The size of human subcutaneous adipocytes, but not adiposity, is associated with inflammation, endoplasmic reticulum stress, and insulin resistance markers.

BACKGROUND: The fat storage capacity of the adipose tissue prevents ectopic lipid deposition, which is one of the risk factors for metabolic abnormalities in obesity. This capacity depends upon the adipogenic gene expression and blood supply provision for tissue expansion through angiogenesis. Here, we studied hyperplasia/hypertrophy of subcutaneous white adipose tissue (scWAT) concerning adipogenic gene expression, angiogenic status, and metabolic parameters in non-obese and different classes of obese individuals. METHODS: The scWAT samples were collected from 80 individuals. The anthropometric parameters, adipose tissue cell size, serum biochemistry, ER stress-induced XBP1 splicing, PPARγ2, SFRP1, WNT10B, and VEGFA gene expression levels were studied. In addition, the CD31 level was investigated by Western blotting. RESULTS: The obese individuals had greater waist circumferences and higher serum TG, TC, insulin, and HOMA-IR than the non-obese group. However, the largest adipocyte size, increased TNFα, insulin, and HOMA-IR, and the highest expression level of sXBP1, WNT10B, and VEGFA were observed in Class I obese individuals. It means that inflammation, insulin resistance, and ER stress accompany hypertrophic scWAT adipocytes with limited adipose tissue expansion ability. Furthermore, the Class II + III obese individuals showed high PPARγ2 expression and CD31 levels. There is adipogenesis through hyperplasia in this group. The SFRP1 expression was not significantly different in the studied groups. CONCLUSION: The results suggest that the capability of adipogenesis with inadequate angiogenesis is related to the metabolic status, inflammation, and ER function. Therefore, therapeutic strategies that support both angiogenesis and adipogenesis can effectively prevent the complications of obesity.

Protective effect of saffron carotenoids against amyloid beta-induced neurotoxicity in differentiated PC12 cells via the unfolded protein response and autophagy.

The preventive effect of saffron against Alzheimer's disease (AD) has been reported. Herein, we studied the effect of Cro and Crt, saffron carotenoids, on the cellular model of AD. The MTT assay, flow cytometry, and elevated p-JNK, p-Bcl-2, and c-PARP indicated the AßOs-induced apoptosis in differentiated PC12 cells. Then, the protective effects of Cro/Crt on dPC12 cells against AßOs were investigated in preventive and therapeutic modalities. Starvation was used as a positive control. RT-PCR and Western blot results revealed the reduced eIF2α phosphorylation and increased spliced-XBP1, Beclin1, LC3II, and p62, which indicate the AßOs-induced autophagic flux defect, autophagosome accumulation, and apoptosis. Cro and Crt inhibited the JNK-Bcl-2-Beclin1 pathway. They altered Beclin1 and LC3II and decreased p62 expressions, leading cells to survival. Cro and Crt altered the autophagic flux by different mechanisms. So, Cro increased the rate of autophagosome degradation more than Crt, while Crt increased the rate of autophagosome formation more than Cro. The application of 4µ8C and chloroquine as the inhibitors of XBP1 and autophagy, respectively, confirmed these results. So, augmentation of the survival branches of UPR and autophagy is involved and may serve as an effective strategy to prevent the progression of AßOs toxicity.

Effects of crocin and saffron aqueous extract on gene expression of SIRT1, AMPK, LOX1, NF-κB, and MCP-1 in patients with coronary artery disease: A randomized placebo-controlled clinical trial.

This trial evaluated the potential impacts of saffron aqueous extract (SAE) and its main carotenoid on some of the atherosclerosis-related gene expression and serum levels of oxidized low-density cholesterol (ox-LDL) and Monocyte chemoattractant protein 1 (MCP-1) in patients with coronary artery disease (CAD). Participants of this randomized controlled trial included 84 CAD patients who categorized into three groups: Group 1 received crocin (30 mg/day), Group 2 SAE (30 mg/day), and Group 3 placebo for 8 weeks. Gene expression of Sirtuin 1 (SIRT1), 5'-adenosine monophosphate-activated protein kinase (AMPK), Lectin-like oxidized LDL receptor 1 (LOX1), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and MCP-1 in peripheral blood mononuclear cells assessed by real-time PCR. Furthermore, serum ox-LDL and MCP-1 levels measured at the beginning and end of the intervention. Compared with the placebo group, gene expression of SIRT1 and AMPK increased significantly in the crocin group (p = .001), and the expression of LOX1 and NF-κB decreased significantly (p = .016 and .004, respectively). Serum ox-LDL levels decreased significantly in the crocin group after the intervention (p = .002) while MCP-1 levels decreased both in crocin and SAE groups (p = .001). Crocin may have beneficial effects on CAD patients by increasing the gene expression of SIRT1 and AMPK and decreasing the expression of LOX1 and NF-κB.

Saffron and natural carotenoids: Biochemical activities and anti-tumor effects.

Saffron, a spice derived from the flower of Crocus sativus, is rich in carotenoids. Two main natural carotenoids of saffron, crocin and crocetin, are responsible for its color. Preclinical studies have shown that dietary intake of some carotenoids have potent anti-tumor effects both in vitro and in vivo, suggesting their potential preventive and/or therapeutic roles in several tissues. The reports represent that the use of carotenoids without the potential for conversion to vitamin A may provide further protection and avoid toxicity. The mechanisms underlying cancer chemo-preventive activities of carotenoids include modulation of carcinogen metabolism, regulation of cell growth and cell cycle progression, inhibition of cell proliferation, anti-oxidant activity, immune modulation, enhancement of cell differentiation, stimulation of cell-to-cell gap junction communication, apoptosis and retinoid-dependent signaling. Taken together, different hypotheses for the antitumor actions of saffron and its components have been proposed such as a) the inhibitory effect on cellular DNA and RNA synthesis, but not on protein synthesis; b) the inhibitory effect on free radical chain reactions; c) the metabolic conversion of naturally occurring carotenoids to retinoids; d) the interaction of carotenoids with topoisomerase II, an enzyme involved in cellular DNA-protein interaction. Furthermore, the immunomodulatory activity of saffron was studied on driving toward Th1 and Th2 limbs of the immune system. In this mini-review, we briefly describe biochemical and immunological activities and chemo-preventive properties of saffron and natural carotenoids as an anticancer drug.

Chemo-immunotherapy using saffron and its ingredients followed by E7-NT (gp96) DNA vaccine generates different anti-tumor effects against tumors expressing the E7 protein of human papillomavirus.

Saffron and its components have been suggested as promising candidates for cancer prevention. Carotenoids and monoterpene aldehydes are two potent ingredients of saffron. The goal of the current study was to investigate the anti-tumor effect of chemo-immunotherapy using saffron and its ingredients followed by E7-NT (gp96) DNA vaccine against tumors expressing the E7 protein of human papillomavirus. The in vitro cytotoxic and apoptotic effects of aqueous saffron extract and its components were evaluated in malignant TC-1 and non-malignant COS-7 cell lines. Then, multimodality treatments using E7-NT (gp96) DNA vaccine combined with saffron extract and its ingredients as well as single-modality treatments were tested for their efficacy in inhibiting large and bulky tumor growth. Saffron and its components exerted a considerable anti-tumor effect through prevention of cell growth and stimulation of programmed cell death. Furthermore, 100 % of mice treated with crocin were tumor-free, in contrast to DNA vaccine alone (~66.7 %) and DNA + crocin (~33.3 %) indicating the high potency of crocin as a chemotherapeutic agent. Interestingly, the multimodality treatment using DNA vaccine along with picrocrocin augmented the anti-tumor effects of picrocrocin. Thus, the combination of DNA vaccine with saffron extract and crocin at certain concentrations did not potentiate protective and therapeutic effects compared to mono-therapies for the control of TC-1 tumors.

Design and characterization of a chimeric multiepitope construct containing CfaB, heat-stable toxoid, CssA, CssB, and heat-labile toxin subunit B of enterotoxigenic Escherichia coli: a bioinformatic approach.

Enterotoxigenic Escherichia coli (ETEC) strains are the most common cause of bacterial diarrhea in children in developing countries and travelers to these areas. Enterotoxins and colonization factors (CFs) are two key virulence factors in ETEC pathogenesis, and the heterogeneity of the CFs is the bottleneck in reaching an effective vaccine. In this study, a candidate subunit vaccine, which is composed of CfaB, CssA and CssB, structural subunits of colonization factor antigen I and CS6 CFs, labile toxin subunit B, and the binding subunit of heat-labile and heat-stable toxoid, was designed to provide broad-spectrum protection against ETEC. The different features of chimeric gene, its mRNA stability, and chimeric protein properties were analyzed by using bioinformatic tools. The optimized chimeric gene was chemically synthesized and expressed successfully in a prokaryotic host. The purified protein was used for assessment of bioinformatic data by experimental methods.

Saffron aqueous extract prevents metabolic syndrome in patients with schizophrenia on olanzapine treatment: a randomized triple blind placebo controlled study.

OBJECTIVE: The aim of this study was to assess whether saffron aqueous extract (SAE) or its active constituent, crocin, prevents olanzapine-induced metabolic syndrome (MetS) and insulin resistance in patients with schizophrenia. METHODS: 66 patients diagnosed with schizophrenia who were on olanzapine treatment (5-20 mg daily) were randomly allocated to receive a capsule of SAE (n=22; 30 mg daily), crocin (n=22; 30 mg daily) or placebo (n=22) in a 12-week triple-blind trial. Patients were screened not to have MetS at baseline and further assessment was done at weeks 6 and 12. Measurement of fasting blood glucose (FBS) and serum lipids were repeated at weeks 2, 6 and 12. Fasting blood levels of insulin and HbA1c were also measured at baseline and week 12. HOMA-IR and HOMA-ß were determined to evaluate insulin resistance. RESULTS: 61 patients completed the trial and no serious adverse effects were reported. Time-treatment interaction showed a significant difference in FBS in both SAE and crocin groups compared to placebo (p=0.004). In addition, SAE could effectively prevent reaching the criteria of metabolic syndrome (0 patients) compared to crocin (9.1%) and placebo (27.3%) as early as week 6. CONCLUSION: SAE could prevent metabolic syndrome compared to crocin and placebo. Furthermore, both SAE and crocin prevented increases in blood glucose during the study.

Co-administration of "L-Lysine, Vitamin C, and Zinc" increased the antioxidant activity, decreased insulin resistance, and improved lipid profile in streptozotocin-induced diabetic rats.

PURPOSE: We previously showed the beneficial effect of L-Lysine (Lys), a chemical chaperone, on reducing diabetic complications in diabetic rats and type 2 diabetic patients. Herein, we evaluated the effect of Lys co-administration with Vitamin C and Zinc (Lys+VC+Zn), in diabetic rats. METHODS: The streptozotocin (50 mg/Kg) was injected into male adult Wistar rats to induce diabetes. Then, different groups of normal and diabetic rats were treated with Lys and Lys+VC+Zn for five months. So, there were 0.1 % Lys in the drinking water of both groups. The control groups received water alone. During the experiment, the body weight, and various parameters were determined in the blood, serum/plasma, and urine of the rats. RESULTS: The determination of biochemical indexes confirmed diabetes induction and its complications in rats. Treatment with either Lys or Lys+VC+Zn resulted in reduced blood glucose and protein glycation (decreasing AGEs and HbA1c), increased insulin secretion, alleviated insulin resistance and HOMA-IR, improved lipid profile and HDL functionality (LCAT and PON1), enhanced antioxidant status (FRAP and AOPP), improved kidney function (decreased microalbuminuria, serum urea, and creatinine), and increased chaperone capacity (HSP70). Lys+VC+Zn showed better effects on these parameters than Lys alone. CONCLUSIONS: The results of this study indicated that co-administration of Lys, a chemical chaperone, with two antioxidants (VC and Zn) potentiates its antidiabetic effects and prevent diabetic complications in rat model of diabetes.

Cadmium nanocluster as a safe nanocarrier: biodistribution in BALB/c mice and application to carry crocin to breast cancer cell lines.

Aim: Metal nanoclusters are emerging nanomaterials applicable for drug delivery. Here, the toxicity and oxidative stress induction of divalent cationic cadmium (Cd2+) was compared with a Cd in the form of nanocluster. Then, it was used for targeted drug delivery into breast cancer cell lines. Methods: Using a green chemistry route, a Cd nanocluster (Cd-NC) was synthesized based on bovine serum albumin. After characterization, its genotoxicity and oxidative stress induction were studied in both in vitro and in vivo. After that, it was conjugated with hyaluronic acid (HA). The efficiency of hyaloronized-Cd-CN (HA-Cd-NC) for loading and releasing crocin (Cro), an anticancer phytochemical, was studied. Finally, it was applied for cell death induction in a panel of breast cancer cell lines. Results: The comet assay results indicated that, unlike Cd2+ and potassium permanganate (KMnO4), no genotoxicity and oxidative stress was induced by Cd-NC in vitro. Then, the pharmacokinetics of this Cd-NC was studied in vivo. The data showed that Cd-NC has accumulated in the liver and excreted from the feces of mice. Unlike Cd2+, no toxicity and oxidative stress were induced by this Cd-NC in animal tissues. Then, the Cd-NC was targeted toward breast cancer cells by adding HA, a ligand for the CD44 cell surface receptor. After that, Cro was loaded on HA-Cd-NC and it was used for the treatment of a panel of human breast cancer cell lines with varying degrees of CD44. The half-maximal drug inhibitory concentration (IC50) of Cro was significantly decreased when it was loaded on HA-Cd-NC, especially in MDA-MB-468 with a higher degree of CD44 at the surface. These results indicate the higher toxicity of Cro toward breast cancers when carried out by HA-Cd-NC. Conclusions: The Cd-NC was completely safe and is a promising candidate for delivering anticancer drugs/phytochemicals into the targeted breast tumors.

Anticancer effects of crocetin in both human adenocarcinoma gastric cancer cells and rat model of gastric cancer.

This study investigated the therapeutic effect of crocetin, a carotenoid derived from saffron, on gastric adenocarcinoma (AGS) cells and 1-methyl-3-nitro-1-nitrosoguanidine (MNNG)-induced gastric cancer in rats. An MTT assay showed a significant dose- and time-dependent inhibition of AGS cell proliferation as a result of crocetin administration. Flow cytometry and caspases activity assays revealed apoptosis had been induced in these cells; RT-PCR and Western blot analyses revealed the suppression of Bcl-2 and up-regulation of Bax expression in AGS cells treated with crocetin. These changes were not observed in normal human fibroblast (HFSF-PI3) cells. Pathological study of the tumor tissue in MNNG-induced gastric cancer in rats indicated the dose-dependent inhibition of tumor progression. In addition, crocetin reversed some changed biochemical parameters, including serum antioxidant activity and lactate dehydrogenase in rat serum. The present study demonstrates the antioxidant, anti-proliferative, and apoptotic activities of crocetin against gastric cancer that may benefit human stomach cancer treatment.

The protective effect of crocin on the amyloid fibril formation of Aß42 peptide in vitro.

Aß is the main constituent of the amyloid plaque found in the brains of patients with Alzheimer's disease. There are two common isoforms of Aß: the more common form, Aß40, and the less common but more amyloidogenic form, Aß42. Crocin is a carotenoid from the stigma of the saffron flower and it has many medicinal properties, including antioxidant effects. In this study, we examined the potential of crocin as a drug candidate against Aß42 amyloid formation. The thioflavin T-binding assay and electron microscopy were used to examine the effects of crocin on the extension and disruption of Aß42 amyloids. To further investigate the relationship between crocin and Aß42 structure, we analyzed peptide conformation using the ANS-binding assay and circular dichroism (CD) spectroscopy. An increase in the thioflavin T fluorescence intensity upon incubation revealed amyloid formation in Aß42. It was found that crocin has the ability to prevent amyloid formation by decreasing the fluorescence intensity. Electron microscopy data also indicated that crocin decreased the amyloid fibril content of Aß. The ANS-binding assay showed that crocin decreased the hydrophobic area in incubated Aß42. CD spectroscopy results also showed that the peptide undergoes a structural change to α-helical and ß-turn. Our study shows that the anti-amyloidogenic effect of crocin may be exerted not only by the inhibition of Aß amyloid formation but also by the disruption of amyloid aggregates. Therefore, crocin could be essential in the search for therapies inhibiting aggregation or disrupting aggregation.

Effect of fractionation on treatment outcome in local dual-frequency sonication and Dox-encapsulated nanomicelles.

PURPOSE: The goal of this study was to localize drug release from nanomicelles using dual-frequency sonication at low levels of acoustic intensity. METHODS: In this study, the antitumor effect of simultaneous dual-frequency sonication (28 kHz and 3 MHz) at low levels of acoustic intensity in combination with doxorubicin and micellar doxorubicin injection was assessed in a spontaneous model of breast adenocarcinoma in female Balb/c mice. Sixty-three tumor-bearing mice were randomly grouped into control, sham, dual-frequency sonication, doxorubicin injection with and without dual-frequency sonication, and micellar doxorubicin injection with and without dual-frequency sonication groups. RESULTS: The results of volume change relative to initial volume showed that in the micellar doxorubicin injection with sonication group, this parameter was significantly different from that of the control, sham, sonication, and doxorubicin injection groups (P < 0.05). In addition, the volume began to increase on the 15th day after the start of treatment, which is a good indication to repeat treatment; therefore, another group received an extra treatment on day 15. The animal life span in the micellar doxorubicin with sonication and repeated treatment groups was significantly higher than that in all the other experimental groups except for the micellar doxorubicin injection group (P < 0.05). CONCLUSION: It was concluded that dual-frequency sonication with micellar doxorubicin injection extends the life span relative to doxorubicin injection or dual-frequency sonication alone, and that repeating this treatment on day 15 decreases the rate of tumor growth significantly.

The Effect of Ultrasonication on the Fibrillar/ Oligomeric Structures of Aß1-42 at Different Concentrations.

The number of disease states linked the aberrant regular protein conformations to oligomers and amyloid fibrils. Amyloid beta 1-42 (Aß1-42) peptide is very hydrophobic and quickly forms the ß-rich structure and fibrillar protein aggregates in some solutions and buffer conditions. Ultrasonication pulses can disrupt amyloid fibrils to smaller fragments and produce Aß1-42 peptides of different sizes and oligomers. Herein, we investigated the effects of buffer and ultrasonication on Aß1-42 structure at low and high concentrations. After ultrasonication, the Western blot results showed that Aß1-42 fibrils were disaggregated into different sizes. The transmission electron microscopy results indicated Aß1-42 at low concentration (25 µM) in Ham's/F12 phenol red-free culture medium formed short-size fragments and oligomers. In comparison, Aß1-42 at higher concentration (100 µM) formed fibrils that break down into smaller fragments after ultrasonication. However, after regrowth, it formed mature fibrils again. Cell viability assay indicated that Aß1-42 oligomers formed at a low concentration (25 µM) were more toxic to PC12 cells than other forms. In conclusion, by applying ultrasonication pulses and controlling peptide concentration and buffer condition, we can rich Aß1-42 aggregates with a particular size and molecular structure.

Saffron carotenoids reversed the UCMS-induced depression and anxiety in rats: Behavioral and biochemical parameters, and hippocampal BDNF/ERK/CREB and NR2B signaling markers.

BACKGROUND: Depression is a debilitating condition that affects the mind and the individual's body. The improving effects of saffron on depression and anxiety have long been discussed, with limited information about the molecular mechanism of action. HYPOTHESIS/PURPOSE: Investigating the effect of saffron carotenoids, Crocin and Crocetin, on depression and anxiety in rats by emphasizing some signaling pathways involved. STUDY DESIGN: Depression and anxiety were induced in rats via unpredictable chronic mild stress (UCMS). Then different rat groups were treated with Crocin, Crocetin, Fluoxetine, and vehicle. Behavioral tests were done before and after treatment. METHODS: The serum Serotonin and Corticosterone and the expression of some hippocampal signaling proteins were studied. Furthermore, bioinformatics tools were used to predict the interactions of Crocin/ Crocetin with the Serotonin transporter and NMDA receptor subunit NR2B. Then, the patch-clamp was used to study the interaction of Crocetin with the NMDA receptor. RESULTS: Various behavioral tests confirmed the induction of depression and the improvement of depression by these natural carotenoids. In addition, Crocin/ Crocetin significantly increased the decreased serum Serotonin and reduced the increased serum Corticosterone in the depressed groups. They also increased or caused a trend of increase in the CREB, ERK, BAD, BDNF, p11, and 5-HT1B expression in the hippocampus of the depressed groups. In addition, there were an increase or a trend in p-CREB/CREB, p-ERK1/2 /ERK1/2, and p-BAD/BAD ratios in the Crocin/ Crocetin treated depressed groups. However, the NR2B and FOXO3a expression showed a trend of decrease in depressed groups after treatment. The bioinformatics data indicated that Crocin/ Crocetin could bind to the Serotonin transporter (SLC6A4) and NR2B subunit of the NMDA receptor. Both carotenoids bind to the same site as Fluoxetine in the SLC6A4. However, they bound to different sites on the NR2B. So, Crocetin binds to NR2B at the same site as Ifenprodil. But Crocin bound to another site. The whole cell patch-clamp recording on the normal rat hippocampus revealed a significant decrease in the NMDA peak amplitude after Crocetin treatment, indicating its inhibitory effect on this receptor. CONCLUSION: The antidepressant activities of Crocin/ Crocetin are possibly due to their effects on Serotonin and Corticosterone serum concentrations, NR2B expression, and the downstream signaling pathways. Furthermore, these natural carotenoids, like Fluoxetine, induced an increasing tendency in p11 and 5HT1B in depressed rats.

Glycine therapy inhibits the progression of cataract in streptozotocin-induced diabetic rats.

PURPOSE: The purpose of this paper was to investigate the effect of the oral administration of L-glycine (Gly) on the development of diabetic cataract induced by streptozotocin (STZ) in rats. METHODS: Two groups of male Wistar rats were intraperitoneally injected with a single dose of STZ (65 mg/kg bodyweight). Then, one group of diabetic rats and a control group were administered with 1% of Gly in drinking water for three months, ad libitum. Cataract development was monitored biweekly through ophthalmoscope inspection and was classified into four stages. At the end of 12 weeks, the animals were sacrificed and some biochemical parameters were determined in their lenses. The parameters include advanced glycation end products (AGEs), glycated proteins, total and soluble protein, glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), aldose reductase (AR), and sorbitol dehydrogenase (SDH). Some parameters were also determined in the serum and blood of the rats. RESULTS: Diabetic cataract gradually progressed in the STZ-administered group with no other treatment. Consequently, up to the end of the experiment, 2/3 of the animals in this group reached to the last stage of the cataract (mature cataract). The progress of this process was much slower in the diabetic group that was treated with Gly. At the end of the study, the visual cataract score was significantly lower in the diabetic group treated with Gly compared to those administered with STZ. Some lens parameters, including glycated proteins, AGEs, SOD, and AR activities, were increased while some others, including soluble and total protein, GSH level, and CAT activity, were decreased due to diabetes induction. After Gly treatment, all the above-named parameters had reverse changes except for the CAT activity. The SDH activity in the lenses had no changes due to diabetes or treatment. In addition, this treatment significantly decreased the amount of serum glucose (Glc), serum AGEs, and glycated hemoglobin (HbA1c) in the diabetic rats. Gly also increased the ferric reducing antioxidant power (FRAP) in the serum of diabetic rats. However, the decreased bodyweight of animals due to diabetes induction was not compensated by Gly administration. It is important to note that Gly had no effect on normal rat parameters. CONCLUSIONS: The results indicated that the oral administration of Gly significantly delayed the onset and the progression of diabetic cataract in rats. These effects were due to its antiglycating action and to a lesser extent, due to the inhibition of oxidative stress and polyol pathway.

The in silico mechanism of hVKOR interaction with acetaminophen and its metabolite, as well as N-acetyl cysteine: caution on application in COVID-19 patients.

Acetaminophen and N-acetyl cysteine (NAC) are being used as supportive care in patients suffering from coronavirus disease 2019 (COVID-19). The coagulopathy and cerebral hemorrhage have been recently reported in these patients. Prolonged acetaminophen use increases the international normalized ratio (INR) and the risk of bleeding among patients taking anti-coagulants. Inhibition of vitamin K epoxide reductase (VKOR) by acetaminophen and NAC in chronic applications has been reported, however, detailed knowledge of the molecular mechanism and binding sites are not clear. Herein, we built the homology model of human VKOR (hVKOR) using ITASSER server, confirmed, and applied it for docking analysis of its interaction with acetaminophen and its metabolite, N-acetyl-p-benzoquinone imine (NAPQI), and NAC. We also calculated the lipophilicity and predicted the blood-brain-barrier (BBB) permeation of NAPQI by Swiss ADME. Our analysis showed that NAPQI and NAC, but not acetaminophen, bind strongly to the similar sites in hVKOR via both hydrogen and van der Waals bonding; particularly with Cys135. Thus, it interrupted the vitamin K reducing electron transfer pathway. Further, molecular dynamic (MD) simulation study revealed that the interactions of the ligands with hVKOR are stable. In conclusion, our analysis shed a light on the molecular mechanism of acetaminophen-induced coagulopathy previously reported in some clinical cases with chronic acetaminophen use. Furthermore, considering the anti-coagulopathy of NAPQI and NAC but not acetaminophen, the BBB permeation potency of these agents, and the risk of coagulopathy in COVID-19, we suggest a regular prothrombin time (PT) and INR monitoring of these patients taking acetaminophen and/or NAC.Communicated by Ramaswamy H. Sarma.

Possible role of WNT10B in increased proliferation and tubule formation of human umbilical vein endothelial cell cultures treated with hypoxic conditioned medium from human adipocytes.

Regulation of angiogenesis plays an important role in adipose tissue expansion and function. The Wnt pathway and WNT10B, the main member of Wnt family, participate in angiogenesis in cancer tumors, but there is limited evidence to support the regulatory role of WNT10B in human adipose tissue angiogenesis. Subcutaneous white adipose tissue (scWAT) of 80 participants including obese and non-obese subjects was obtained and the expression of WNT10B and VEGFA genes were evaluated using qPCR. Human adipose-derived stem cells (hADSC) were differentiated to adipocytes and incubated under either hypoxic or normoxic conditions. The conditioned media of these adipocytes were collected and used as growth media for human umbilical vein endothelial cells (HUVEC) in Matrigel. We evaluated the proliferation, cell cycle phases, tubule formation and ß-catenin activation of these treated cells. We found a significant correlation between WNT10B and VEGFA expression in the scWAT of both obese and non-obese subjects. Proliferation and tubule formation of HUVEC treated with conditioned media of hypoxic adipocytes (hCM) in the S-phase were increased significantly compared to the HUVEC treated with the conditioned media of normoxic adipocytes (nCM). The expression of WNT10B and VEGFA was enhanced in hypoxic adipocytes compared to normoxic adipocytes; also, activation and nuclear translocation of ß-catenin was enhanced in the HUVEC treated with hCM compared to nCM. WNT10B acts as an angiogenic protein in scWAT under hypoxic conditions. Hypoxia induced WNT10B increases VEGFA expression and causes tube formation by HUVECs and angiogenesis in adipose tissue via the canonical Wnt/ß-catenin pathway.

The Role of BiP and the IRE1α-XBP1 Axis in Rhabdomyosarcoma Pathology.

BACKGROUND: Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma in children, and is associated with a poor prognosis in patients presenting with recurrent or metastatic disease. The unfolded protein response (UPR) plays pivotal roles in tumor development and resistance to therapy, including RMS. METHODS: In this study, we used immunohistochemistry and a tissue microarray (TMA) on human RMS and normal skeletal muscle to evaluate the expression of key UPR proteins (GRP78/BiP, IRE1α and cytosolic/nuclear XBP1 (spliced XBP1-sXBP1)) in the four main RMS subtypes: alveolar (ARMS), embryonal (ERMS), pleomorphic (PRMS) and sclerosing/spindle cell (SRMS) RMS. We also investigated the correlation of these proteins with the risk of RMS and several clinicopathological indices, such as lymph node involvement, distant metastasis, tumor stage and tumor scores. RESULTS: Our results revealed that the expression of BiP, sXBP1, and IRE1α, but not cytosolic XBP1, are significantly associated with RMS (BiP and sXBP1 p-value = 0.0001, IRE1 p-value = 0.001) in all of the studied types of RMS tumors (n = 192) compared to normal skeletal muscle tissues (n = 16). In addition, significant correlations of BiP with the lymph node score (p = 0.05), and of IRE1α (p value = 0.004), cytosolic XBP1 (p = 0.001) and sXBP1 (p value = 0.001) with the stage score were observed. At the subtype level, BiP and sXBP1 expression were significantly associated with all subtypes of RMS, whereas IRE1α was associated with ARMS, PRMS and ERMS, and cytosolic XBP1 expression was associated with ARMS and SRMS. Importantly, the expression levels of IRE1α and sXBP1 were more pronounced in ARMS than in any of the other subtypes. The results also showed correlations of BiP with the lymph node score in ARMS (p value = 0.05), and of sXBP1 with the tumor score in PRMS (p value = 0.002). CONCLUSIONS: In summary, this study demonstrates that the overall UPR is upregulated and, more specifically, that the IRE1/sXBP1 axis is active in RMS. The subtype and stage-specific dependency on the UPR machinery in RMS may open new avenues for the development of novel targeted therapeutic strategies and the identification of specific tumor markers in this rare but deadly childhood and young-adult disease.

New applications and mechanisms of action of saffron and its important ingredients.

Saffron (Crocus sativus L.) has been an important subject of interest for research teams in the past two decades because of its various biological properties. Chemical analysis has shown the presence of more than 150 components in saffron stigmas. Here, we review the medicinal and industrial applications of saffron. Then, the new findings from different research groups about its medicinal properties and various cellular and molecular mechanisms of action will be discussed. The methods used for this study included searching Web of Science and Medline for saffron and its constituent's applications. The results show that in recent years saffron's application in a variety of disorders involving neuronal, cardiovascular and other systems, as well as cancer have been investigated. The cellular and molecular mechanisms of its action are also under study. The more powerful components of saffron are carotenoids and monoterpene aldehydes. Structure-function relationship studies show that some properties are related to deglycosylated derivatives, while others belong to more glycosylated ones. Our study concludes that saffron has a wide range of usefulness in medicine, cosmetics, and coloring industries, so it can be used for new drug designs. However, more research about its mechanism of action is needed.

The effect of hot-tub therapy on serum Hsp70 level and its benefit on diabetic rats: a preliminary report.

PURPOSE: To carry out a preliminary study examining the efficacy of long-term hot-tub therapy (HTT) in the improvement of diabetic complications on streptozotocin-induced diabetic rats. MATERIALS AND METHODS: Male Wistar rats were immersed mid-sternum in a circulating water bath (42 degrees C for 30 min) to obtain a core body temperature of 41 degrees C; this process was repeated three times a week for 5 months. The blood was collected every month. Multiple parameters were examined for all rats including heat shock protein (Hsp70) level, serum glucose and insulin concentrations, advanced glycation end product (AGE) and glycated haemoglobin (HbA1c) formation, lipid profile and antioxidant defence system. Additionally, the chaperoning capacity of glycated Hsp70 was evaluated based on in vitro studies in which the refolding of denatured luciferase was compared to refolding by native Hsp70. RESULTS: HTT-treated diabetic rats showed a significant improvement in lipid profile, antioxidant capacity, insulin secretion and serum Hsp70 level and a significant decrease in AGE formation compared to the untreated diabetic rats. However, HTT had a borderline significant effect on weight and fasting blood glucose. Glycated Hsp70 lost its chaperoning ability to reactivate the denatured luciferase. CONCLUSION: A decrease in complications in diabetic rats after hot-tub therapy is shown here. An increase in the extracellular Hsp70 level due to HTT was observed. This increase may serve to protect the structure of proteins (e.g. preventing AGE formation), and the observed beneficial effects may be related to it.