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Background and objectives: C3 glomerulopathy and idiopathic Ig-associated membranoproliferative GN are kidney diseases characterized by abnormal glomerular complement C3 deposition. These conditions are heterogeneous in outcome, but approximately 50% of patients develop kidney failure within 10 years. Design, setting, participants, & measurements: To improve identification of patients with poor prognosis, we performed a detailed analysis of percutaneous kidney biopsies in a large cohort of patients. Using a validated histologic scoring system, we analyzed 156 native diagnostic kidney biopsies from a retrospective cohort of 123patients with C3 glomerulopathy and 33 patients with Ig-associated membranoproliferative GN. We used linear regression, survival analysis, and Cox proportional hazards models to assess the relationship between histologic and clinical parameters with outcome. Results: Frequent biopsy features were mesangial expansion and hypercellularity, glomerular basement membrane double contours, and endocapillary hypercellularity. Multivariable analysis showed negative associations between eGFR and crescents, interstitial inflammation, and interstitialfibrosis/tubular atrophy. Proteinuria positively associated with endocapillary hypercellularity and glomerular basement membrane double contours. Analysis of second native biopsies did not demonstrate associations between immunosuppression treatment and improvement in histology. Using a composite outcome, risk of progression to kidney failure associated with eGFR and proteinuria at the time of biopsy, cellular/fibrocellular crescents, segmental sclerosis, and interstitial fibrosis/tubular atrophy scores. Conclusions: Our detailed assessment of kidney biopsy data indicated that cellular/fibrocellular crescents and interstitial fibrosis/tubular atrophy scores were significant determinants of deterioration in kidney function.

AIM Hyperphosphataemia is associated with increased adverse outcomes, including mortality. Re-examining this association using up-to-date data reflecting current and real-world practices, across different global regions and in both haemodialysis and peritoneal dialysis patients, is important. METHODS We describe the association between serum phosphate and all-cause and cardiovascular mortality in incident dialysis patients between 2008 and 2018 using the Australia and New Zealand Dialysis and Transplant (ANZDATA) Registry. Time-dependent Cox proportionate hazards models were used. Models were adjusted for available covariates and fitted for the overall cohort, and also each dialysis modality. RESULTS 31 989 patients were followed over 97 122 person-years at risk (mean age at first dialysis 61 years, 38% female, 67% haemodialysis). We observed a U-shaped association between serum phosphate and all-cause mortality. In the fully adjusted model, categories of serum phosphate above and below 1.25-1.99 mmol/L were associated with progressively higher risk, reaching a hazard ratio of 2.13 (95% CI 1.93-2.36, p

Skeletal muscle regenerates through the activation of resident stem cells. Termed satellite cells, these normally quiescent cells are induced to proliferate by wound-derived signals1. Identifying the source and nature of these cues has been hampered by an inability to visualize the complex cell interactions that occur within the wound. Here we use muscle injury models in zebrafish to systematically capture the interactions between satellite cells and the innate immune system after injury, in real time, throughout the repair process. This analysis revealed that a specific subset of macrophages 'dwell' within the injury, establishing a transient but obligate niche for stem cell proliferation. Single-cell profiling identified proliferative signals that are secreted by dwelling macrophages, which include the cytokine nicotinamide phosphoribosyltransferase (Nampt, which is also known as visfatin or PBEF in humans). Nampt secretion from the macrophage niche is required for muscle regeneration, acting through the C-C motif chemokine receptor type 5 (Ccr5), which is expressed on muscle stem cells. This analysis shows that in addition to their ability to modulate the immune response, specific macrophage populations also provide a transient stem-cell-activating niche, directly supplying proliferation-inducing cues that govern the repair process that is mediated by muscle stem cells. This study demonstrates that macrophage-derived niche signals for muscle stem cells, such as NAMPT, can be applied as new therapeutic modalities for skeletal muscle injury and disease.

There is increasing global incidence of acute kidney injury (AKI) and significant short- and long-term impacts on patients.To determine incidence and outcomes of community-acquired AKI (CA-AKI) and hospital-acquired AKI (HA-AKI) among inpatients in the Australian healthcare setting using modern health information systems.A retrospective cohort study of adult patients admitted to a quaternary hospital in Melbourne, Australia, between 1 January 2018 and 31 December 2019 utilising an electronic data warehouse. Participants included adult patients admitted for24 h who had more than one serum creatinine level recorded during admission. Kidney transplant and maintenance dialysis patients were excluded. Main outcomes measured included AKI, as classified by the Kidney Disease Improving Global Outcomes (KDIGO) criteria, hospital length of stay and 30-day mortality.A total of 6477 AKI episodes was identified across 43 791 admissions. Of all AKI episodes, 77% (n = 5011), 15% (n = 947) and 8% (n = 519) were KDIGO stage 1, 2 and 3 respectively. HA-AKI accounted for 55.9% episodes. Patients required intensive care unit admission in 22.7% (n = 1100) of CA-AKI and 19.3% (n = 935) of HA-AKI, compared with 7.5% (n = 2815) of patients with no AKI (P = 0.001). Patients with AKI were older with more co-morbidities, particularly chronic kidney disease (CKD). Length of stay was longer in CA-AKI (8.8 days) and HA-AKI (11.8 days) compared with admissions without AKI (4.9 days; P 0.001). Thirty-day mortality was increased with CA-AKI (10.2%) and HA-AKI (12.8%) compared with no AKI (3.7%; P 0.001).The incidence of AKI detected by the electronic data warehouse was higher than previously reported. Patients who experienced AKI had greater morbidity and mortality. CKD was an important risk factor for AKI in hospitalised patients.

There is a growing appreciation of the role of lung stem/progenitor cells in the development and perpetuation of chronic lung disease including idiopathic pulmonary fibrosis. Human amniotic epithelial cells (hAECs) were previously shown to improve lung architecture in bleomycin-induced lung injury, with the further suggestion that hAECs obtained from term pregnancies possessed superior anti-fibrotic properties compared with their preterm counterparts. In the present study, we aimed to elucidate the differential effects of hAECs from term and preterm pregnancies on lung stem/progenitor cells involved in the repair. Here we showed that term hAECs were better able to activate bronchioalveolar stem cells (BASCs) and type 2 alveolar epithelial cells (AT2s) compared with preterm hAECs following bleomycin challenge. Further, we observed that term hAECs restored TGIF1 and TGFβ2 expression levels, while increasing c-MYC expression despite an absence of significant changes to Wnt/β-catenin signaling. In vitro, term hAECs increased the average size and numbers of BASC and AT2 colonies. The gene expression levels of Wnt ligands were higher in term hAECs, and the expression levels of BMP4, CCND1 and CDC42 were only increased in the BASC and AT2 organoids co-cultured with hAECs from term pregnancies but not preterm pregnancies. In conclusion, term hAECs were more efficient at activating the BASC niche compared with preterm hAECs. The impact of gestational age and/or complications leading to preterm delivery should be considered when applying hAECs and other gestational tissue-derived stem and stem-like cells therapeutically.

Plasma approaches metastability with respect to its calcium and phosphate content, with only minor perturbations in ionic activity needed to sustain crystal growth once nucleated. Physiologically, calcium and phosphate are intermittently absorbed from the diet each day, yet plasma concentrations of these ions deviate minimally post-prandially. This implies the existence of a blood-borne mineral buffer system to sequester calcium phosphates and minimise the risk of deposition in the soft tissues. Calciprotein particles (CPP), endogenous mineral-protein colloids containing the plasma protein fetuin-A, may fulfill this function but definitive evidence linking dietary mineral loading with their formation is lacking. Here we demonstrate that CPP are formed as a normal physiological response to feeding in healthy adults and that this occurs despite minimal change in conventional serum mineral markers. Further, in individuals with Chronic Kidney Disease (CKD), in whom mineral handling is impaired, we show that both fasting and post-prandial levels of CPP precursors are markedly augmented and strongly inversely correlated with kidney function. This study highlights the important, but often neglected, contribution of colloidal biochemistry to mineral homeostasis and provides novel insight into the dysregulation of mineral metabolism in CKD.

Excess sodium is a major modifiable contributor to hypertension and cardiovascular risk. Knowledge of sodium storage and metabolism has derived mainly from indirect measurements of dietary sodium intake and urinary sodium excretion, however both attempt to measure body sodium and fluid in a two-compartment model of intracellular and extracellular spaces. Our understanding of total body sodium has recently included a storage pool in tissues. In the last two decades, sodium-23 magnetic resonance imaging (23 Na MRI) has allowed dynamic quantification of tissue sodium in vivo. Tissue sodium is independently associated with cardiovascular dysfunction and inflammation. This review explores (i) The revolution of our understanding of sodium physiology, (ii) The development and potential clinical adoption of 23 Na MRI to provide improved measurement of total body sodium in CKD and (iii) How we can better understand mechanistic and clinical implications of tissue sodium in hypertension, cardiovascular disease and immune dysregulation, especially in the CKD population.

Excess sodium intake is a leading but modifiable risk factor for mortality, with implications on hypertension, inflammation, cardiovascular disease, and chronic kidney disease (CKD). This review will focus mainly on the limitations of current measurement methods of sodium balance particularly in patients with CKD who have complex sodium physiology. The suboptimal accuracy of sodium intake and excretion measurement is seemingly more marked with the evolving understanding of tissue (skin and muscle) sodium. Tissue sodium represents an extrarenal influence on sodium homeostasis with demonstrated clinical associations of hypertension and inflammation. Measurement of tissue sodium has been largely unexplored in patients with CKD. Development and adoption of more comprehensive and dynamic assessment of body sodium balance is needed to better understand sodium physiology in the human body and explore therapeutic strategies to improve the clinical outcomes in the CKD population.

DNA mismatch repair (MMR) deficiency has been associated with a higher risk of developing colorectal, endometrial, and ovarian cancer, and confers resistance in conventional chemotherapy. In addition to the lack of treatment options that work efficaciously on these MMR-deficient cancer patients, there is a great need to discover new drug leads for this purpose. In this study, we screened through a library of commercial and semisynthetic natural compounds to identify potential synthetic lethal drugs that may selectively target MLH1 mutants using MLH1 isogenic colorectal cancer cell lines and various cancer cell lines with known MLH1 status. We identified a novel diarylpentanoid analogue, 2-benzoyl-6-(2,3-dimethoxybenzylidene)-cyclohexenol, coded as AS13, that demonstrated selective toxicity toward MLH1-deficient cancer cells. Subsequent analysis suggested AS13 induced elevated levels of oxidative stress, resulting in DNA damage where only the proficient MLH1 cells were able to be repaired and hence escaping cellular death. While AS13 is modest in potency and selectivity, this discovery has the potential to lead to further drug development that may offer better treatment options for cancer patients with MLH1 deficiency.

Lymphangiogenesis, the formation of new lymphatic vessels from pre-existing vasculature, plays critical roles in disease, including in cancer metastasis and chronic inflammation. Preclinical and recent clinical studies have now demonstrated therapeutic utility for several anti-lymphangiogenic agents, but optimal agents and efficacy in different settings remain to be determined. We tested the anti-lymphangiogenic property of 3,4-Difluorobenzocurcumin (CDF), which has previously been implicated as an anti-cancer agent, using zebrafish embryos and cultured vascular endothelial cells. We used transgenic zebrafish labelling the lymphatic system and found that CDF potently inhibits lymphangiogenesis during embryonic development. We also found that the parent compound, Curcumin, does not inhibit lymphangiogenesis. CDF blocked lymphatic and venous sprouting, and lymphatic migration in the head and trunk of the embryo. Mechanistically, CDF impaired VEGFC-VEGFR3-ERK signalling in vitro and in vivo. In an in vivo pathological model of Vegfc-overexpression, treatment with CDF rescued endothelial cell hyperplasia. CDF did not inhibit the kinase activity of VEGFR3 yet displayed more prolonged activity in vivo than previously reported kinase inhibitors. These findings warrant further assessment of CDF and its mode of action as a candidate for use in metastasis and diseases of aberrant lymphangiogenesis.

Chronic wounds are a major clinical problem where wound closure is prevented by pathologic factors, including immune dysregulation. To design efficient immunotherapies, an understanding of the key molecular pathways by which immunity impairs wound healing is needed. Interleukin-1 (IL-1) plays a central role in regulating the immune response to tissue injury through IL-1 receptor (IL-1R1). Generating a knockout mouse model, we demonstrate that the IL-1–IL-1R1 axis delays wound closure in diabetic conditions. We used a protein engineering approach to deliver IL-1 receptor antagonist (IL-1Ra) in a localised and sustained manner through binding extracellular matrix components. We demonstrate that matrix-binding IL-1Ra improves wound healing in diabetic mice by re-establishing a pro-healing microenvironment characterised by lower levels of pro-inflammatory cells, cytokines and senescent fibroblasts, and higher levels of anti-inflammatory cytokines and growth factors. Engineered IL-1Ra has translational potential for chronic wounds and other inflammatory conditions where IL-1R1 signalling should be dampened., Tan, Lash et al. show that blocking the Interleukin-1 receptor promotes rapid wound closure in diabetic mice. They engineered a matrix binding form of Interleukin-1 receptor antagonist (IL-1Ra) that has the ability of precise localisation and retention in the tissue. IL-1Ra holds the potential for expediting wound healing in diabetic patients.

There is ample support for developmental regulation of glioblastoma stem cells (GSCs). To examine how cell lineage controls GSC function we have performed a cross-species epigenome analysis of mouse and human GSC cultures. We have analyzed and compared the chromatin-accessibility landscape of nine mouse GSC cultures of defined cell of origin and 60 patient-derived GSC cultures by assay for transposase-accessible chromatin using sequencing (ATAC-seq). This uncovered a variability of both mouse and human GSC cultures that was different from transcriptome analysis and better at predicting functional subgroups. In both species the chromatin accessibility-guided clusters were predominantly determined by distal regulatory element (DRE) regions, displayed contrasting sets of transcription factor binding motifs, and exhibited different functional and drug-response properties. Cross-species analysis of DRE regions in accessible chromatin revealed conserved epigenetic regulation of mouse and human GSCs. Human ATAC-seq data produced three distinct clusters with significant overlap to our previous mouse cell of origin- based stratification, where two of the clusters displayed significantly different patient survival. We conclude that epigenetic regulation of GSCs primarily is dictated by developmental origin which controls key GSC properties and affects therapeutic response.

Glioblastoma (GBM) is the most common and lethal primary malignant brain tumor which lacks efficient treatment and predictive biomarkers. Expression of the epithelial stem cell marker Leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5) has been described in GBM, but its functional role has not been conclusively elucidated. Here, we have investigated the role of LGR5 in a large repository of patient-derived GBM stem cell (GSC) cultures. The consequences of LGR5 overexpression or depletion have been analyzed using in vitro and in vivo methods, which showed that, among those with highest LGR5 expression (LGR5high ), there were two phenotypically distinct groups: one that was dependent on LGR5 for its malignant properties and another that was unaffected by changes in LGR5 expression. The LGR5-responding cultures could be identified by their significantly higher self-renewal capacity as measured by extreme limiting dilution assay (ELDA), and these LGR5high -ELDAhigh cultures were also significantly more malignant and invasive compared to the LGR5high -ELDAlow cultures. This showed that LGR5 expression alone would not be a strict marker of LGR5 responsiveness. In a search for additional biomarkers, we identified LPAR4, CCND2, and OLIG2 that were significantly upregulated in LGR5-responsive GSC cultures, and we found that OLIG2 together with LGR5 were predictive of GSC radiation and drug response. Overall, we show that LGR5 regulates the malignant phenotype in a subset of patient-derived GSC cultures, which supports its potential as a predictive GBM biomarker. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Natural products are prolific producers of diverse chemical scaffolds, which have yielded several clinically useful drugs. However, the complex features of natural products present challenges for identifying bioactive molecules using high-throughput screens. For most assays, measured endpoints are either colorimetric or luminescence based. Thus, the presence of the major metabolites, tannins, and chlorophylls, in natural products could potentially interfere with these measurements to give either false-positive or false-negative hits. In this context, zebrafish phenotypic assays provide an alternative approach to bioprospect naturally occurring bioactive compounds. Whether tannins and/or chlorophylls interfere in zebrafish phenotypic assays, is unclear. In this study, we evaluated the interference potential of tannins and chlorophylls against efficacy of known small-molecule inhibitors that are known to cause phenotypic abnormalities in developing zebrafish embryos. First, we fractionated tannin-enriched fraction (TEF) and chlorophyll-enriched fraction (CEF) from Camellia sinensis and cotreated them with PD0325901 [mitogen-activated protein kinase-kinase (MEK) inhibitor] and sunitinib malate (SM; anti-[lymph]angiogenic drug). While TEF and CEF did not interfere with phenotypic or molecular endpoints of PD0325901, TEF at 100 μg/mL partially masked the antiangiogenic effect of SM. On the other hand, CEF (100 μg/mL) was toxic when treated up to 6 dpf. Furthermore, CEF at 100 μg/mL potentially enhanced the activity of γ-secretase inhibitors, resulting in toxicity of treated embryos. Our study provides evidence that the presence of tannin and/or chlorophyll in natural products do interfere with zebrafish phenotype assays used for identifying potential hits. However, this may be target/assay dependent and thus requiring additional optimization steps to assess interference potential of tannins and chlorophylls before performing any screening assay.

Bronchopulmonary dysplasia (BPD) is a chronic lung disease that mainly affects premature babies who require ventilator support. The pathogenesis of BPD is complex but includes vascular maldevelopment, alveolarization arrest, and lung inflammation. There is no cure for BPD. Clinical care is limited to supportive respiratory measures. A population of stem-like cells derived from placental membranes, human amnion epithelial cells (hAECs), has shown therapeutic promise in preclinical models of BPD. With a view to future efficacy trials, we undertook a first-in-human clinical trial of hAECs in babies with BPD to assess the safety of these cells. In a single-center, open-label phase I trial, we administered allogeneic hAECs (1 × 106 per kilogram bodyweight) by intravenous infusion to six premature babies with BPD. The primary outcomes of the study were focused on safety, including local site reaction, anaphylaxis, infection, features of rejection, or tumor formation. Outcomes to discharge from neonatal unit were studied. The hAECs were well tolerated. In the first baby, there was transient cardiorespiratory compromise during cell administration consistent with a pulmonary embolic event. Following changes to cell administration methods, including introduction of an inline filter, and reducing the cell concentration and the rate of cell infusion, no such events were observed in the subsequent five babies. We did not see evidence of any other adverse events related to cell administration. Allogeneic hAECs can be safely infused into babies with established BPD. Future randomized clinical trials to assess efficacy in this patient population are justified.

Idiopathic pulmonary fibrosis (IPF) is characterized by chronic inflammation, severe scarring, and stem cell senescence. Stem cell‐based therapies modulate inflammatory and fibrogenic pathways by release of soluble factors. Stem cell‐derived extracellular vesicles should be explored as a potential therapy for IPF. Human amnion epithelial cell‐derived exosomes (hAEC Exo) were isolated and compared against human lung fibroblasts exosomes. hAEC Exo were assessed as a potential therapy for lung fibrosis. Exosomes were isolated and evaluated for their protein and miRNA cargo. Direct effects of hAEC Exo on immune cell function, including macrophage polarization, phagocytosis, neutrophil myeloperoxidase activity and T cell proliferation and uptake, were measured. Their impact on immune response, histological outcomes, and bronchioalveolar stem cell (BASC) response was assessed in vivo following bleomycin challenge in young and aged mice. hAEC Exo carry protein cargo enriched for MAPK signaling pathways, apoptotic and developmental biology pathways and miRNA enriched for PI3K‐Akt, Ras, Hippo, TGFβ, and focal adhesion pathways. hAEC Exo polarized and increased macrophage phagocytosis, reduced neutrophil myeloperoxidases, and suppressed T cell proliferation directly. Intranasal instillation of 10 μg hAEC Exo 1 day following bleomycin challenge reduced lung inflammation, while treatment at day 7 improved tissue‐to‐airspace ratio and reduced fibrosis. Administration of hAEC Exo coincided with the proliferation of BASC. These effects were reproducible in bleomycin‐challenged aged mice. The paracrine effects of hAECs can be largely attributed to their exosomes and exploitation of hAEC Exo as a therapy for IPF should be explored further. Stem Cells Translational Medicine 2018;7:180–196

Background Arterial stiffness is an independent predictor of all-cause and cardiovascular mortality in patients with chronic kidney disease (CKD). There are limited prospective data however on progression of arterial stiffness in CKD, including evaluating associations with bone mineral markers such as fibroblast growth factor 23 (FGF23) and soluble α-klotho (sKl). Methods In this prospective, single-center, observational study, arterial stiffness [measured by pulse wave velocity (PWV)] and hormones influencing mineral homeostasis, including serum FGF23 and sKl, were compared between non-dialysis CKD stages 4/5 and healthy controls at baseline and 12 months (12 m). Abdominal aortic calcification (AAC) was quantitated using lateral lumbar radiography at baseline. Results Forty patients with CKD [mean estimated glomerular filtration rate (eGFR) 19.5 ± 6.7 mL/min/1.73m2] and 42 controls (mean eGFR 88.6 ± 12.9 mL/min/1.73m2) completed follow-up. There were no differences in age, gender and body mass index between groups. A significant increase in FGF23 [240.6 (141.9–1129.8) to 396.8 (160.3–997.7) pg/mL, p = 0.001] was observed in the CKD group but serum phosphate, corrected calcium, parathyroid hormone and sKl did not change significantly over 12 m. At baseline, CKD subjects had higher AAC prevalence [83.8% versus (vs.) 43.6%, p = 0.002] and higher aortic PWV [9.7(7.6–11.7) vs. 8.1 (7.2–9.7) m/s, p = 0.047] compared to controls. At 12 m, aortic PWV increased by 1.3 m/s (95% confidence interval, 0.56 to 2.08, p 10 m/s). Serum FGF23 was associated with AAC, abnormal PWV and progression of PWV at 12 m. Conclusions Arterial stiffness and serum FGF23, both of which are associated with increased cardiovascular risk, increased over one year in individuals with CKD. Additionally, a significant association was found between serum FGF23 and arterial calcification and stiffness. Larger clinical studies and further experimental work are warranted to delineate the temporal relationship as well as the pathological mechanisms linking FGF23 and vascular disease. Electronic supplementary material The online version of this article (10.1186/s12882-017-0705-4) contains supplementary material, which is available to authorized users.

Mineralisation paradox is prevalent in chronic kidney disease and ageing where increased vascular calcification is accompanied by reduced bone mineralisation and osteopenia. Secondary calciprotein particles (CPP2), colloidal nanoparticles containing hydroxyapatite crystal stabilised by a protein shell, have been implicated in vascular calcification in chronic kidney disease. Here, we describe the effect of CPP2 on osteoblasts and vascular smooth muscle cells (VSMC) mineralisation in an in vitro model system. The mineralisation paradox can be simulated in vitro by the addition of phosphate ions (Pi, 3 mM) and CPP2 (10 µg/ml of Ca equivalent). Pi alone induced osteoblast mineralisation but had no effect on VSMC mineralisation. CPP2 alone had no effect on mineralisation in either cell line, but when combined with elevated Pi, reduced osteoblast-like mineralisation (P

Klotho is predominantly expressed in the kidney and reported to have antioxidant and antifibrotic properties. Soluble Klotho (sKl), the circulating protein cleaved from membrane-bound Klotho, is reduced significantly with kidney disease and inversely associated with mortality. sKl has not been thoroughly evaluated prospectively after kidney transplantation. Incident kidney transplant recipients (KTRs) were prospectively evaluated pretransplantation, 1, 12 and 52 weeks post-transplantation. Basic biochemistry, sKl and intact FGF23 were measured. Within-subject comparisons were evaluated using repeat-measure anova or Friedman's analysis. Effects of immunosuppression and biochemical parameters on sKl and FGF-23 over time were analysed using mixed-effects modelling. Median serum creatinine (sCr) at 1 week was 116 (92-142) μmol/l, and at 52 weeks, all 29 KTRs had a functioning graft with median sCr of 111 (97-131) μmol/l. Compared with baseline, sKl was increased at 52 weeks following an initial decline at 1 week (P < 0.005 and P < 0.01, respectively), while FGF23 was considerably reduced at 52 weeks (P < 0.001). In a mixed-effects model, an increased sKl was not associated with reduction in immunosuppression or evaluated biochemical parameters. Modest increase in sKl is observed one-year postkidney transplantation with excellent early graft function suggesting factors beyond renal capacity may influence circulating sKl. FGF23 normalization was observed. Longer term evaluation in transplantation, specifically addressing the effects of immunosuppression, is required to understand the pathophysiology of the sKl/FGF23 axis and potential for modification.

The current management of Chronic Kidney Disease-Mineral Bone Disorder (CKD-MBD) relies largely on clinical judgement and assessment of biochemical parameters including serum calcium, phosphate and intact parathyroid hormone concentrations. In the past two decades, there has been a leap in the understanding of the pathophysiology of CKD-MBD, leading to the discovery of novel biomarkers. The potential utility of these markers in this clinical setting is an area of intense investigation. In the absence of any guidelines aiding the clinician's understanding and application of these markers, we summarise the current available literature surrounding fibroblast growth factor-23, α-Klotho, sclerostin and serum calcification propensity testing and their respective assays in the context of CKD-MBD management.

Glioblastoma (GBM) is the most frequent and deadly primary malignant brain tumor. Hallmarks are extensive intra-tumor and inter-tumor heterogeneity and highly invasive growth, which provide great challenges for treatment. Efficient therapy is lacking and the majority of patients survive less than 1 year from diagnosis. GBM progression and recurrence is caused by treatment-resistant glioblastoma stem cells (GSCs). GSC cultures are considered important models in target identification and drug screening studies. The current state-of-the-art method, to isolate and maintain GSC cultures that faithfully mimic the primary tumor, is to use serum-free (SF) media conditions developed for neural stem cells (NSCs). Here we have investigated the outcome of explanting 218 consecutively collected GBM patient samples under both SF and standard, serum-containing media conditions. The frequency of maintainable SF cultures (SFCs) was most successful, but for a subgroup of GBM specimens, a viable culture could only be established in serum-containing media, called exclusive serum culture (ESC). ESCs expressed nestin and SOX2, and displayed all functional characteristics of a GSC, that is, extended proliferation, sustained self-renewal and orthotopic tumor initiation. Once adapted to the in vitro milieu they were also sustainable in SF media. Molecular analyses showed that ESCs formed a discrete group that was most related to the mesenchymal GBM subtype. This distinct subgroup of GBM that would have evaded modeling in SF conditions only provide unique cell models of GBM inter-tumor heterogeneity.

There is ample support for developmental control of glioblastoma stem cells (GSCs), and a deeper knowledge of their epigenetic regulation could be central to more efficient glioblastoma (GBM) therapies. For this purpose, we analyzed the chromatin-accessibility landscape of nine mouse GSC cultures of defined cell of origin and 60 patient-derived GSC cultures by assay for transposase-accessible chromatin using sequencing (ATAC-seq). This uncovered an epigenetic variability of both mouse and human GSC cultures that differed from transcriptome clusters. Both mouse and human chromatin accessibility-guided clusters were predominantly determined by distal regulatory elements, displayed unique sets of transcription factor motif enrichment, and exhibited different functional and drug-response properties. Cross-species analysis of distal regulatory element regions in accessible chromatin of mouse and human cultures revealed conserved epigenetic regulation of GSCs.

Iron and phosphate are both vital to many biological cellular processes with central roles in energy metabolism, cellular proliferation and nucleic acid synthesis. Regulatory pathways in some of these metabolic pathways may intersect at fibroblast growth factor 23 (FGF23), a major phosphate regulatory hormone. Iron is reported to induce hypophosphataemia in rare cases, and recent reports suggest that iron deficiency may upregulate FGF23 synthesis by mechanisms involving hypoxia-inducible factor 1α (HIF1α). Our objective was to evaluate the effect of administration of intravenous iron polymaltose on intact and c-terminal FGF23 (i:cFGF23) ratios in two independent cohorts of patients, iron-deficient but non-inflamed patients and haemodialysis (HD)-dependent patients, and to examine the balance of synthesis and degradation. We studied biochemical effects of intravenous iron polymaltose on both iFGF23 and cFGF23 fragments and their ratios in two patient groups: iron-deficient patients with normal renal function (ID-norm) and HD patients receiving iron supplementation (HD-ESKD) at a single institution. Patients were tested at baseline, day 4 and day 12 post iron administration. Parenteral iron polymaltose resulted in increased i:cFGF23 ratios in ID-norm patients where circulating cFGF23 levels decreased with no appreciable effect on iFGF23, whereas no significant changes in i:cFGF23 ratios were observed in HD-ESKD patients following intravenous administration of 100mg iron polymaltose. Dysregulation of intracellular FGF23-processing mechanisms may be related to iron deficiency per se rather than iron repletion with iron polymaltose. In ID-norm, i:cFGF23 ratios altered with iron administration without significant clinical alterations in mineral parameters, implying that other regulatory mechanisms may be important. Finally, iron supplementation in HD-ESKD patients does not appear to significantly affect i:cFGF23 ratios already disturbed by a chronic inflammatory or functionally iron-deficient state.

Human amnion epithelial cells (hAECs) have been shown to possess potent immunomodulatory properties across a number of disease models. Recently, we reported that hAECs influence macrophage polarization and activity, and that this step was dependent on regulatory T cells. In this study, we aimed to assess the effects of hAEC-derived proresolution lipoxin-A4 (LXA4) on T-cell, macrophage, and neutrophil phenotype and function during the acute phase of bleomycin-induced lung injury. Using C57Bl6 mice, we administered 4 million hAECs intraperitoneally 24 hours after bleomycin challenge. Outcomes were measured at days 3, 5, and 7. hAEC administration resulted in significant changes to T-cell, macrophage, dendritic cell, and monocyte/macrophage infiltration and phenotypes. Endogenous levels of lipoxygenases, LXA4, and the lipoxin receptor FPR2 were elevated in hAEC-treated animals. Furthermore, we showed that the effects of hAECs on macrophage phagocytic activity and T-cell suppression are LXA4 dependent, whereas the inhibition of neutrophil-derived myleoperoxidase by hAECs is independent of LXA4. This study provides the first evidence that lipid-based mediators contribute to the immunomodulatory effects of hAECs and further supports the growing body of evidence that LXA4 is proresolutionary in lung injury. This discovery of LXA4-dependent communication between hAECs, macrophages, T cells, and neutrophils is important to the understanding of hAEC biodynamics and would be expected to inform future clinical applications.

Zebrafish is gaining prominence as an important vertebrate model for investigating various human diseases. Zebrafish provides unique advantages such as optical clarity of embryos, high fecundity rate, and low cost of maintenance, making it a perfect complement to the murine model equivalent in biomedical research. Due to these advantages, researchers in Malaysia are starting to take notice and incorporate the zebrafish model into their research activities. However, zebrafish research in Malaysia is still in its infancy stage and many researchers still remain unaware of the full potential of the zebrafish model or have limited access to related tools and techniques that are widely utilized in many zebrafish laboratories worldwide. To overcome this, we organized the First Malaysia Zebrafish Disease Model Workshop in Malaysia that took place on 11th and 12th of November 2015. In this workshop, we showcased how the zebrafish model is being utilized in the biomedical field in international settings as well as in Malaysia. For this, notable international speakers and those from local universities known to be carrying out impactful research using zebrafish were invited to share some of the cutting edge techniques that are used in their laboratories that may one day be incorporated in the Malaysian scientific community.

Poor prognosis of most cancer patients is in part, due to limited therapeutic options. Furthermore, as chemotherapy remains the standard-of-care for several cancers, partial or lack of response remains a concern and compounding this are the adverse side effects of the treatment that severely impacts the quality of life and survival. In pursuit of improving treatment options, we have opted to investigate the unique chemical skeleton of natural compounds as anticancer therapies. In this study, from an initial screen of 31 crude methanol extracts from -15 plant species using HL60 cells, the root extract of Bruceajavanica (L.) Merr indicated the presence of bioactive compounds. Subsequent bioassay-guided purification on the root extract yielded two alkaloids canthin-6-one (1) and bruceolline J (2), which were further investigated for their bioactivity in representative human cancer lines and normal phenotypic counterparts. MTT assay demonstrated ED50 values from 34.7-72.9 gM for 1 and 16.0-54.0 gM for 2 for the cancer cell lines panel. NP69 cells also demonstrated sensitivity to. both compounds (9.3 piM and 4.5 pM). As amount of 2 isolated were limiting, we focused on 1 to further identify novel anticancer properties in PC3 and HeLa cancer lines. We observed at 30 gM, I induced a G2/M phase arrest coinciding with decreased cell proliferation. Furthermore, I was able to synergize the cytotoxic effect of cisplatin when used in combination, suggesting the potential of combination therapy for those less responsive lesions to standard chemotherapy.

Perivascular mesenchymal stem/stromal cells can be isolated from the human endometrium using the surface marker SUSD2, and are being investigated for use in tissue repair. Mesenchymal stem/stromal cells from other tissues modulate T cell responses via mechanisms including interleukin-10, prostaglandin E2, TGF-β1 and regulatory T cells. Animal studies demonstrate that endometrial mesenchymal stem/stromal cells can also modify immune responses to implanted mesh, but the mechanism/s they employ have not been explored. We examined the immunomodulatory properties of human endometrial mesenchymal stem/stromal cells on lymphocyte proliferation using mouse splenocyte cultures. Endometrial mesenchymal stem/stromal cells inhibited mitogen-induced lymphocyte proliferation in vitro in a dose-dependent manner. Inhibition of lymphocyte proliferation was not affected by blocking the mouse interleukin-10 receptor or inhibiting prostaglandin production. Endometrial mesenchymal stem/stromal cells continued to restrain lymphocyte proliferation in the presence of an inhibitor of TGF-β receptors, despite a reduction in regulatory T cells. Thus the in vitro inhibition of mitogen-induced lymphocyte proliferation by endometrial mesenchymal stem/stromal cells occurs by a mechanism distinct from the interleukin-10, prostaglandin E2, TGF-β1, and regulatory T cell-mediated mechanisms employed by MSC from other tissues. eMSC were shown to produce interleukin-17A and Dickkopf-1 which may contribute to their immunomodulatory properties. In contrast to MSC from other sources, systemic administration of endometrial mesenchymal stem/stromal cells did not inhibit swelling in a T cell-mediated model of skin inflammation. We conclude that, while endometrial mesenchymal stem/stromal cells can modify immune responses, their immunomodulatory repertoire may not be sufficient to restrain some T cell-mediated inflammatory events.

Glioblastoma (GBM) is the most common and lethal primary malignant brain tumor which lacks efficient treatment and predictive biomarkers. Expression of the epithelial stem cell marker Leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5) has been described in GBM, but its functional role has not been conclusively elucidated. Here, we have investigated the role of LGR5 in a large repository of patient-derived GBM stem cell (GSC) cultures. The consequences of LGR5 overexpression or depletion have been analyzed using in vitro and in vivo methods, which showed that, among those with highest LGR5 expression (LGR5

The amount of electronics within vehicles continue to increase with the drive towards autonomous driving and green vehicles through improved engine controls and hybrid or full electric vehicles. Engine control units require electronic components which are located very close to the engine, and thus must operate within a high temperature ambient environment, at least meeting AEC Grade 1 or Grade 0 requirements. This paper reviews the bill of material selection for quad-flat package (QFP), in particularly, mold compound and copper wire, with high density leadframes. Palladium coated copper wire (PCC) is the current preferred wire of choice due to the improved manufacturability. Although PCC wire is susceptible to galvanic corrosion due to the presence of sulfur in the molding compound, the level of corrosion is minimal and impact is cosmetic only. Wire sweep is a major factor in the mold compound selection, especially for high density leadframes. Wire sweep percentage differs greatly from row to row, and mold compound type contributes significantly for a robust process on high density leadframes. Furthermore, on large body sized QFPs, package warpage is another main consideration in the selection of mold compound type as warpage directly impacts the lead co-planarity. It is shown that additional heat treatment post assembly, such as during burn-in, may contribute significantly towards warpage degradation. As such, it is necessary to select the molding compound with sufficiently low warpage through repeated heat treatment on the package. Lastly, the selected mold compound needs to meet the delamination requirements as per AEC-Q006 for copper wire interconnects.

In this work, the potential of targeting griseofulvin (GF) into the skin by topical application was evaluated by in-vitro studies. Griseofulvin loaded lipid nanoparticles (GF-LN) were prepared using high pressure homogenization method without any penetration enhancer or solubilizing solvent. The formulations were freeze dried to increase stability of the lipid nanoparticles (LN). The optimum formulation showed a mean particle size of 179.8 nm with polydispersity index of 0.306 and zeta potential of −27.9 mV and the loading of griseofulvin was 0.77%. Morphology and the degree of crystallinity of the LN were investigated using transmission electron microscope (TEM) and differential scanning calorimetry (DSC). In-vitro cytotoxicity studies showed the LN has better safety profile in human keratinocyte cells (HaCaT) with four-folds reduction in cytotoxicity (IC 50 30 ± 2.37 μg/mL). In the in-vitro antifungal susceptibility tests, the GF-LN demonstrated comparable antifungal activity against Trichophyton rubrum (MIC 0.5 μg/mL) and Trichophyton mentagrophytes (MIC 0.25 μg/mL). In-vitro skin penetration and retention studies were carried out on porcine skin. The GF-LN demonstrated sufficient skin permeation with penetration flux of 0.067 ± 0.003 μg/cm 2 /hr. The retention of griseofulvin was found four-folds higher in the epidermis (14.9 ± 0.483 μg/cm 2 ) on comparison to the dermis.

Aim Cinacalcet is effective in reducing parathyroid hormone (PTH) in patients on dialysis. Reports of biochemical profiles and other clinical outcomes in patients discontinuing cinacalcet at time of renal transplantation are limited. Methods A retrospective study assessing markers of mineral metabolism, graft and patient outcomes in renal transplant recipients to determine differences in patients discontinuing cinacalcet (C+) compared with patients not treated with cinacalcet (C−) at time of transplantation. To allow for differences between groups in pre-transplant biochemical parameters, we also analysed a matched cohort of C− with C+ recipients (2:1), matched for age, calcium and PTH levels at transplantation. Results Five hundred thirty-two recipients (460 C−, 72 C+), transplanted January 2006–December 2012, were analysed, mean age 48.0 ± 12.7 years and 64.3% were men. At a median 42.9 months follow up, there were 10 deaths (1.9%), 56 allograft loss (10.6%) and 5 parathyroidectomies post-transplant (0.8%). Median PTH immediately pre-transplant was higher in C+ versus C− (50.7(25.4–75.2) versus 28.3(13.9–49.7) pmol/L, P

Ethnopharmacological relevance Several members of the genus Lignosus , which are collectively known as cendawan susu rimau (in Malay) or tiger׳s milk mushrooms (TMM), are regarded as important local medicine particularly by the indigenous communities in Malaysia. The mushroom sclerotia are purportedly effective in treating cancer, coughs, asthma, fever, and other ailments. The most commonly encountered Lignosus spp. in Malaysia was authenticated as Lignosus rhinocerotis (Cooke) Ryvarden (synonym: Polyporus rhinocerus ), which is also known as hurulingzhi in China and has been used by Chinese physicians to treat liver cancer, gastric ulcers, and chronic hepatitis. In spite of growing interest in the therapeutic potential of TMM, there is no compilation of scientific evidence that supports the ethnomedicinal uses of these mushrooms. Therefore, the present review is intended (i) to provide a comprehensive, up-to-date overview of the ethnomedicinal uses, pharmacological activities, and cultivation of TMM in general and L. rhinocerotis in particular, (ii) to demonstrate how recent scientific findings have validated some of their traditional uses, and (iii) to identify opportunities for future research and areas to prioritize for TMM bioprospecting. Materials and methods A detailed literature search was conducted via library search (books, theses, reports, newspapers, magazines, and conference proceedings) and electronic search (Web of Science, PubMed, and Google Scholar) for articles published in peer-reviewed journals. These sources were scrutinized for information on TMM and specifically for L. rhinocerotis . Results Ethnomycological knowledge about TMM, with an emphasis on cultural associations and use as local medicine, has been comprehensively and systematically compiled for the first time. Some of the reported medicinal properties of TMM have been validated by scientific studies. The anti-tumor, immuno-modulatory, anti-inflammatory, anti-oxidative, anti-microbial, neurite outgrowth stimulation, and other pharmacological activities of L. rhinocerotis sclerotial extracts have been explored. The nature of sclerotial bioactive components, such as proteins, polysaccharides, and/or polysaccharide–protein complexes, has been identified, whereas the low-molecular-weight constituents remain poorly studied. The artificial cultivation of L. rhinocerotis via solid substrate and liquid fermentations successfully yielded fruiting bodies, sclerotium, mycelium, and culture broth that could be exploited as substitutes for the wild resources. The cultivated sclerotium and mycelium were shown to be safe from a toxicological point of view. Other research areas, e.g. , chemical studies, genomics, and proteomics, have been employed to gain insights into the medicinal properties of TMM. Conclusions This review clarified the medicinal properties of TMM as recorded in various ethnomycological records, and it simultaneously highlighted the current efforts to provide scientific evidence by using various in vitro and in vivo models. Thus far, only the anti-tumor and immuno-modulatory effects of L. rhinocerotis sclerotial aqueous extracts have been extensively investigated, and other medicinal properties relevant to their traditional uses, e.g. , anti-tussive and anti-pyretic properties, have yet to be validated. Further studies focusing on (i) the isolation and characterization of active components, (ii) the elucidation of their modes of action, and (iii) an evaluation of their safety and efficacy, when compared with the crude aqueous preparations, are warranted to accelerate potential drug discovery from TMM.

Epithelial–mesenchymal transition (EMT) is a vital process that takes place during embryogenesis and cancer progression. Epithelial cells shed their adhesive characteristics to acquire mesenchymal properties that allow them to become motile. Tumor cells undergoing EMT are conferred aggressive traits, such as motility, invasiveness, stem cell-like features, immunosuppression and therapeutic resistance. However in recent years, increasing evidence suggests that the existence of intermediate EMT states (i.e., part epithelial and part mesenchymal) has been associated with worse prognosis compared to full mesenchymal phenotype. The EMT program is more dynamic and plastic than previously thought.

We have recently identified LGR5 as a marker of an immature, neural stem cell-like cell of origin for glioblastoma (GBM). Here we have further investigated the role of LGR5 in GBM. We have found that in a panel of 61 patient-derived GBM stem cell lines, LGR5 was differentially expressed and significantly enriched in those of the proneural subtype. High LGR5 expression in proneural GBM lines (LGR5high-PN) was associated with increased self-renewal and tumorigenicity compared to low expressing (LGR5low) and high expressing mesenchymal (LGR5high-MS) GBM cell lines. Knock-down of LGR5 gene expression in LGR5high-PN lines resulted in decreased self-renewal, while over-expression in both LGR5high-PN and LGR5low lines produced a higher self-renewal capacity of the cells. Furthermore, by comparing LGR5high-PN cells with and without si/shLGR5 we found that LGR5 also could regulate tumor cell invasion in vitro and in vivo. When analyzing transcriptome changes upon LGR5 depletion we detected a transition from the proneural to the mesenchymal subtype in LGR5high-PN GBM cells treated with shLGR5 that was coupled to an upregulation of genes of the TNFA/NFKB pathway. Taken together our data suggests that LGR5 could be an essential regulator of a subset of GBMs. To stratify patients carrying LGR5-dependent GBMs we have identified a number of additional candidate biomarkers, which together with LGR5 could help defining LGR5-driven GBM cells.

Palm-based lipid nanoparticle formulation loaded with griseofulvin was prepared by solvent-free hot homogenization method. The griseofulvin loaded lipid nanoparticles were prepared via stages of optimisation, by altering the high pressure homogenisation (HPH) parameters, screening on palm-based lipids and Tween series surfactants and selection of lipid to surfactant ratios. A HPLC method has been validated for the drug loading capacity study. The optimum HPH parameter was determined to be 1500 bar with 5 cycles and among the palm-based lipid materials; Lipid C (triglycerides) was selected for the preparation of lipid nanoparticles. Tween 80 was chosen from the Tween series surfactants for its highest saturated solubility of griseofulvin at 53.1 ± 2.16 µg/mL. The optimum formulation of the griseofulvin loaded lipid nanoparticles demonstrated nano-range of particle size (179.8 nm) with intermediate distribution index (PDI) of 0.306, zeta potential of -27.9 mV and drug loading of 0.77%. The formulation was stable upon storage for 1 month at room temperature (25 °C) and 45 °C with consistent drug loading capacity.

Glioblastoma multiforme is a brain malignancy characterized by high heterogeneity, invasiveness, and resistance to current therapies, attributes related to the occurrence of glioma stem cells (GSCs). Transforming growth factor β (TGFβ) promotes self-renewal and bone morphogenetic protein (BMP) induces differentiation of GSCs. BMP7 induces the transcription factor Snail to promote astrocytic differentiation in GSCs and suppress tumor growth in vivo. We demonstrate that Snail represses stemness in GSCs. Snail interacts with SMAD signaling mediators, generates a positive feedback loop of BMP signaling and transcriptionally represses the TGFB1 gene, decreasing TGFβ1 signaling activity. Exogenous TGFβ1 counteracts Snail function in vitro, and in vivo promotes proliferation and re-expression of Nestin, confirming the importance of TGFB1 gene repression by Snail. In conclusion, novel insight highlights mechanisms whereby Snail differentially regulates the activity of the opposing BMP and TGFβ pathways, thus promoting an astrocytic fate switch and repressing stemness in GSCs.

In kidney disease, higher circulating levels of the mineral-regulating hormone fibroblast growth factor (FGF)-23 are predictive of disease progression but direct pathogenic effects on the kidney are unknown. We sought evidence of local renal synthesis in response to unilateral ureteric obstruction in the mouse, and pro-fibrotic actions of FGF23 on the fibroblast in vitro. Acute tubulointerstitial injury due to unilateral ureteric obstruction stimulated renal FGF23 synthesis by tubules, and downregulated inactivating proprotein convertases, without effects on systemic mineral metabolism. In vitro, FGF23 had divergent effects on fibroblast activation in cells derived from normal and obstructed kidneys. While FGF23 failed to stimulate fibrogenesis in normal fibroblasts, in those primed by injury, FGF23 induced pro-fibrotic signalling cascades via activation of TGF-β pathways. Effects were independent of α-klotho. Tubule-derived FGF23 may amplify myofibroblast activation in acute renal injury, and might provide a novel therapeutic target in renal fibrosis.

The current management of Chronic Kidney Disease-Mineral Bone Disorder (CKD-MBD) relies largely on clinical judgement and assessment of biochemical parameters including serum calcium, phosphate and intact parathyroid hormone concentrations. In the past two decades, there has been a leap in the understanding of the pathophysiology of CKD-MBD, leading to the discovery of novel biomarkers. The potential utility of these markers in this clinical setting is an area of intense investigation. In the absence of any guidelines aiding the clinician's understanding and application of these markers, we summarise the current available literature surrounding fibroblast growth factor-23, α-Klotho, sclerostin and serum calcification propensity testing and their respective assays in the context of CKD-MBD management.