoption of ...plasmid vector assembly standards and parts libraries has greatly enhanced the reproducibility of research and the exchange of parts between different labs and biological systems. However, a standardized modular cloning (MoClo) system is not yet available for cyanobacteria, which lag behind other prokaryotes in synthetic biology despite their huge potential regarding biotechnological applications. By building on the assembly library and syntax of the Plant Golden Gate MoClo kit, we have developed a versatile system called CyanoGate that unites cyanobacteria with plant and algal systems. Here, we describe the generation of a suite of parts and acceptor vectors for making (1) marked/unmarked knock-outs or integrations using an integrative acceptor vector, and (2) transient multigene expression and repression systems using known and previously undescribed replicative vectors. We tested and compared the CyanoGate system in the established model cyanobacterium
sp. PCC 6803 and the more recently described fast-growing strain
UTEX 2973. The UTEX 2973 fast-growth phenotype was only evident under specific growth conditions; however, UTEX 2973 accumulated high levels of proteins with strong native or synthetic promoters. The system is publicly available and can be readily expanded to accommodate other standardized MoClo parts to accelerate the development of reliable synthetic biology tools for the cyanobacterial community.
Abstract
Many photosynthetic species have evolved CO2-concentrating mechanisms (CCMs) to improve the efficiency of CO2 assimilation by Rubisco and reduce the negative impacts of photorespiration. ...However, the majority of plants (i.e. C3 plants) lack an active CCM. Thus, engineering a functional heterologous CCM into important C3 crops, such as rice (Oryza sativa) and wheat (Triticum aestivum), has become a key strategic ambition to enhance yield potential. Here, we review recent advances in our understanding of the pyrenoid-based CCM in the model green alga Chlamydomonas reinhardtii and engineering progress in C3 plants. We also discuss recent modeling work that has provided insights into the potential advantages of Rubisco condensation within the pyrenoid and the energetic costs of the Chlamydomonas CCM, which, together, will help to better guide future engineering approaches. Key findings include the potential benefits of Rubisco condensation for carboxylation efficiency and the need for a diffusional barrier around the pyrenoid matrix. We discuss a minimal set of components for the CCM to function and that active bicarbonate import into the chloroplast stroma may not be necessary for a functional pyrenoid-based CCM in planta. Thus, the roadmap for building a pyrenoid-based CCM into plant chloroplasts to enhance the efficiency of photosynthesis now appears clearer with new challenges and opportunities.
Diet norms are the shared social behaviours and beliefs about diets. In many societies, including the UK, these norms are typically linked to unhealthy diets and impede efforts to improve food ...choices. Social interactions that could influence one another's food choices, were highly disrupted during the lockdowns in response to the COVID-19 pandemic. A return to workplaces and re-establishment of eating networks may present an opportunity to influence dietary norms by introducing minimum dietary standards to in workplaces, which could then spread through wider home and workplace networks.
An agent-based model was constructed to simulate a society reflecting the structure of a city population (1000 households) to explore changes in personal and social diet-related norms. The model tracked individual meal choices as agents interact in home, work or school settings and recorded changes in diet quality (range 1 to 100). Scenarios were run to compare individuals' diet quality with the introduction of minimum dietary standards with degrees of working from home.
The more people mixed at work the greater the impact of minimum standards on improving diet norms. Socially isolated households remained unaffected by minimum standards, whereas household members exposed directly, in workplaces or schools, or indirectly, influenced by others in the household, had a large and linear increase in diet quality in relation to minimum standards (0.48 95% CI 0.34, 0.62 per unit increase in minimum standards). Since individuals regressed to the new population mean, a small proportion of diets decreased toward lower population norms. The degree of return to work influenced the rate and magnitude of change cross the population (-2.4 points -2.40, -2.34 in mean diet quality per 20% of workers isolating).
These model results illustrate the qualitative impact social connectivity could have on changing diets through interventions. Norms can be changed more in a more connected population, and social interactions spread norms between contexts and amplified the influence of, for example, workplace minimum standards beyond those directly exposed. However, implementation of minimum standards in a single type of setting would not reach the whole population and in some cases may decrease diet quality. Any non-zero standard could yield improvements beyond the immediate adult workforce and this could spill between social contexts, but would be contingent on population connectivity.
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This study examines the relationship between synovial hypoxia and cellular bioenergetics with synovial inflammation.
Primary rheumatoid arthritis synovial fibroblasts (RASF) were cultured with ...hypoxia, dimethyloxalylglycine (DMOG) or metabolic intermediates. Mitochondrial respiration, mitochondrial DNA mutations, cell invasion, cytokines, glucose and lactate were quantified using specific functional assays. RASF metabolism was assessed by the XF24-Flux Analyzer. Mitochondrial structural morphology was assessed by transmission electron microscopy (TEM). In vivo synovial tissue oxygen (tpO
mmHg) was measured in patients with inflammatory arthritis (n=42) at arthroscopy, and markers of glycolysis/oxidative phosphorylation (glyceraldehyde 3-phosphate dehydrogenase (GAPDH), PKM2, GLUT1, ATP) were quantified by immunohistology. A subgroup of patients underwent contiguous MRI and positron emission tomography (PET)/CT imaging. RASF and human dermal microvascular endothelial cells (HMVEC) migration/angiogenesis, transcriptional activation (HIF1α, pSTAT3, Notch1-IC) and cytokines were examined in the presence of glycolytic inhibitor 3-(3-Pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO).
DMOG significantly increased mtDNA mutations, mitochondrial membrane potential, mitochondrial mass, reactive oxygen species and glycolytic RASF activity with concomitant attenuation of mitochondrial respiration and ATP activity (all p<0.01). This was coupled with altered mitochondrial morphology. Hypoxia-induced lactate levels (p<0.01), which in turn induced basic fibroblast growth factor (bFGF) secretion and RASF invasiveness (all p<0.05). In vivo glycolytic markers were inversely associated with synovial tpO
levels <20 mm Hg, in contrast ATP was significantly reduced (all p<0.05). Decrease in GAPDH and GLUT1 was paralleled by an increase in in vivo tpO
in tumour necrosis factor alpha inhibitor (TNFi) responders. Novel PET/MRI hybrid imaging demonstrated close association between metabolic activity and inflammation. 3PO significantly inhibited RASF invasion/migration, angiogenic tube formation, secretion of proinflammatory mediators (all p<0.05), and activation of HIF1α, pSTAT3 and Notch-1IC under normoxic and hypoxic conditions.
Hypoxia alters cellular bioenergetics by inducing mitochondrial dysfunction and promoting a switch to glycolysis, supporting abnormal angiogenesis, cellular invasion and pannus formation.
Psoriatic arthritis (PsA) is a chronic inflammatory disease, characterised by synovitis and destruction of articular cartilage/bone. Janus-kinase and signal transducer and activator of transcription ...(JAK-STAT) signalling pathway is implicated in the pathogenesis of PsA.
To examine the effect of tofacitinib (JAK inhibitor) on proinflammatory mechanisms in PsA.
Primary PsA synovial fibroblasts (PsAFLS) and ex vivo PsA synovial explants were cultured with tofacitinib (1 µM). PhosphoSTAT3 (pSTAT3), phosphoSTAT1 (pSTAT1), suppressor of cytokine signaling-3 (SOCS3), protein inhibitor of activated Stat3 (PIAS3) and nuclear factor kappa B cells (NFκBp65) were quantified by western blot. The effect of tofacitinib on PsAFLS migration, invasion, Matrigel network formation and matrix metallopeptidase (MMP)2/9 was quantified by invasion/migration assays and zymography. Interleukin (IL)-6, IL-8, IFN-gamma-inducible protein 10 (IP-10) monocyte chemoattractant protein (MCP)-1, IL-17, IL-10, MMP3 and tissue inhibitor of metalloproteinases 3 (TIMP3) were assessed by ELISA.
Tofacitinib significantly decreased pSTAT3, pSTAT1, NFκBp65 and induced SOCS3 and PIAS3 expression in PsAFLS and synovial explant cultures (p<0.05). Functionally, PsAFLS invasion, network formation and migration were inhibited by tofacitinib (all p<0.05). In PsA explant, tofacitinib significantly decreased spontaneous secretion of IL-6, IL-8, MCP-1, MMP9/MMP2, MMP3 (all p<0.05) and decreased the MMP3/TIMP3 ratio (p<0.05), with no effect observed for IP-10 or IL-10.
This study further supports JAK-STAT inhibition as a therapeutic target for the treatment of PsA.
Highly porous metal-organic frameworks (MOFs), which have undergone exciting developments over the past few decades, show promise for a wide range of applications. However, many studies indicate that ...they suffer from significant stability issues, especially with respect to their interactions with water, which severely limits their practical potential. Here we demonstrate how the presence of 'sacrificial' bonds in the coordination environment of its metal centres (referred to as hemilability) endows a dehydrated copper-based MOF with good hydrolytic stability. On exposure to water, in contrast to the indiscriminate breaking of coordination bonds that typically results in structure degradation, it is non-structural weak interactions between the MOF's copper paddlewheel clusters that are broken and the framework recovers its as-synthesized, hydrated structure. This MOF retained its structural integrity even after contact with water for one year, whereas HKUST-1, a compositionally similar material that lacks these sacrificial bonds, loses its crystallinity in less than a day under the same conditions.
Most of Earth's fresh surface water is consolidated in just a few of its largest lakes, and because of their unique response to environmental conditions, lakes have been identified as climate change ...sentinels. While the response of lake surface water temperatures to climate change is well documented from satellite and summer in situ measurements, our understanding of how water temperatures in large lakes are responding at depth is limited, as few large lakes have detailed long-term subsurface observations. We present an analysis of three decades of high frequency (3-hourly and hourly) subsurface water temperature data from Lake Michigan. This unique data set reveals that deep water temperatures are rising in the winter and provides precise measurements of the timing of fall overturn, the point of minimum temperature, and the duration of the winter cooling period. Relationships from the data show a shortened winter season results in higher subsurface temperatures and earlier onset of summer stratification. Shifts in the thermal regimes of large lakes will have profound impacts on the ecosystems of the world's surface freshwater.
Coordination complexes of lanthanide metals with tris-1-naphthylphosphine oxide (Nap3PO, L) have not been previously reported in the literature. We describe here the formation of lanthanide(III) ...nitrate complexes Ln(NO3)3L4 (Ln = Eu to Lu) and the structures of Ln(NO3)3L2·2L (Ln = Eu, Dy, Ho, Er) and L. The core structure of the complexes is an eight-coordinate Ln(NO3)3L2 with the third and fourth ligands H-bonded via their oxygen atoms to one of the naphthyl rings. The structures are compared with those of the analogous complexes of triphenylphosphine oxide and show that the Ln-O(P) bond in the Nap3PO complexes is slightly longer than expected on the basis of differences in coordination numbers. The reaction solutions, investigated by 31P and 13C NMR spectroscopy in CD3CN, show that coordination of L occurs across the lanthanide series, even though complexes can only be isolated from Eu onwards. Analysis of the 31P NMR paramagnetic shifts shows that there is a break in the solution structures with a difference between the lighter lanthanides (La–Eu) and heavier metals (Tb–Lu) which implies a minor difference in structures. The isolated complexes are very poorly soluble, but in CDCl3, NMR measurements show dissociation into Ln(NO3)3L2 and 2L occurs.
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