Hypoxia has been suggested as an important pathophysiological feature in varicocele disease. On the other hand, the expression of hypoxia-inducible factor 1-alpha (HIF1-α) is associated with the incidence of hypoxia. In this study, we investigated the expression of HIF1-α in varicocele disease through a comprehensive systematic review. We searched PubMed, Scopus, Web of Science, and Embase databases to identify the related studies published up to February 2021. Human studies have demonstrated an increase in the HIF-1α protein expression in the internal spermatic vein (ISV) of the varicocele testicle. HIF-1α mRNA expression in the seminal plasma was significantly higher in infertile varicocele patient compared with fertile ones. Similarly, most animal studies demonstrated a significant increase in HIF-1α gene and protein expression in varicocele testicular tissue compared with control groups. The studies illustrated that hypoxia followed by increased expression of hypoxia-inducible factor 1-alpha (HIF1-α) mRNA and protein occurs in varicocele disease. Expression of HIF-1α regulates the expression of many genes, including VEGF, p53, GLUT, Bax, and Caspase-3, that could be involved in many of the varicocele pathophysiological effects such as DNA fragmentation and apoptosis of sperm cells. Further studies with a large number of patients are necessary and can provide more definitive evidence.

Keywords: HIF1-α; Hypoxia; Systematic review; Varicocele.

Galaxolide and tonalide are well-known polycyclic musks whose intensive use without limitations in numerous cleaning, hygiene, and personal care products has resulted in widespread direct human exposure via absorption, inhalation, and oral ingestion. Latest data shows that long-term, low-dose exposure to toxic chemicals can induce unpredictable harmful effects in a variety of living systems, however, interactions between synthetic musks and brain tumours remain largely unexplored. Glioblastoma (GB) accounts for nearly half of all tumours of the central nervous system and is characterized by very poor prognosis. The aims of this study were (1) to investigate the potential effect of long-term (20-generation) single and combined application of galaxolide and tonalide at sub-lethal doses (5-2.5 u M) on the angiogenesis, invasion, and migration of human U87 cells or tumour spheroids, and (2) to explore the underlying molecular mechanisms. Random amplified polymorphic DNA assays revealed significant DNA damage and increased total mutation load in galaxolide- and/or tonalide-treated U87 cells. In those same groups, we also detected remarkable tumour spheroid invasion and up-regulation of both HIF1-α/VEGF/MMP9 and IL6/JAK2/STAT3 signals, known to have important roles in hypoxia-related angiogenesis and/or proliferation. Prolonged musk treatment further altered angio-miRNA expression in a manner consistent with poor prognosis in GB. We also detected significant over-expression of the genes Slug, Snail, ZEB1, and Vimentin, which are biomarkers of epithelial to mesenchymal transition. In addition, matrigel, transwell, and wound healing assays clearly showed that long-term sub-lethal exposure to galaxolide and/or tonalide induced invasion and migration proposing a high metastatic potential. Our results suggest that assessing expression of HIF-1a, VEGF, STAT3, and the miR-17-92 cluster in biopsy samples of GB patients who have a history of possible long-term exposure to galaxolide or tonalide could be beneficial for deciding a therapy regime. Additionally, we recommend that extensively-used hygiene and cleaning materials be selected from synthetic musk-free products, especially when used in palliative care processes for GB patients.

Keywords: Angio-miRs; Galaxolide (HHCB); Glioblastoma; Metastatic potential; Tonalide (AHTN); U87 MG cells.

Reoxygenation of hypoxic cardiac myocytes can paradoxically induce myocardial injury and affect the recovery processes. Pharmacological postconditioning is an efficient strategy used in clinical practice that protects cardiomyocytes from hypoxia/reoxygenation (HR) injury. Natural products or foods have been known to possess effective cardioprotective properties. Majonoside-R2 (MR2) is a dominant saponin component of Vietnamese ginseng that has several biological effects. In this study, we evaluated the protective effect of MR2 on HR-stimulated cardiomyocytes and investigated the related molecular mechanisms. H9C2 cardiomyocytes were exposed to HR conditions with or without MR2 supplementation. Samples from experimental groups were used to analyze the expression of apoptosis- and activating reperfusion injury salvage kinase (RISK)-related factors in response to HR injury by using enzyme-linked immunosorbent assay, real-time polymerase chain reaction, and western blotting. Post-treatment, MR2 enhanced cell viability under HR conditions. We found that MR2 suppressed the expression of hypoxia-inducible factor 1-alpha (HIF1α) and transforming growth factor beta 1 (TGFβ1), modulated Akt/GSK3ß/cAMP response element-binding signaling, and regulated gene expression related to apoptosis (B cell lymphoma-extra-large [Bcl-xl], Bcl-2 homologous killer [Bak], Bcl-2 associated X [Bax], and connexin 43 [Cnx43]). Thus, the present findings demonstrate that MR2 protects cardiomyocytes against HR injury by suppressing the expression of HIF1α and activating the RISK pathway.

Keywords: antiapoptosis; cardioprotection; hypoxic injury; majonoside-R2.

Basal-like breast cancer (BLBC) is the most aggressive subtype of breast tumors with poor prognosis and limited molecular-targeted therapy options. We show that BLBC cells have a high Cys demand and reprogrammed Cys metabolism. Patient-derived BLBC tumors from four different cohorts exhibited elevated expression of the transsulfuration enzyme cystathione β-synthetase (CBS). CBS silencing (shCBS) made BLBC cells less invasive, proliferate slower, more vulnerable to oxidative stress and cystine (CySSCy) deprivation, prone to ferroptosis, and less responsive to HIF1-α activation under hypoxia. shCBS xenograft tumors grew slower than controls and exhibited impaired angiogenesis and larger necrotic areas. Sulfur metabolite profiling suggested that realigned sulfide/persulfide-inducing functions of CBS are important in BLBC tumor progression. Supporting this, the exclusion of serine, a substrate of CBS for producing Cys but not for producing sulfide/persulfide, did not exacerbate CySSCy deprivation-induced ferroptosis in shCBS BLBC cells. Impaired Tyr phosphorylation was detected in shCBS cells and xenografts, likely due to persulfidation-inhibited phosphatase functions. Overexpression of cystathione γ-lyase (CSE), which can also contribute to cellular sulfide/persulfide production, compensated for the loss of CBS activities, and treatment of shCBS xenografts with a CSE inhibitor further blocked tumor growth. Glutathione and protein-Cys levels were not diminished in shCBS cells or xenografts, but levels of Cys persulfidation and the persulfide-catabolizing enzyme ETHE1 were suppressed. Finally, expression of enzymes of the oxidizing Cys catabolism pathway was diminished, but expression of the persulfide-producing CARS2 was elevated in human BLBC tumors. Hence, the persulfide-producing pathways are major targetable determinants of BLBC pathology that could be therapeutically exploited.

Keywords: basal-like breast cancer; cystathione β-synthetase; hydrogen sulfide; persulfide; transsulfuration.

Although doxorubicin (DOX) is used in many cancer treatments, it causes neurotoxicity. In this study, the effect of thymoquinone (THQ), a powerful antioxidant, on DOX-induced neurotoxicity was evaluated. In total, 40 rats were used and 5 groups were formed. Group I: control group (n = 8); Group II: olive oil group (n = 8); Group III: the THQ group (n = 8); THQ 10 mg/kg per day was given intraperitoneally (i.p.) throughout the experiment; group IV: DOX group (n = 8); On Day 7 of the experiment, a single dose of 15 mg/kg intraperitoneally DOX injected; group V: DOX + THQ group (n = 8); Throughout the experiment, 10 mg/kg THQ per day and intraperitoneally 15 mg/kg DOX on Day 7 were injected. Immunohistochemically, tumor necrosis factor-α (TNF-α), interleukin-17 (IL-17), hypoxia-inducible factor 1α (HIF1-α), glucose regulatory protein 78 (GRP78), and the gene inducible by growth arrest and DNA damage 153 (GADD153) proteins were evaluated in the brain cortex, medulla, and hippocampus regions. Total oxidant status (TOS) levels and total antioxidant status (TAS) in the brain tissue were measured. TNF-α, IL-17, HIF1-α, GRP78, and GADD153 immunoreactivities significantly increased in the DOX group in the study. THQ significantly reduced these values. THQ increased the TAS level significantly and decreased the TOS level significantly compared to the DOX group. THQ may play a role as a neuroprotective agent in DOX-induced neurotoxicity in the cortex, medulla, and hippocampus regions of the brain.

Keywords: doxorubicin; endoplasmic reticulum stress; inflammation; neurotoxicity; thymoquinone.

Maximum and minimum Critical thermal limits (CTMax and CTMin) have been studied extensively to assess thermal tolerance in ectotherms by means of ramping assays. Notothenioid fish have been proposed as particularly sensitive to temperature increases related to global climate change. However, there are large gaps in our understanding of the thermal responses of these extreme cold-adapted fish in assays with heating rates. We evaluated the effects of two commonly used heating rates (0.3 and 1 °C/min) on the cellular stress responses in the intertidal Antarctic fish Harpagifer antarcticus immediately after CTMax was reached, and at 2 and 4 h of recovery time in ambient water. We compared CTMax values, the relative transcript expression of genes relvant to heat shock response (Hsc70, Hsp70, Grp78), hypoxia (Hif1-α, LDHa, GR), ubiquitination (Ube2), and apoptosis (SMAC/DIABLO), and five plasma parameters - glucose, lactate, total protein, osmolality and cortisol. CTMax values between the two heating rates are not significantly different, and both rates elicited a similar stress response at molecular and physiological levels. We found a lack of up-regulated response of heat shock proteins, consistent with other Antarctic notothenioids. The general transcriptional pattern trended to downregulation, which was more evident in the slower 0.3 °C/min rate, and instances of upregulation were mainly related to ubiquitination. The faster 1 °C/min rate, rarely used for Antarctic fish, can be suitable for studying cold-adapted stenothermic fish without overestimating thermal tolerance or inducing damage from longer heat exposure.

Keywords: Ectotherms; Global climate change; Physiology; Plunder fish; mRNA.

Purpose: Diabetic retinopathy (DR) is one of the leading causes of blindness in working-aged people. Few studies were on the relationship between S100 Calcium Binding Protein A9 (S100A9) protein and DR, and none on endothelial cells induced by tasquinimod in high glucose. Therefore, we assessed the relationship between tasquinimod and S100A9 in DR.

Methods: DR pathogenesis was simulated using high-glucose-induced human retinal endothelial cells (HRECs) to study the mRNA expression of s100a9, thrombospondin-1 (tsp-1), hypoxia-inducible factor 1-alpha (hif1-α), intercellular adhesion molecule 1 (icam-1), and vascular endothelial growth factor (vegf) after tasquinimod treatment. The protein expression of S100A9, TSP-1, extracellular signal-regulated kinase (ERK), ICAM-1 and VEGF was also analyzed.

Result: A total of 28 eyes of 26 patients were included in this experiment. A significantly higher expression of S100A9 as well as enhanced proliferation and mobility was observed in the high-glucose-treated HRECs compared with that in low-glucose-treated cells. However, these were significantly inhibited when treated with high glucose with 50 μM tasquinimod. The mRNA expression of tsp-1 was increased, whereas that of hif1-α, icam-1 and vegf was decreased after tasquinimod treatment. Western blot indicated the increased TSP-1 but decreased ERK, ICAM-1 and VEGF expression after treating with tasquinimod.

Conclusion: High glucose promoted the expression of s100a9, S100A9 protein in DR patients and HRECs. Tasquinimod inhibited the proliferation, migration and lumen formation of HRECs under a high glucose environment. Tasquinimod might play a vital role in inhibiting angiogenesis through inducing TSP-1 and inhibiting VEGF, ICAM-1 and ERK.

Keywords: Diabetic retinopathy; Human retinal endothelial cell; S100A9; Tasquinimod.

Hypoxia is a universal pathological feature of solid tumors. Hypoxic tumor cells acquire metastatic and lethal phenotypes primarily through the activities of hypoxia-inducible factor 1 alpha (HIF1α). Therefore, HIF1α is considered as a promising therapeutic target. However, HIF inhibitors have not proven to be effective in clinical testing. The underlying mechanism is unclear. We report that oncogenic protein ID1 is upregulated in hypoxia by HIF1α shRNA or pharmacological inhibitors. In turn, ID1 supports tumor growth in hypoxia in vitro and in xenografts in vivo, conferring adaptive survival response and resistance. Mechanistically, ID1 proteins interfere HIF1-mediated gene transcription activation, thus ID1 protein degradation is accelerated by HIF1α-dependent mechanisms in hypoxia. Inhibitions of HIF1α rescues ID1, which compensates the loss of HIF1α by the upregulation of GLS2 and glutamine metabolism, thereby switching the metabolic dependency of HIF1α -inhibited cells from glucose to glutamine.

Keywords: HIF1; ID1; hypoxia; resistance; targeted-treatment.

The aim of this study was to investigate differences in skeletal muscle gene expression of highly trained endurance and strength athletes in comparison to untrained individuals at rest and in response to either an acute bout of endurance or strength exercise. Endurance (ET, n = 8, VO₂max 67 ± 9 mL/kg/min) and strength athletes (ST, n = 8, 5.8 ± 3.0 training years) as well as untrained controls (E-UT and S-UT, each n = 8) performed an acute endurance or strength exercise test. One day before testing (Pre), 30 min (30'Post) and 3 h (180'Post) afterwards, a skeletal muscle biopsy was obtained from the m. vastus lateralis. Skeletal muscle mRNA was isolated and analyzed by Affymetrix-microarray technology. Pathway analyses were performed to evaluate the effects of training status (trained vs. untrained) and exercise mode-specific (ET vs. ST) transcriptional responses. Differences in global skeletal muscle gene expression between trained and untrained were smaller compared to differences in exercise mode. Maximum differences between ET and ST were found between Pre and 180'Post. Pathway analyses showed increased expression of exercise-related genes, such as nuclear transcription factors (NR4A family), metabolism and vascularization (PGC1-α and VEGF-A), and muscle growth/structure (myostatin, IRS1/2 and HIF1-α. The most upregulated genes in response to acute endurance or strength exercise were the NR4A genes (NR4A1, NR4A2, NR4A3). The mode of acute exercise had a significant effect on transcriptional regulation Pre vs. 180'Post. In contrast, the effect of training status on human skeletal muscle gene expression profiles was negligible compared to strength or endurance specialization. The highest variability in gene expression, especially for the NR4A-family, was observed in trained individuals at 180'Post. Assessment of these receptors might be suitable to obtain a deeper understanding of skeletal muscle adaptive processes to develop optimized training strategies.

Keywords: endurance exercise; microarray; molecular muscle adaptations; pathway analysis; strength exercise; training status; transcriptional regulation.

Background: A low tissue oxygen level, < 1% O₂, is a typical characteristic inside of solid tumors in head and neck cancer (HNSCC) affecting a wide array of cell populations, such as macrophages. However, the mechanisms of how hypoxia influences macrophages are not yet fully elucidated. Our research aimed to study the effect of soluble mediators produced by hypoxic cancer cells on macrophage polarization. Furthermore, we studied the effect of a hypoxic microenvironment on the expression of tumorigenic toll-like receptor 9 (TLR9) and the consecutive macrophage polarization.

Methods: Conditioned media (CM_NOX or CM_HOX) from cell lines UT-SCC-8, UT-SCC-74A, FaDu, MDA-MB-231 and HaCat cultured under normoxic (21% O₂) and hypoxic (1% O₂) conditions were used to polarize human monocyte-derived macrophages. Macrophage polarization was measured by flow cytometry and the production of cytokine mRNA using Taqman qPCR. To study the role of TLR9 in macrophage polarization, the lentiviral CRISPR/Cas9 method was used to establish a stable FaDu^TLR9def clone.

Results: Our results demonstrate that the soluble mediators produced by the cancer cells under normoxia polarize macrophages towards a hybridized M1/M2a/M2c phenotype. Furthermore, the results suggest that hypoxia has a limited role in altering the array of cancer-produced soluble factors affecting macrophage polarization and cytokine production. Our data also indicates that increased expression of TLR9 due to hypoxia in malignant cells does not markedly influence the polarization of macrophages. TLR9 transcriptional response to hypoxia is dissimilar to a HIF1-α-regulated LDH-A. This may indicate a context-dependent expression of TLR9 under hypoxia.

Conclusions: HNSCC cell lines affect both macrophage activity (polarization) and functionality (cytokines), but with exception to iNOS expression, the effects appear independent of hypoxia and TLR9.

Keywords: Anti-cancer immunomodulation; Head and neck squamous cell carcinoma; Hypoxia; Immune evasion; Immunoediting; Innate immune response; Macrophage polarization; TLR9.

Hypoxic conditions induce the activation of hypoxia-inducible factor-1α (HIF-1α) to restore the supply of oxygen to tissues and cells. Activated HIF-1α translocates into the nucleus and binds to hypoxia response elements to promote the transcription of target genes. Cathepsin L (CTSL) is a lysosomal protease that degrades cellular proteins via the endolysosomal pathway. In this study, we attempted to determine if CTSL is a hypoxia responsive target gene of HIF-1α, and decipher its role in melanocytes in association with the autophagic pathway. The results of our luciferase reporter assay showed that the expression of CTSL is transcriptionally activated through the binding of HIF1-α at its promoter. Under autophagy-inducing starvation conditions, HIF-1α and CTSL expression is highly upregulated in melan-a cells. The mature form of CTSL is closely involved in melanosome degradation through lysosomal activity upon autophagosome-lysosome fusion. The inhibition of conversion of pro-CTSL to mature CTSL leads to the accumulation of gp100 and tyrosinase in addition to microtubule-associated protein 1 light chain 3 (LC3) II, due to decreased lysosomal activity in the autophagic pathway. In conclusion, we have identified that CTSL, a novel target of HIF-1α, participates in melanosome degradation in melanocytes through lysosomal activity during autophagosome-lysosome fusion.

Keywords: Cathepsin L; autophagy; hypoxia-inducible factor-1-alpha; melanosome.

Background: ZMYND8 (Zinc finger MYND (Myeloid, Nervy and DEAF-1)-type containing 8) has been known to play an important role in tumor regulation in various types of cancer. However, the results of ZMYND8 expression and their clinical significance in hepatocellular carcinoma (HCC) have not yet been published. In the present study, we investigate the expression of ZMYND8 protein and mRNA in HCC and elucidate its prognostic significance.

Methods: ZMYND8 protein and mRNA expression in 283 and 234 HCCs were investigated using immunohistochemistry and microarray gene expression profiling data. The relationships between ZMYND8 expression with clinicopathologic features and prognosis of HCC patients were evaluated. Furthermore, we performed the invasion, migration, apoptosis, soft agar formation assay and sphere formation assay in HCC cell lines, and evaluated tumorigenicity in a nude mouse model, after ZMYND8 knockdown.

Results: Overexpression of ZMYND8 protein and mRNA was observed in 20.5% and 26.9% of HCC cases, respectively. High ZMYND8 expression showed significant correlations with microvascular invasion, high Edmondson grade, advanced American Joint Committee on Cancer, and increased alpha-fetoprotein level. ZMYND8 mRNA overexpression was an independent prognostic factor for predicting early recurrence as well as short recurrence-free survival (RFS). Downregulation of ZMYND8 reduced migration and invasion of HCC cells, and promoted apoptosis of HCC cells in an in vitro model. In a xenograft nude mouse model, knockdown of ZMYND8 significantly reduced tumor growth.

Conclusion: ZMYND8 mRNA overexpression could be a prognostic marker of shorter RFS in HCC patients after curative resection. ZMYND8 might play an important role in the proliferation and progression of HCC and could be a promising candidate for targeted therapy.

Keywords: HIF1; Hepatocellular carcinoma; Prognosis; RACK7; Target; ZMYND8.

Muscle is a contractile tissue responsible for maintaining posture and the movement of all parts of the body. Prolonged oxidizative stress can lead to the damage of cells, tissues, and organs. In this study, we investigated the possibility of oxidative stress in the process of myoblast differentiation of C2C12 cells. First, the myoblast differentiation model of C2C12 cells was constructed and verified by Giemsa staining. The expression of hypoxia inducible factor1-alpha (HIF1-α), hypoxia inducible factor1-beta (HIF1-β), Von Hippel-Lindau (VHL), lysyl oxidase (Lox), EGL-9 family hypoxia-inducible factor 1 (EGLN1), proline 4-hydroxylase alpha 1 (P4HA1) and heme oxygenase-1 (HOMX1) in the process of myoblast differentiation was verified by in vitro experiments and Gene Expression Omnibus (GEO) bioinformatic analysis. We found that with the increased expression of myogenic factor 5 (MYF5), myogenic differentiation 1 (MYOD1), and Desmin, myotube fusion became more obvious during the process of C2C12 cell differentiation. Both experimental and GEO analysis indicated that the expression of HIF1-α, HIF1-β, VHL, LOX, EGLN1 and P4HA1 increased, and the expression of HOMX1 decreased during myogenic differentiation. Therefore, we suggest that the myoblast differentiation of C2C12 cells may be related to oxidative stress. Their possible relationship was proposed, though further studies are needed.

Keywords: C2C12 cells; myoblast differentiation; oxidative stress.

Delayed injured nerve regeneration remains a clinical problem, partly ascribing to the lack of regulation of regenerative microenvironment, topographical cues, and blood nourishment. Functional electrospun conduits have been established as an efficacious strategy to facilitate nerve regeneration by providing structural guidance, regulating the regenerative immune microenvironment, and improving vascular regeneration. However, the synthetic polymers conventionally used to fabricate electrospinning scaffolds, such as poly(L-lactic acid), poly(glycolic acid), and poly(lactic-co-glycolic acid), can cause aseptic inflammation due to acidic degradation products. Therefore, a poly[3(S)-methyl-morpholine-2,5-dione-co-lactic] [P(MMD-co-LA)] containing alanine units with good mechanical properties and reduced acid degradation products, was obtained by melt ring-opening polymerization (ROP). Here, we aimed to explore the effect of oriented nanofiber/Deferoxamine (DFO, a hydrophilic angiogenic drug) scaffold in the rapid construction of a favorable regenerative microenvironment, including cell bridge, polarized vascular system, and immune microenvironment. In vitro studies have shown that the scaffold can sustainably release DFO, which accelerates the migration and tube formation of human umbilical vein endothelial cells (HUVECs), as well as the expression of genes related to angiogenesis. The physical clues provided by the arranged nanofibers can regulate the polarization of macrophages and reduce the expression of inflammatory factors. Furthermore, the in vivo results demonstrated a higher M2 polarization level of the oriented nanofibrous scaffold treatment group with reducedinflammation reaction in the injured nerve. Moreover, the in-situ release of DFO up-regulated the expression of HIF1-α and SDF-1α genes, as well as the expression of HIF1-α's target gene VEGF, further promoting revascularization and enhancing nerve regeneration at the defect site. The obtained results provide essential insights on accelerating the creation of the nerve regeneration microenvironment by combining the physiological processes of nerve regeneration with topographical cues and chemical signal induction.

Keywords: Aligned nanofibers; Angiogenesis; Deferoxamine; Macrophage polarization; Nerve regeneration; P(MMD-co-LA).

Background: Biological markers expressed in cancer cells and the surrounding cancer-associated fibroblasts (CAF) can be used for prediction of patient prognosis in colorectal cancer (CRC). Here, we used immunohistochemical techniques to evaluate cancer cells' expression of specific biomarkers that are closely associated with neoplastic progression.

Methods: Immunohistochemical markers included Ki-67, p53, β-catenin, MMP7, E-cadherin and HIF1-α. We also characterized microenvironmental markers expressed by CAF, including expression of α-smooth muscle actin, CD10, podoplanin, fibroblast specific protein 1, platelet derived growth factor β, fibroblast association protein, tenascin-C (TNC), ZEB1 and TWIST1. The study population consisted of 286 CRC patients with stage II and III disease. Stage II and III CRC were divided into a first and a second cohort (for validation). The CRCs were stratified using cluster analysis. To identify the utility of prognostic markers in stage II and III CRC, univariate and multivariate analyses were performed in both cohorts.

Results: Stage II and III CRCs were stratified into 3 subgroups. Specific subgroups were significantly correlated to disease-free survival using univariate and multivariate analyses in the first cohort. High expression of TNC was identified as a single prognostic marker in both cohorts by univariate and multivariate analyses.

Conclusions: We suggest that the presence of a specific subgroup defined by multiple markers can be used for prediction of CRC outcome in stages II and III. In addition, we showed that high expression of TNC was correlated with a poorer prognosis in stages II and III of CRC.

Keywords: cancer-associated fibroblast; cluster analysis; colorectal cancer; prognostic marker; tenascin-C.

Purpose: Although radiation is one of the basic methods commonly used in cancer treatment, it inevitably enters the field of treatment in healthy tissues and is adversely affected by the acute and chronic side effects of radiation. This study evaluated the possible protective effects of quercetin, an antioxidant agent, against liver and kidney damage in rats exposed to a whole-body single dose of radiation (10 Gy of gamma-ray).

Materials and methods: The study groups were formed as control, sham, quercetin, radiation, quercetin + radiation and radiation + quercetin using 60 male Wistar albino (200-250 g, 3 months old) rats, including 10 rats in each group. The gamma-ray provided by the Co60 teletherapy machine was given to the whole body as external irradiation. According to the groups, quercetin was administered to rats at 50 mg/kg/day via oral gavage before or after radiation administration. The rats were sacrificed the day after irradiation and the extracted tissue samples from all groups were compared histologically and immunohistochemically. DNA damage was determined by the neutral comet assay technique. Also, malondialdehyde (MDA) and glutathione peroxidase (GSH) were evaluated in liver and kidney tissues by the ELISA method.

Results: Histopathological changes were observed altered morphology of liver and kidney tissues in the radiation groups. Sinusoidal dilatations, vacuolization, and hepatic parenchyma necrosis in the liver, while in kidneys, glomerular shrinkage, widened Bowman's space, tubular dilatation, and inflammation were evident. TNF-α, IL1-α, HIF1-α, and caspase 3 immunoreactivities in tissues were determined by immunohistochemistry. High caspase 3 positive cell number confirmed apoptosis, the comet parameters were decreased in the quercetin + radiation group. When compared to the control group, the exposure to radiation showed a marked elevation in MDA which was accompanied by high GSH. This damage was reduced in the quercetin + radiation group.

Conclusions: With the results obtained from the study; Quercetin is thought to have a protective potential against radiation-induced liver and kidney damage due to its radioprotective effect.

Keywords: Radiation; apoptosis; comet assay; cytokines; quercetin.

Intracellular metabolism of excess glucose induces mitochondrial dysfunction and diversion of glycolytic intermediates into branch pathways, leading to cell injury and inflammation. Hyperglycemia-driven overproduction of mitochondrial superoxide was thought to be the initiator of these biochemical changes, but accumulating evidence indicates that mitochondrial superoxide generation is dispensable for diabetic complications development. Here we tested the hypothesis that hypoxia inducible factor (HIF)-1α and related bioenergetic changes (Warburg effect) play an initiating role in glucotoxicity. By using human endothelial cells and macrophages, we demonstrate that high glucose (HG) induces HIF-1α activity and a switch from oxidative metabolism to glycolysis and its principal branches. HIF1-α silencing, the carbonyl-trapping and anti-glycating agent ʟ-carnosine, and the glyoxalase-1 inducer trans-resveratrol reversed HG-induced bioenergetics/biochemical changes and endothelial-monocyte cell inflammation, pointing to methylglyoxal (MGO) as the non-hypoxic stimulus for HIF1-α induction. Consistently, MGO mimicked the effects of HG on HIF-1α induction and was able to induce a switch from oxidative metabolism to glycolysis. Mechanistically, methylglyoxal causes HIF1-α stabilization by inhibiting prolyl 4-hydroxylase domain 2 enzyme activity through post-translational glycation. These findings introduce a paradigm shift in the pathogenesis and prevention of diabetic complications by identifying HIF-1α as essential mediator of glucotoxicity, targetable with carbonyl-trapping agents and glyoxalase-1 inducers.

Keywords: Warburg effect; carnosine; cellular energetics; diabetes; glycolysis; hyperglycemia; inflammation; methylglyoxal; prolyl 4-hydroxylase 2; trans-resveratrol.

Retinoblastoma (RB) is the most common intraocular tumor among children. Leucine-rich pentatricopeptide repeat (PPR)-motif-containing protein (LRPPRC), a suppressor gene of autophagy, has been proven to play a regulatory role in tumor progression. However, little is known about functional roles and mechanisms of LRPPRC in RB progression. First, we performed a detailed analysis for RB and normal control. The expression of LRPPRC in the RB tissues was significantly higher than that in normal tissues. Moreover, LRPPRC suppression could repress tumor cell migration, invasion, glycolysis, and reactive oxygen species (ROS)/hypoxia-inducible factor-1α (HIF1-α) pathway activation by mediating autophagy. Furthermore, overexpression of HIF1-α partially reversed the above changes induced by LRPPRC knockdown. The regulation of LRPPRC on tumor metastasis and glycolysis was also validated by a xenograft tumor assay. In summary, LRPPRC could regulate metastasis and glycolysis of RB by mediating autophagy suppression and further activating the ROS/HIF1-α pathway, and LRPPRC could be a promising prognostic biomarker for RB.

Keywords: LRPPRC; ROS/HIF1-α pathway; autophagy; glycolysis; metastasis; retinoblastoma.

Metformin is a first-line drug in the treatment of type-2 diabetes mellitus (T2DM). In addition to its antigluconeogenic and insulin-sensitizing properties, metformin has emerged as a potent inhibitor of the chronic inflammatory response of macrophages. In particular, metformin treatment has been shown to reduce expression of interleukin (IL-) 1β during long-term exposure to the pro-inflammatory stimulus lipopolysaccharide (LPS) through a reduction in reactive oxygen species (ROS), which decreases the levels of the hypoxia-inducible factor (HIF) 1-α, and through enhanced expression of IL-10. However, the effect of metformin on the acute inflammatory response, before significant levels of ROS accumulate in the cell, has not been explored. Here, we show that metformin alters the acute inflammatory response through its activation of AMP-activated protein kinase (AMPK), but independently of HIF1-α and IL-10, in primary macrophages and two macrophage-like cell lines. Thus, metformin changes the acute and the chronic inflammatory response through fundamentally distinct mechanisms. Furthermore, RNA-seq analysis reveals that metformin pretreatment affects the levels of a large yet selective subset of inflammatory genes, dampening the response to short-term LPS exposure and affecting a wide range of pathways and biological functions. Taken together, these findings reveal an unexpected complexity in the anti-inflammatory properties of this widely used drug.

Objective: To evaluate whether gestational diabetes mellitus (GDM) is associated with increased risks of autistic traits and attention deficit/hyperactivity disorder (ADHD) among offspring and whether placental inflammatory and oxidative stress cytokines play an intermediary role.

Methods: Based on a prospective cohort study from China, namely, the Ma'anshan Birth Cohort study (MABC), 3260 mother-child pairs were included. Autistic traits and ADHD symptoms among children were assessed at 18 months and 36 months, respectively. The mRNA expression levels of fourteen placental cytokines were determined using PCR. Logistic regression analysis was used to examine the associations between GDM and the risks of autistic traits or ADHD symptoms. Mediation analysis was used to assess the potential mediation effects of certain placental inflammatory factors.

Results: Of the 3260 children, 419 (12.85%) were exposed to GDM. The prevalence rates of autistic traits and ADHD symptoms were 13.86% and 6.4%, respectively. A 48.6% increased risk of autistic traits was observed among offspring born to mothers with GDM [odds ratio (OR) = 1.49, 95% confidence interval (95%CI): 1.11-2.00)], while no significant association was found in terms of ADHD symptoms. There were significant positive associations between GDM and IL-10 expression and between HIF1-α and CRP mRNA expression and a significant negative association between GDM and CD206 mRNA expression. The expression of MCP-1 mRNA was negatively associated with the risk of autistic traits [adjusted OR = 0.73 (95%CI: 0.73-0.55)]. The levels of TNF-α were positively associated with the risk of ADHD symptoms [OR = 2.11 (95%CI: 1.39-3.21)], while GRP78 was inversely associated with it [OR = 0.64 (95%CI: 0.44-0.94)]. However, none of the 14 placental cytokines was involved as a key mediator.

Conclusion: Our findings suggest that GDM may act as a risk factor for autistic traits in offspring, while the biological mechanisms may not involve the 14 placental cytokines studied. No significant association between GDM and ADHD symptoms was observed.

Keywords: Attention deficit/hyperactivity disorder; Autism; Gestational diabetes mellitus; Placental cytokines.

Objective: To investigate the relationship between hypoxia inducible factor 1 (HIF1α) and Wilms' tumor 1associating protein (WTAP) expression level in t(8;21) acute myeloid leukemia cells.

Methods: The t(8;21) acute myeloid leukemia cell lines, including SKNO-1 and Kasumi-1 were treated by Echinomycin for 24 h, RT-qPCR and Western blot were used to detect the expression levels of WTAP mRNA and the protein. The CoCl ₂ was used to induce the hypoxia of the cells for 24 h, the expression levels of HIF1α, WTAP protein were detected by Western blot.

Results: The expression level of WTAP mRNA and the protein in the echinomycin treated group was significantly lower than those in the control group (P<0.01). The expression level of WTAP protein in the CoCl₂ treated group was significantly higher than those in the control group (P<0.05).

Conclusion: The inhibition of HIF1-α could down-regulates the expression of WTAP, while the up-regulation of HIF1α could up-regulates the expression of WTAP, which shows that there is a positive correlation of HIF1α and WTAP expression. This result suggesting that HIF1α may be involves in the expression regulation of WTAP gene.

题目: t(8;21)急性髓系白血病细胞中HIF1α与WTAP表达关系研究.

目的: 探讨t(8;21)急性髓系白血病细胞中HIF1α与WTAP表达的关系。.

方法: 用HIF1α特异性抑制剂棘霉素（Echinomycin）处理t(8;21)阳性急性髓系白血病细胞系SKNO-1、Kasumi-1 24 h， 采用RT-qPCR及Western blot检测WTAP mRNA及蛋白的表达水平。经CoCl₂诱导细胞缺氧24 h，采用Western blot检测HIF1α、WTAP蛋白的表达水平。.

结果: Echinomycin处理组WTAP mRNA及蛋白表达水平显著低于对照组（P<0.01），CoCl₂处理组WTAP蛋白表达水平显著高于对照组（P<0.05）。.

结论: 抑制HIF1α可使WTAP的表达下调，而上调HIF1α则可使WTAP的表达上调，HIF1α与WTAP表达水平呈正相关，表明HIF1α可能参与了对WTAP的调控。.

This study was intended (1) to develop a robust animal model for hepatocellular carcinoma (HCC) research, in which HCC tumors develop in a background of fibrosis or cirrhosis; and (2) to explore time-dependent regulatory changes in key molecular markers during disease advancement and HCC development. With the aim of establishing such HCC model, male Sprague-Dawley rats were injected with diethylnitrosamine (DEN) at a dose of 30 mg/kg twice a week for 10 weeks then once a week from 12th to 16th weeks. The rats were kept under observation until 18th week. At defined time intervals (2nd, 4th, 12th, and 18th week), serum biomarkers and microscopic components of tissue samples were used to investigate the chronic progression of liver disease, while gene and protein analysis was used to monitor expression patterns during HCC development. DEN-intoxicated rats manifested inflammation at week 4, fibrosis at week 12 and cirrhosis with early HCC tumors at week 18. Molecular analysis revealed that key markers of inflammation (Il-1β, Il-6, and Tnf-α), fibrosis (Tgf-β1, Col1ααHif1-α and Vegf) were promptly (P ≤ 0.001) up-regulated at week 4, week 12 and week 18, respectively. Oxidative stress (iNos, Cyp2e1, and Sod1) and pro-apoptotic (Bax) markers showed significant upregulation from week 4 to week 12. However, Sod1 and Bax expressions dropped after week 12 and reached a minimum at 18th week. Strikingly, expressions of anti-apoptotic (Bcl-2) and cell proliferation (Pcna, Hgf, and Afp) markers were abruptly increased at week 18. Collectively, we describe an 18-week HCC model in DEN-intoxicated rats that exhibit chronic inflammation, oxidative imbalance, advance fibrosis/cirrhosis, halted apoptosis, and angiogenic sprouting, progressively.

Keywords: Cytokines; Diethylnitrosamine; Hepatocellular carcinoma; Molecular markers; Pathogenesis; Rat model.

Background: In recent years, the endometriosis has overcome a noteworthy renaissance in the recognition of its potential. In certain patients, a demonstrable malignant progression of ectopic foci leading to development of ovarian cancer is seen. The knowledge of endometriosis overthrow background into endometriosis associated ovarian cancer is of paramount importance for selection of patients at risk. The goal of the presented study was to review a malignant potential of the endometriosis and to specify predictive factors of endometriosis progression into ovarian cancer. Altogether 189 patients were included in the study. Conventional cytogenetics as well as measurement of transcriptional activity of CTNNB1 (β-catenin) and HIF1A (HIF1-α) genes were prospectively studied in 60 endometriosis patients and 50 control group patients. The retrospective histopathological analysis was performed in 19 endometriosis associated ovarian cancer patients and 60 patients with histologically confirmed endometriosis.

Results: Five endometriosis patients showed a deviation from normal cytogenetics finding without affecting of their phenotype. In 6 cases of endometriosis associated ovarian cancer ectopic endometrium was not confirmed. The remaining 13 cases demonstrated either benign or atypical endometriosis or even structures of borderline carcinoma. Atypical endometriosis was histologically confirmed in 20% of 60 endometriosis patients. Determination of gene expression (CTNNB1, HIF1A) formed two subgroups. Transcriptionally incipient endometriosis subgroup with insignificant genes expression compared to control group. In transcriptionally evident endometriosis subgroup were genes expressions significantly higher compared to control group (p < 0.01) as well as transcriptionally incipient endometriosis subgroup (p < 0.05).

Conclusions: Significant structural abnormalities of chromosomes are not included in genetic rigging of endometriosis patients. Atypical endometriosis represents a histopathologically detectable intermediate of endometriosis progression. Determination of genes expression CTNNB1 and HIF1A helps to allocate risk patients with endometriosis where more precise management is needed.

Keywords: Atypical endometriosis; CTNNB1; Conventional cytogenetics; Endometriosis; Endometriosis associated ovarian cancer; HIF1A.

Heat stress (HS) triggers oxidative stress, systemic inflammation, and disrupts growth efficiency of livestock. β-adrenergic agonists supplemented to ruminant livestock improve growth performance, increase skeletal muscle mass and decrease carcass fat. The objective of this study was to understand the independent and interacting effects of HS and zilpaterol hydrochloride (ZH) supplementation on the transcriptome of subcutaneous white adipose tissue and the longissimus dorsi muscle in steers. Twenty-four Red Angus-based steers were assigned to thermoneutral (TN; Temperature Humidity Index (THI)=68) or HS (THI=73-85) conditions and were not supplemented or supplemented with ZH (8.33 mg/kg/day) for 21 d in a 2x2 factorial. Steers in the TN condition were pair-fed to the average daily feed intake of HS steers. RNA was isolated from adipose tissue and skeletal muscle samples collected via biopsy on 3, 10, and 21 d and sequenced using 3' Tag-Seq to an achieved average depth of 3.6 million reads/sample. Transcripts, mapped to ARS-UCD1.2, were quantified. Differential expression (DE) analyses were performed in DESeq2 with a significance threshold for false discovery rate of 0.05. In adipose, 4 loci (MISP3, APOL6, SLC25AAAD, ALB) were DE due to the interaction of HS and ZH on d 10 (Padj<0.05). In muscle, 40 loci (including TENM4 and OAZ1) were DE due to ZH on d 10 and 6 loci (HIF1A, LOC101903734, PDZD9, HNRNPU, MTUS1, TMCO6) were DE due to environment on d 21 (Padj<0.05). To explore biological pathways altered by environment, supplement, and their interaction, loci with DE (Praw<0.05) were evaluated in Ingenuity Pathway Analysis. In adipose, 509 pathways were predicted to be altered (P<0.01): 202 due to HS, 126 due to ZH, and 181 due to the interaction; these included inflammatory pathways predicted to be upregulated due to HS but downregulated due to the interaction of HS and ZH. In muscle, 113 pathways were predicted to be altered (P<0.01): 23 due to HS, 66 due to ZH, and 24 due to the interaction of HS and ZH. Loci and pathway data in muscle suggest HS induced oxidative stress and that the stress response was moderated by ZH. Metabolic pathways were predicted to be altered due to HS, ZH, and their interaction in both tissues. These data provide evidence that HS and ZH interact to alter expression of genes in metabolic and immune function pathways and that ZH moderates some adverse effects of HS.

Keywords: Bos taurus; HIF1-A; RNA-Seq; efficiency; metabolism; pathway analysis.