Complex patterns of dopamine‐related gene expression in the ventral tegmental area of male zebra finches relate to dyadic interactions with long‐term female partners

Dopaminergic projections from the ventral tegmental area (VTA) to multiple efferent targets are implicated in pair bonding, yet the role of the VTA in the maintenance of long‐term pair bonds is not well characterized. Complex interactions between numerous neuromodulators modify activity in the VTA, suggesting that individual differences in patterns of gene expression in this region may explain individual differences in long‐term social interactions in bonded pairs. To test this hypothesis we used RNA‐seq to measure expression of over 8000 annotated genes in male zebra finches in established male‐female pairs. Weighted gene co‐expression network analysis identified a gene module that contained numerous dopamine‐related genes with TH found to be the most connected gene of the module. Genes in this module related to male agonistic behaviors as well as bonding‐related behaviors produced by female partners. Unsupervised learning approaches identified two groups of males that differed with respect to expression of numerous genes. Enrichment analyses showed that many dopamine‐related genes and modulators differed between these groups, including dopamine receptors, synthetic and degradative enzymes, the avian dopamine transporter and several GABA‐ and glutamate‐related genes. Many of the bonding‐related behaviors closely associated with VTA gene expression in the two male groups were produced by the male's partner, rather than the male himself. Collectively, results highlight numerous candidate genes in the VTA that can be explored in future studies and raise the possibility that the molecular/genetic organization of the VTA may be strongly shaped by a social partner and/or the strength of the pair bond.     

Metabolic phenotyping to monitor chronic enteritis canceration

Introduction

Untargeted metabolomics intends to objectively analyze a wide variety of compounds. Their diverse physicochemical properties make it difficult to choose an appropriate reconstitution solvent after sample evaporation without influencing the chromatography or hamper column sorbent integrity.

Objectives

The study aimed to identify the most appropriate reconstitution solvent for blood plasma samples in terms of feature recovery, four endogenous compounds, and one selected internal standard.

Methods

We investigated several reconstitution solvent mixtures containing acetonitrile and methanol to resolve human plasma extract and evaluated them concerning the peak areas of tryptophan-d₅, glucose, creatinine, palmitic acid, and the phophatidylcholine PC(P-16:0/P-16:0), as well as the total feature count

Results

Results indicated that acetonitrile containing 30% methanol was best suited to match all tested criteria at least for human blood plasma samples.

Conclusion

Despite identifying the mixture of acetonitrile and methanol being suitable as solvent for human blood plasma extracts, we recommend to systematically test for an appropriate reconstitution solvent for each analyzed biomatrix.

     

Kruppel‐like factor 4 improves obesity‐related nephropathy through increasing mitochondrial biogenesis and activities

Obesity is positively linked to multiple metabolic complications including renal diseases. Several studies have demonstrated Kruppel‐like factor 4 (KLF4) participated in renal dysfunction and structural disorders in acute kidney injuries, but whether it affected the process of chronic kidney diseases was unknown. Therefore, present study was to disclose the role of renal KLF4 in dietary‐induced renal injuries and underlying mechanisms in obesity. Through utilizing high‐fat diet‐fed mice and human renal biopsies, we provided the physiological roles of KLF4 in protecting against obesity‐related nephropathy. Decreased levels of renal KLF4 were positively correlated with dietary‐induced renal dysfunction, including increased levels of creatinine and blood urea nitrogen. Overexpression of renal KLF4 suppressed inflammatory response in palmitic acid‐treated mouse endothelial cells. Furthermore, overexpressed KLF4 also attenuated dietary‐induced renal functional disorders, abnormal structural remodelling and inflammation. Mechanistically, KLF4 maintained renal mitochondrial biogenesis and activities to combat obesity‐induced mitochondrial dysfunction. In clinical renal biopsies and plasma, the renal Klf4 level was negatively associated with circulating levels of creatinine but positively associated with renal creatinine clearance. In conclusions, the present findings firstly supported that renal KLF4 played an important role in combating obesity‐related nephropathy, and KLF4/mitochondrial function partially determined the energy homeostasis in chronic kidney diseases.     

A stroma-related lncRNA panel for predicting recurrence and adjuvant chemotherapy benefit in patients with early-stage colon cancer

The heterogeneity in prognoses and chemotherapeutic responses of colon cancer patients with similar clinical features emphasized the necessity for new biomarkers that help to improve the survival prediction and tailor therapies more rationally and precisely. In the present study, we established a s troma-related l ncRNA s ignature (SLS) based on 52 lncRNAs to comprehensively predict clinical outcome. The SLS model could not only distinguish patients with different recurrence and mortality risks through univariate analysis, but also served as an independent factor for relapse-free and overall survival. Compared with the conventionally used TNM stage system, the SLS model clearly possessed higher predictive accuracy. Moreover, the SLS model also effectively screened chemotherapy-responsive patients, as only patients in the low-SLS group could benefit from adjuvant chemotherapy. The following cell infiltration and competing endogenous RNA (ceRNA) network functional analyses further confirmed the association between the SLS model and stromal activation-related biological processes. Additionally, this study also identified three phenotypically distinct colon cancer subtypes that varied in clinical outcome and chemotherapy benefits. In conclusion, our SLS model may be a significant determinant of survival and chemotherapeutic decision-making in colon cancer and may have a strong clinical transformation value.     

Metformin attenuates trauma‐induced heterotopic ossification via inhibition of Bone Morphogenetic Protein signalling

AMP‐activated protein kinase (AMPK) is an intracellular sensor of energy homoeostasis that is activated under energy stress and suppressed in energy surplus. AMPK activation leads to inhibition of anabolic processes that consume ATP. Osteogenic differentiation is a process that highly demands ATP during which AMPK is inhibited. The bone morphogenetic proteins (BMPs) signalling pathway plays an essential role in osteogenic differentiation. The present study examines the inhibitory effect of metformin on BMP signalling, osteogenic differentiation and trauma‐induced heterotopic ossification. Our results showed that metformin inhibited Smad1/5 phosphorylation induced by BMP6 in osteoblast MC3T3‐E1 cells, concurrent with up‐regulation of Smad6, and this effect was attenuated by knockdown of Smad6. Furthermore, we found that metformin suppressed ALP activity and mineralization of the cells, an event that was attenuated by the dominant negative mutant of AMPK and mimicked by its constitutively active mutant. Finally, administration of metformin prevented the trauma‐induced heterotopic ossification in mice. In conjuncture, AMPK activity and Smad6 and Smurf1 expression were enhanced by metformin treatment in the muscle of injured area, concurrently with the reduction of ALK2. Collectively, our study suggests that metformin prevents heterotopic ossification via activation of AMPK and subsequent up‐regulation of Smad6. Therefore, metformin could be a potential therapeutic drug for heterotopic ossification induced by traumatic injury.     

Extracellular vesicle‐mediated crosstalk between NPCE cells and TM cells result in modulation of Wnt signalling pathway and ECM remodelling

Primary open‐angle glaucoma is a leading cause of irreversible blindness, often associated with increased intraocular pressure. Extracellular vesicles (EVs) carry a specific composition of proteins, lipids and nucleotides have been considered as essential mediators of cell‐cell communication. Their potential impact for crosstalk between tissues responsible for aqueous humour production and out‐flow is largely unknown. The study objective was to investigate the effects of EVs derived from non‐pigmented ciliary epithelium (NPCE) primary cells on the expression of Wnt proteins in a human primary trabecular meshwork (TM) cells and define the mechanism underlying exosome‐mediated regulation that signalling pathway. Consistent with the results in TM cell line, EVs released by both primary NPCE cells and NPCE cell line showed diminished pGSK3β phosphorylation and decreased cytosolic levels of β‐catenin in primary TM cells. At the molecular level, we showed that NPCE exosome treatment downregulated the expression of positive GSKβ regulator‐AKT protein but increased the levels of GSKβ negative regulator‐PP2A protein in TM cells. NPCE exosome protein analysis revealed 584 miRNAs and 182 proteins involved in the regulation of TM cellular processes, including WNT/β‐catenin signalling pathway, cell adhesion and extracellular matrix deposition. We found that negative modulator of Wnt signalling miR‐29b was abundant in NPCE exosomal samples and treatment of TM cells with NPCE EVs significantly decreased COL3A1 expression. Suggesting that miR‐29b can be responsible for decreased levels of WNT/β‐catenin pathway. Overall, this study highlights a potential role of EVs derived from NPCE cells in modulating ECM proteins and TM canonical Wnt signalling.     

Mesenchymal stromal cell‐derived factors promote the colonization of collagen 3D scaffolds with human skin cells

The development of stem cell technology in combination with advances in biomaterials has opened new ways of producing engineered tissue substitutes. In this study, we investigated whether the therapeutic potential of an acellular porous scaffold made of type I collagen can be improved by the addition of a powerful trophic agent in the form of mesenchymal stromal cells conditioned medium (MSC‐CM) in order to be used as an acellular scaffold for skin wound healing treatment. Our experiments showed that MSC‐CM sustained the adherence of keratinocytes and fibroblasts as well as the proliferation of keratinocytes. Moreover, MSC‐CM had chemoattractant properties for keratinocytes and endothelial cells, attributable to the content of trophic and pro‐angiogenic factors. Also, for the dermal fibroblasts cultured on collagen scaffold in the presence of MSC‐CM versus serum control, the ratio between collagen III and I mRNAs increased by 2‐fold. Furthermore, the gene expression for α‐smooth muscle actin, tissue inhibitor of metalloproteinase‐1 and 2 and matrix metalloproteinase‐14 was significantly increased by approximately 2‐fold. In conclusion, factors existing in MSC‐CM improve the colonization of collagen 3D scaffolds, by sustaining the adherence and proliferation of keratinocytes and by inducing a pro‐healing phenotype in fibroblasts.     

Aldosterone enhances high phosphate–induced vascular calcification through inhibition of AMPK‐mediated autophagy

It remains unclear whether the necessity of calcified mellitus induced by high inorganic phosphate (Pi) is required and the roles of autophagy plays in aldosterone (Aldo)‐enhanced vascular calcification (VC) and vascular smooth muscle cell (VSMC) osteogenic differentiation. In the present study, we found that Aldo enhanced VC both in vivo and in vitro only in the presence of high Pi, alongside with increased expression of VSMC osteogenic proteins (BMP2, Runx2 and OCN) and decreased expression of VSMC contractile proteins (α‐SMA, SM22α and smoothelin). However, these effects were blocked by mineralocorticoid receptor inhibitor, spironolactone. In addition, the stimulatory effects of Aldo on VSMC calcification were further accelerated by the autophagy inhibitor, 3‐MA, and were counteracted by the autophagy inducer, rapamycin. Moreover, inhibiting adenosine monophosphate‐activated protein kinase (AMPK) by Compound C attenuated Aldo/MR‐enhanced VC. These results suggested that Aldo facilitates high Pi‐induced VSMC osteogenic phenotypic switch and calcification through MR‐mediated signalling pathways that involve AMPK‐dependent autophagy, which provided new insights into Aldo excess‐associated VC in various settings.     

Emperipolesis mediated by CD8+ T cells correlates with biliary epithelia cell injury in primary biliary cholangitis

Primary biliary cholangitis (PBC) is an autoimmune disease characterized by chronic destruction of the bile ducts. A major unanswered question regarding the pathogenesis of PBC is the precise mechanisms of small bile duct injury. Emperipolesis is one of cell‐in‐cell structures that is a potential histological hallmark associated with chronic hepatitis B. This study aimed to clarify the pathogenesis and characteristics of emperipolesis in PBC liver injury. Sixty‐six PBC patients, diagnosed by liver biopsy combined with laboratory test, were divided into early‐stage PBC (stages I and II, n = 39) and late‐stage PBC (stages III and IV, n = 27). Emperipolesis was measured in liver sections stained with haematoxylin‐eosin. The expressions of CK19, CD3, CD4, CD8, CD20, Ki67 and apoptosis of BECs were evaluated by immunohistochemistry or immunofluorescence double labelling. Emperipolesis was observed in 62.1% of patients with PBC, and BECs were predominantly host cells. The number of infiltrating CD3⁺ and CD8⁺ T cells correlated with the advancement of emperipolesis (R² = 0.318, P < .001; R² = 0.060, P < .05). The cell numbers of TUNEL‐positive BECs and double staining for CK19 and Ki67 showed a significant positive correlation with emperipolesis degree (R² = 0.236, P < .001; R² = 0.267, P < .001). We conclude that emperipolesis mediated by CD8⁺ T cells appears to be relevant to apoptosis of BEC and thus may aggravate the further injury of interlobular bile ducts.     

Novel mutations in ADAMTS13 CUB domains cause abnormal pre-mRNA splicing and defective secretion of ADAMTS13

Hereditary thrombotic thrombocytopenic purpura (TTP) is an autosomal recessive thrombosis disorder, caused by loss-of-function mutations in ADAMTS13. Mutations in the CUB domains of ADAMTS13 are rare, and the exact mechanisms through which these mutations result in the development of TTP have not yet been fully elucidated. In this study, we identified two novel mutations in the CUB domains in a TTP family with an acceptor splice-site mutation (c.3569−1, G>A, intron 25) and a point missense mutation (c.3923, G>A, exon 28), resulting in a glycine to aspartic acid substitution (p.G1308D). In vitro splicing analysis revealed that the intronic mutation resulted in abnormal pre-mRNA splicing, and an in vitro expression assay revealed that the missense mutation significantly impaired ADAMTS13 secretion. Although both the patient and her brother displayed significantly reduced ADAMTS13 activity and increased levels of ultra-large VWF (ULVWF) multimers in plasma, only the female developed acute episodes of TTP. Our findings indicate the importance of the CUB domains for the protein stability and extracellular secretion of ADAMTS13.