Toxicokinetics of α- and ß-Amanitin in Mice Following Single and Combined Administrations: Simulating In Vivo Amatoxins Processes in Clinical Cases.

α-Amanitin and ß-amanitin, two of the most toxic amatoxin compounds, typically coexist in the majority of Amanita mushrooms. The aim of this study was to use a newly developed ultra-performance liquid chromatography-mass spectrometry (UPLC-MS/MS) method to determine the toxicokinetics and tissue distribution of α- and ß-amanitin following single or combined oral (po) administration in mice. α-Amanitin and ß-amanitin administered at 2 or 10 mg/kg doses showed similar toxicokinetic profiles, except for peak concentration (Cmax). The elimination half-life (t1/2) values of α-amanitin and ß-amanitin in mice were 2.4-2.8 h and 2.5-2.7 h, respectively. Both α- and ß-amanitin were rapidly absorbed into the body, with times to reach peak concentration (Tmax) between 1.0 and 1.5 h. Following single oral administration at 10 mg/kg, the Cmax was significantly lower for α-amanitin (91.1 µg/L) than for ß-amanitin (143.1 µg/L) (p < 0.05). The toxicokinetic parameters of α-amanitin, such as t1/2, mean residence time (MRT), and volume of distribution (Vz/F) and of ß-amanitin, such as Vz/F, were significantly different (p < 0.05) when combined administration was compared to single administration. Tissues collected at 24 h after po administration revealed decreasing tissue distributions for α- and ß-amanitin of intestine > stomach > kidney > lung > spleen > liver > heart. The substantial distribution of toxins in the kidney corresponds to the known target organs of amatoxin poisoning. The content in the stomach, liver, and kidney was significantly higher for of ß-amanitin than for α-amanitin at 24 h following oral administration of a 10 mg/kg dose. No significant difference was detected in the tissue distribution of either amatoxin following single or combined administration. After po administration, both amatoxins were primarily excreted through the feces. Our data suggest the possibility of differences in the toxicokinetics in patients poisoned by mushrooms containing both α- and ß-amanitin than containing a single amatoxin. Continuous monitoring of toxin concentrations in patients' blood and urine samples is necessary in clinical practice.

Efficacy of IAPP suppression in mouse and human islets by GLP-1 analogue conjugated antisense oligonucleotide.

Insulin resistance is the major risk factor for Type 2 diabetes (T2D). In vulnerable individuals, insulin resistance induces a progressive loss of insulin secretion with islet pathology revealing a partial deficit of beta cells and islet amyloid derived from islet amyloid polypeptide (IAPP). IAPP is co-expressed and secreted with insulin by beta cells, expression of both proteins being upregulated in response to insulin resistance. If IAPP expression exceeds the threshold for clearance of misfolded proteins, beta cell failure occurs exacerbated by the action of IAPP toxicity to compromise the autophagy lysosomal pathway. We postulated that suppression of IAPP expression by an IAPP antisense oligonucleotide delivered to beta cells by the GLP-1 agonist exenatide (eGLP1-IAPP-ASO) is a potential disease modifying therapy for T2D. While eGLP1-IAPP-ASO suppressed mouse IAPP and transgenic human IAPP expression in mouse islets, it had no discernable effects on IAPP expression in human islets under the conditions studied. Suppression of transgenic human IAPP expression in mouse islets attenuated disruption of the autophagy lysosomal pathway in beta cells, supporting the potential of this strategy.

IAPP-induced beta cell stress recapitulates the islet transcriptome in type 2 diabetes.

AIMS/HYPOTHESIS: Type 2 diabetes is characterised by islet amyloid and toxic oligomers of islet amyloid polypeptide (IAPP). We posed the questions, (1) does IAPP toxicity induce an islet response comparable to that in humans with type 2 diabetes, and if so, (2) what are the key transcriptional drivers of this response? METHODS: The islet transcriptome was evaluated in five groups of mice: beta cell specific transgenic for (1) human IAPP, (2) rodent IAPP, (3) human calpastatin, (4) human calpastatin and human IAPP, and (5) wild-type mice. RNA sequencing data was analysed by differential expression analysis and gene co-expression network analysis to establish the islet response to adaptation to an increased beta cell workload of soluble rodent IAPP, the islet response to increased expression of oligomeric human IAPP, and the extent to which the latter was rescued by suppression of calpain hyperactivation by calpastatin. Rank-rank hypergeometric overlap analysis was used to compare the transcriptome of islets from human or rodent IAPP transgenic mice vs humans with prediabetes or type 2 diabetes. RESULTS: The islet transcriptomes in humans with prediabetes and type 2 diabetes are remarkably similar. Beta cell overexpression of soluble rodent or oligomer-prone human IAPP induced changes in islet transcriptome present in prediabetes and type 2 diabetes, including decreased expression of genes that confer beta cell identity. Increased expression of human IAPP, but not rodent IAPP, induced islet inflammation present in prediabetes and type 2 diabetes in humans. Key mediators of the injury responses in islets transgenic for human IAPP or those from individuals with type 2 diabetes include STAT3, NF-κB, ESR1 and CTNNB1 by transcription factor analysis and COL3A1, NID1 and ZNF800 by gene regulatory network analysis. CONCLUSIONS/INTERPRETATION: Beta cell injury mediated by IAPP is a plausible mechanism to contribute to islet inflammation and dedifferentiation in type 2 diabetes. Inhibition of IAPP toxicity is a potential therapeutic target in type 2 diabetes.

Symptomatic spinal cord compression: an uncommon symptom in pseudohypoparathyroidism.

We describe symptomatic spinal cord compression associated with pseudohypoparathyroidism (PHP) in a young female patient and reviewed similar cases previously reported in the literature. The characteristics of these cases were analyzed from etiology, clinical subtypes, symptoms, treatment, and prognosis. Neurological examination revealed functional upper extremities with bilateral lower extremity paraplegia. Laboratory tests showed hypocalcemia, hyperphosphatemia, and elevated parathyroid hormone; high-throughput sequencing showed a heterozygous GNAS mutation in exon 12, specifically c.1006C > T (p.R336W). Imaging findings showed multilevel spinal stenosis with significant spinal cord compression at the T2-T3 level. Seventeen cases with similar characteristics were reviewed. We found that the primary clinical manifestation of these patients was bilateral lower extremity spastic paraplegia. Multilevel spinal cord compression was commonly observed, especially at the lower cervical and upper thoracic spinal cord. Most of the patients had poor surgical treatment outcome and prognosis. Clinicians should be aware of paraplegia due to spinal cord compression as a rare neurological complication in patients with PHP. Early diagnosis and treatment of PHP is one basis for preventing severe spinal cord-related complications.

A Novel MYH9-RET Fusion Occurrence and EGFR T790M Loss as an Acquired Resistance Mechanism to Osimertinib in a Patient with Lung Adenocarcinoma: A Case Report.

BACKGROUND: Osimertinib is a novel and irreversible epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI) targeting EGFR sensitive mutations and EGFR exon20 p.T790M mutation, which demonstrated superior progression-free survival (PFS) and overall survival (OS). CASE PRESENTATION: We report a patient with lung adenocarcinoma harboring EGFR exon 19 deletion mutant treatment with icotinib. After 6 months, she developed EGFR exon20 p.T790M and then the patient received osimertinib treatment. A novel MYH9 (exon41)-RET (exon12) fusion and EGFR exon20 p.T790M loss were identified using plasma circulation tumor DNA (ctDNA) after osimertinib treatment, which led to rapid progression after osimertinib five months and suggested a potential resistance mechanism. CONCLUSION: Our findings expanded the spectrum of RET arrangement types and provided the basis for this hypothesis: acquired RET rearrangement and EGFR exon20 p.T790M loss potentially serve an additional resistance mechanism to osimertinib in EGFR-mutated non-small-cell lung cancer (NSCLC).

Activation of the HIF1α/PFKFB3 stress response pathway in beta cells in type 1 diabetes.

AIMS/HYPOTHESIS: The conserved hypoxia inducible factor 1 α (HIF1α) injury-response pro-survival pathway has recently been implicated in early beta cell dysfunction but slow beta cell loss in type 2 diabetes. We hypothesised that the unexplained prolonged prediabetes phase in type 1 diabetes may also be, in part, due to activation of the HIF1α signalling pathway. METHODS: RNA sequencing (RNA-Seq) data from human islets with type 1 diabetes or after cytokine exposure in vitro was evaluated for activation of HIF1α targets. This was corroborated by immunostaining human pancreases from individuals with type 1 diabetes for 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), the key effector of HIF1α-mediated metabolic remodelling, and by western blotting of islets and INS-1 832/13 cells exposed to cytokines implicated in type 1 diabetes. RESULTS: HIF1α signalling is activated (p = 4.5 × 10-9) in islets from individuals with type 1 diabetes, and in human islets exposed in vitro to cytokines implicated in type 1 diabetes (p = 1.1 × 10-14). Expression of PFKFB3 is increased fivefold (p < 0.01) in beta cells in type 1 diabetes and in human and rat islets exposed to cytokines that induced increased lactate production. HIF1α attenuates cytokine-induced cell death in beta cells. CONCLUSIONS/INTERPRETATION: The conserved pro-survival HIF1α-mediated injury-response signalling is activated in beta cells in type 1 diabetes and likely contributes to the relatively slow rate of beta cell loss at the expense of early defective glucose-induced insulin secretion.

Publisher Correction: IAPP toxicity activates HIF1α/PFKFB3 signaling delaying ß-cell loss at the expense of ß-cell function.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

IAPP toxicity activates HIF1α/PFKFB3 signaling delaying ß-cell loss at the expense of ß-cell function.

The islet in type 2 diabetes (T2D) is characterized by amyloid deposits derived from islet amyloid polypeptide (IAPP), a protein co-expressed with insulin by ß-cells. In common with amyloidogenic proteins implicated in neurodegeneration, human IAPP (hIAPP) forms membrane permeant toxic oligomers implicated in misfolded protein stress. Here, we establish that hIAPP misfolded protein stress activates HIF1α/PFKFB3 signaling, this increases glycolysis disengaged from oxidative phosphorylation with mitochondrial fragmentation and perinuclear clustering, considered a protective posture against increased cytosolic Ca2+ characteristic of toxic oligomer stress. In contrast to tissues with the capacity to regenerate, ß-cells in adult humans are minimally replicative, and therefore fail to execute the second pro-regenerative phase of the HIF1α/PFKFB3 injury pathway. Instead, ß-cells in T2D remain trapped in the pro-survival first phase of the HIF1α injury repair response with metabolism and the mitochondrial network adapted to slow the rate of cell attrition at the expense of ß-cell function.

Pregnancy in human IAPP transgenic mice recapitulates beta cell stress in type 2 diabetes.

AIMS/HYPOTHESIS: Islet amyloid polypeptide (IAPP) misfolding and toxic oligomers contribute to beta cell loss and stress in type 2 diabetes. Pregnancy-related diabetes predicts subsequent risk for type 2 diabetes but little is known about the impact of pregnancy on beta cell mass, turnover and stress. Availability of human pancreas tissue in pregnancy is limited and most widely used mouse models of type 2 diabetes do not develop pregnancy-related diabetes, possibly because rodent IAPP is not prone to form toxic oligomers. We hypothesised that mice transgenic for human IAPP (hIAPP) are prone to pregnancy-related diabetes with beta cell responses reflective of those in type 2 diabetes. METHODS: We evaluated the impact of a first and second pregnancy on glucose homeostasis, beta cell mass and turnover and markers of beta cell stress in hIAPP transgenic (hTG) mice. RESULTS: Pregnancy induced both endoplasmic reticulum stress and oxidative stress and compromised autophagy in beta cells in hTG mice, which are characteristic of beta cells in type 2 diabetes. Beta cell stress persisted after pregnancy, resulting in subsequent diabetes before or during a second pregnancy. CONCLUSIONS/INTERPRETATION: High expression of hIAPP in response to pregnancy recapitulates mechanisms contributing to beta cell stress in type 2 diabetes. We hypothesise that, in individuals prone to type 2 diabetes, pregnancy-induced increased expression of IAPP inflicts beta cell damage that persists and is compounded by subsequent additive stress such as further pregnancy. The hTG mouse model is a novel model for pregnancy-related diabetes.

Etoposide-loaded nanostructured lipid carriers for gastric cancer therapy.

PURPOSE: Gastric carcinoma is one of the most common cancers and the second most frequent cause of cancer-related deaths. The aim of this study was to prepare and characterize etoposide-loaded nanostructured lipid carriers (ETP-NLCs) and evaluate their antitumor activity in vitro and in vivo. METHODS: Novel ETP-NLCs were constructed. The physicochemical properties of the ETP-NLCs were investigated by particle-size analysis, zeta potential measurement, drug loading, drug entrapment efficiency, stability and in vitro drug release behavior. In vitro cytotoxicity against human gastric cancer cells (SGC7901 cells) was investigated, and in vivo antitumor of NLCs was evaluated on mice bearing SGC7901 cells xenografts. RESULTS: ETP-NLCs have a narrow size distribution at 91 nm, a zeta potential value of +23.1 mV, high drug entrapment efficiency of 78%. The drug release of ETP-NLCs exhibited a sustained behavior, which made it an ideal vehicle for drug delivery. Furthermore, ETP-NLCs could significantly enhance in vitro cytotoxicity and in vivo antitumor effect against SGC7901 cells and gastric cancer animal model compared to the free drug. CONCLUSION: The results demonstrated that the NLCs might be a promising nanomedicine for the treatment of gastric carcinoma.

Downregulation of chemerin and alleviation of endoplasmic reticulum stress by metformin in adipose tissue of rats.

AIMS: To investigate whether metformin regulates chemerin expression in vivo by alleviating ER stress. METHODS: Male Sprague-Dawley rats were fed a high-fat or normal diet for 10 weeks to induce insulin resistance. During the following 6 weeks, the rats were divided into four groups: normal diet without treatment (NC), normal diet with metformin treatment (NM), high-fat diet without metformin (HF), and high-fat diet with metformin (HM). Body weight, fasting glucose, basal insulin level, insulin sensitivity, chemerin expression in serum and adipose tissue, ER stress marker and its pathway were measured. RESULTS: After 6 weeks treatment, metformin reduced the body weight gain and enhanced insulin sensitivity of high-fat fed rats. The basal insulin level in the HM group was lower than in the HF group. Metformin reduced chemerin expression in the HM group compared with HF. Metformin reduced the GRP78 mRNA expression in HM rats. Activation of IRE1 alpha was lower in the HM group than the HF group. CONCLUSIONS: Metformin treatment decreased the chemerin expression and alleviated the ER stress in the visceral adipose tissue of high-fat diet-induced insulin-resistant rats. These data may also provide a further rationale for exploring the use of metformin in the treatment of insulin resistance.

Concurrent expression of aryl hydrocarbon receptor and CYP1A1 but not CYP1A1 MspI polymorphism is correlated with gastric cancers raised in Dalian, China.

The frequency of cancer-associated m2m2- (C-) genotype of CYP1A1 and the factors contributing to the increased CYP1A1 expression in gastric cancers (GCs) are largely unknown. To address theses issues, PCR-restriction fragment length polymorphism (PCR-RFLP) was performed to elucidate the MspI polymorphism in 60 GC cases and 57 normal donor samples. The frequencies of m1m1-, m1m2- and m2m2-genotype were 43.3, 45 and 11.7% among GC patients and 45.6, 49.1 and 5.3% among the normal donors respectively, demonstrating no significant difference of them between cancer and control groups (chi(2)=0.343, P=0.558). The correlation of Aryl hydrocarbon receptor (AhR) with the frequent CYP1A1 expression in stepwise gastrocarcinogenesis was determined by RT-PCR, immunohistochemical staining (IHC) and Western blotting, using GC samples as well as their pre-malignant and non-cancerous counterparts. RT-PCR revealed that the AhR detection rates were 100, 94.12 and 85.17% in GC, pre-malignant and non-cancerous mucosa (P>0.05) respectively but the level of AhR expression in GCs was much higher than that of non-cancerous tissues. IHC showed that the frequencies of AhR detection were 94.87% (37/39) in GCs, 94.12% (16/17) in pre-malignant lesions and 50% (3/6) in non-cancerous mucosa, revealing significant difference in frequencies of AhR detection and levels of AhR expression between GC or pre-malignant group and non-cancerous one (P<0.05). The frequency of AhR nucleus translocation was significantly high in GCs (94.87%; 37/39) than that in pre-malignant (70.59%; 12/17) and especially in non-cancerous group (16.67%; 1/6). Co-existence of AhR nuclear translocation and CYP1A1 expressions were found in 82.70% (43/52) of GCs (r(s)=0.437, P<0.01). Our results suggest (1) that CYP1A1 MspI polymorphism may not contribute to the high gastric cancer risk in Dalian region and (2) that enhanced AhR expression and especially its nuclear translocation may be a favorable factor for GC formation presumably via up-regulating CYP1A1 expression.