Objective
To test whether high‐fat diet (HFD) decreases dopaminergic tone in reward regions of the brain and evaluate whether these changes reverse after removal of the HFD.
Design and Methods
Male ...and female mice were fed a 60% HFD for 12 weeks. An additional group was evaluated 4 weeks after removal of the HFD. These groups were compared with control fed, age‐matched controls. Sucrose and saccharin preference was measured along with mRNA expression of dopamine (DA)‐related genes by Real Time‐quantitative PCR (RT‐qPCR). DA and 3,4‐dihydroxyphenylacetic acid (DOPAC) were measured using high‐performance liquid chromatography. DNA methylation of the dopamine transporter (DAT) promoter was measured by methylated DNA immunoprecipitation and RT‐qPCR.
Results
After chronic HFD, sucrose preference was reduced, and then normalized after removal of the HFD. Decreased expression of DA genes, decreased DA content and alterations in DAT promoter methylation, was observed. Importantly, response to HFD and the persistence of changes depended on sex and brain region.
Conclusions
These data identify diminished DA tone after early‐life chronic HFD with a complex pattern of reversal and persistence that varies by both sex and brain region. Central nervous system changes that did not reverse after HFD withdrawal may contribute to the difficulty in maintaining weight‐loss after diet intervention.
Global species richness patterns of soil micro‐organisms remain poorly understood compared to macro‐organisms. We use a global analysis to disentangle the global determinants of diversity and ...community composition for ectomycorrhizal (EcM) fungi—microbial symbionts that play key roles in plant nutrition in most temperate and many tropical forest ecosystems. Host plant family has the strongest effect on the phylogenetic community composition of fungi, whereas temperature and precipitation mostly affect EcM fungal richness that peaks in the temperate and boreal forest biomes, contrasting with latitudinal patterns of macro‐organisms. Tropical ecosystems experience rapid turnover of organic material and have weak soil stratification, suggesting that poor habitat conditions may contribute to the relatively low richness of EcM fungi, and perhaps other soil biota, in most tropical ecosystems. For EcM fungi, greater evolutionary age and larger total area of EcM host vegetation may also contribute to the higher diversity in temperate ecosystems. Our results provide useful biogeographic and ecological hypotheses for explaining the distribution of fungi that remain to be tested by involving next‐generation sequencing techniques and relevant soil metadata.
See also the Perspective by Kennedy et al
To derive childhood-onset SLE (cSLE) specific remission definitions for future treat-to-target (T2T) trials, observational studies, and clinical practice.
The cSLE International T2T Task Force ...conducted Delphi surveys exploring paediatric perspectives on adult-onset SLE remission targets. A modified nominal group technique was used to discuss, refine, and agree on the cSLE remission target criteria.
The Task Force proposed two definitions of remission: ‘cSLE clinical remission on steroids (cCR)’ and ‘cSLE clinical remission off steroids (cCR-0)’. The common criteria are: (1) Clinical-SLEDAI-2 K = 0; (2) PGA score < 0.5 (0–3 scale); (4) stable antimalarials, immunosuppressive, and biologic therapy (changes due to side-effects, adherence, weight, or when building up to target dose allowed). Criterion (3) in cCR is the prednisolone dose ≤0.1 mg/kg/day (maximum 5 mg/day), whereas in cCR-0 it is zero.
cSLE definitions of remission have been proposed, maintaining sufficient alignment with the adult-SLE definition to facilitate life-course research.
•To date, specific remission definitions in cSLE are lacking.•The cSLE International T2T Task Force has derived two cSLE remission definitions.•These include clinical remission on (cCR) and off steroids (cCR-0).•cSLE definitions align with the aSLE definition, facilitating life-course research.•These definitions will facilitate T2T approaches in clinical practice and trials.
Inhibition of HER2 in HER2-amplified breast cancer has been remarkably successful clinically, as demonstrated by the efficacy of HER-kinase inhibitors and HER2-antibody treatments. Whilst resistance ...to HER2 inhibition is common in the metastatic setting, the specific programs downstream of HER2 driving resistance are not established. Through genomic profiling of 733 HER2-amplified breast cancers, we identify enrichment of somatic alterations that promote MEK/ERK signaling in metastatic tumors with shortened progression-free survival on anti-HER2 therapy. These mutations, including NF1 loss and ERBB2 activating mutations, are sufficient to mediate resistance to FDA-approved HER2 kinase inhibitors including tucatinib and neratinib. Moreover, resistant tumors lose AKT dependence while undergoing a dramatic sensitization to MEK/ERK inhibition. Mechanistically, this driver pathway switch is a result of MEK-dependent activation of CDK2 kinase. These results establish genetic activation of MAPK as a recurrent mechanism of anti-HER2 therapy resistance that may be effectively combated with MEK/ERK inhibitors.
Genetic evidence has implicated multiple pathways in eukaryotic DNA mismatch repair (MMR) downstream of mispair recognition and Mlh1-Pms1 recruitment, including Exonuclease 1 (Exo1)-dependent and ...-independent pathways. We identified 14 mutations in POL30, which encodes PCNA in Saccharomyces cerevisiae, specific to Exo1-independent MMR. The mutations identified affected amino acids at three distinct sites on the PCNA structure. Multiple mutant PCNA proteins had defects either in trimerization and Msh2-Msh6 binding or in activation of the Mlh1-Pms1 endonuclease that initiates excision during MMR. The latter class of mutations led to hyperaccumulation of repair intermediate Mlh1-Pms1 foci and were enhanced by an msh6 mutation that disrupted the Msh2-Msh6 interaction with PCNA. These results reveal a central role for PCNA in the Exo1-independent MMR pathway and suggest that Msh2-Msh6 localizes PCNA to repair sites after mispair recognition to activate the Mlh1-Pms1 endonuclease for initiating Exo1-dependent repair or for driving progressive excision in Exo1-independent repair.
Display omitted
•PCNA mutants disrupt Exo1-independent MMR by two mechanisms•Mutants either poorly bind Msh2-Msh6 or poorly activate the Mlh1-Pms1 endonuclease•Activation mutations are enhanced by loss of the interaction with Msh2-Msh6•Msh2-Msh6 promotes excision by localizing PCNA for Mlh1-Pms1 activation
DNA mismatch repair (MMR) involves Exo1-dependent and -independent pathways for excision of the mispair. Using S. cerevisiae, Goellner et al. identified mutations in the gene encoding PCNA specific to the Exo1-independent pathway and show that PCNA binding to Msh2-Msh6 and activation of the Mlh1-Pms1 endonuclease are critical to Exo1-independent MMR.
To examine whether free fatty acid receptor 4 (FFAR4) activation can protect against choroidal neovascularization (CNV), which is a common cause of blindness, and to elucidate the mechanism ...underlying the inhibition, we used the mouse model of laser-induced CNV to mimic angiogenic aspects of age-related macular degeneration (AMD). Laser-induced CNV was compared between groups treated with an FFAR4 agonist or vehicle, and between FFAR4 wild-type (
Ffar4
+
/
+
) and knock out (
Ffar4
−/−
) mice on a C57BL/6J/6N background. The ex vivo choroid-sprouting assay, including primary retinal pigment epithelium (RPE) and choroid, without retina was used to investigate whether FFAR4 affects choroidal angiogenesis. Western blotting for pNF-ĸB/NF-ĸB and qRT-PCR for
Il-6, Il-1β, Tnf-α, Vegf, and Nf-ĸb
were used to examine the influence of FFAR4 on inflammation, known to influence CNV. RPE isolated from
Ffar4
+
/
+
and
Ffar4
−/−
mice were used to assess RPE contribution to inflammation. The FFAR4 agonist suppressed laser-induced CNV in C57BL/6J mice, and CNV increased in
Ffar4
−/−
compared to
Ffar4
+
/
+
mice. We showed that the FFAR4 agonist acted through the FFAR4 receptor. The FFAR4 agonist suppressed mRNA expression of inflammation markers (
Il-6, Il-1β
) via the NF-ĸB pathway in the retina, choroid, RPE complex. The FFAR4 agonist suppressed neovascularization in the choroid-sprouting ex vivo assay and FFAR4 deficiency exacerbated sprouting. Inflammation markers were increased in primary RPE cells of
Ffar4
−/−
mice compared with
Ffar4
+
/
+
RPE. In this mouse model, the FFAR4 agonist suppressed CNV, suggesting FFAR4 to be a new molecular target to reduce pathological angiogenesis in CNV.
The complex pathology of disease has sparked the development of novel protein expression profiling techniques that require validation in clinical settings. This study focuses on multiplexed analyses ...of adipocytokines and biomarkers linked to the metabolic syndrome, diabetes, and cardiovascular disease.
Multiplexed immunoassays using fluorescent microspheres and the Luminex-100 system were performed on plasma from 80 obese patients (40 with the metabolic syndrome) before and after 6-8 weeks of diet-induced weight loss. Leptin, insulin, C-peptide, monocyte chemoattractant protein-1 (MCP-1), eotaxin, interleukin-8 (IL-8), tumor necrosis factor-alpha (TNF-alpha), and IL-6 concentrations measured with multiplex panels from 3 different manufacturers were compared with results from commercial ELISAs. Detection limits and between- and within-run imprecision were determined for each analyte. Bland-Altman analysis was used to determine agreement between multiplexed immunoassays and ELISAs.
Correlation between the Luminex multiplexed assays and ELISAs was good for leptin (Linco), insulin (Linco), MCP-1 (Biosource and Upstate), and eotaxin (Biosource) with correlation coefficients of 0.711-0.895; fair for eotaxin (Upstate) and C-peptide (Linco) with correlation coefficients of 0.496-0.582; and poor for TNF-alpha, IL-8, and IL-6 (Linco, Biosource, Upstate, and R&D) with correlation coefficients of -0.107 to 0.318. Within- and between-run imprecision values for the multiplex method were generally <15%. Relative changes in plasma leptin and insulin concentrations after diet-induced weight loss were similar whether assessed by multiplex assay or ELISA.
Although this technology appears useful in clinical research studies, low assay sensitivity and poor correlations with conventional ELISA methods for some analytes with very low plasma concentrations should be considered when using the Luminex platform in clinical studies.
There is increasing evidence that proton-coupled oligopeptide transporters (POTs) can transport bacterially derived chemotactic peptides and therefore reside at the critical interface of innate ...immune responses and regulation. However, there is substantial contention regarding how these bacterial peptides access the cytosol to exert their effects and which POTs are involved in facilitating this process. Thus, the current study proposed to determine the (sub)cellular expression and functional activity of POTs in macrophages derived from mouse bone marrow and to evaluate the effect of specific POT deletion on the production of inflammatory cytokines in wild-type,
knockout and
knockout mice. We found that PEPT2 and PHT1 were highly expressed and functionally active in mouse macrophages, but PEPT1 was absent. The fluorescent imaging of muramyl dipeptide-rhodamine clearly demonstrated that PEPT2 was expressed on the plasma membrane of macrophages, whereas PHT1 was expressed on endosomal membranes. Moreover, both transporters could significantly influence the effect of bacterially derived peptide ligands on cytokine stimulation, as shown by the reduced responses in
knockout and
knockout mice as compared with wild-type animals. Taken as a whole, our results point to PEPT2 (at plasma membranes) and PHT1 (at endosomal membranes) working in concert to optimize the uptake of bacterial ligands into the cytosol of macrophages, thereby enhancing the production of proinflammatory cytokines. This new paradigm offers significant insight into potential drug development strategies along with transporter-targeted therapies for endocrine, inflammatory, and autoimmune diseases.
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