Knockout of caspase-7 gene improves the expression of recombinant protein in CHO cell line through the cell cycle arrest in G2/M phase

Background Chinese hamster ovary cell line has been used routinely as a bioproduction factory of numerous biopharmaceuticals. So far, various engineering strategies have been recruited to improve the production efficiency of this cell line such as apoptosis engineering. Previously, it is reported that the caspase-7 deficiency in CHO cells reduces the cell proliferation rate. But the effect of this reduction on the CHO cell productivity remained unclear. Hence, in the study at hand the effect of caspase-7 deficiency was assessed on the cell growth, viability and protein expression. In addition, the enzymatic activity of caspase-3 was investigated in the absence of caspase-7. Results Findings showed that in the absence of caspase-7, both cell growth and cell viability were decreased. Cell cycle analysis illustrated that the CHO knockout (CHO-KO) cells experienced a cell cycle arrest in G2/M phase. This cell cycle arrest resulted in a 1.7-fold increase in the expression of luciferase in CHO-KO cells compared to parenteral cells. Furthermore, in the apoptotic situation the enzymatic activity of caspase-3 in CHO-KO cells was approximately 3 times more than CHO-K1 cells. Conclusions These findings represented that; however, caspase-7 deficiency reduces the cell proliferation rate but the resulted cell cycle arrest leads to the enhancement of recombinant protein expression. Moreover, increasing in the caspase-3 enzymatic activity compensates the absence of caspase-7 in the caspase cascade of apoptosis.

Genome-wide analysis of metallothionein gene family in maize to reveal its role in development and stress resistance to heavy metal

Background Maize (Zea mays L.) is a widely cultivated cereal and has been used as an optimum heavy metal phytoremediation crop. Metallothionein (MT) proteins are small, cysteine-rich, proteins that play important roles in plant growth and development, and the regulation of stress response to heavy metals. However, the MT genes for maize have not been fully analyzed so far. Methods The putative ZmMT genes were identified by HMMER.The heat map of ZmMT genes spatial expression analysis was generated by using R with the log2 (FPKM + 1).The expression profiles of ZmMT genes under three kinds of heavy metal stresses were quantified by using qRT-PCR. The metallothionein proteins was aligned using MAFFT and phylogenetic analysis were constructed by ClustalX 2.1. The protein theoretical molecular weight and pI, subcellular localization, TFs binding sites, were predicted using ProtParam, PSORT, PlantTFDB, respectively. Results A total of 9 ZmMT genes were identified in the whole genome of maize. The results showed that eight of the nine ZmMT proteins contained one highly conserved metallothio_2 domain, while ZmMT4 contained a Metallothio_PEC domain. All the ZmMT proteins could be classified into three major groups and located on five chromosomes. The ZmMT promoters contain a large number of hormone regulatory elements and hormone-related transcription factor binding sites. The ZmMT genes exhibited spatiotemporal specific expression patterns in 23 tissues of maize development stages and showed the different expression patterns in response to Cu, Cd, and Pb heavy metal stresses. Conclusions We identified the 9 ZmMT genes, and explored their conserved motif, tissue expression patterns, evolutionary relationship. The expression profiles of ZmMT genes under three kinds of heavy metal stresses (Cu, Cd, Pb) were analyzed. In summary, the expression of ZmMTs have poteintial to be regulated by hormones. The specific expression of ZmMTs in different tissues of maize and the response to different heavy metal stresses are revealed that the role of MT in plant growth and development, and stress resistance to heavy metals.

Diosmetin inhibits cell proliferation and promotes apoptosis through STAT3/c-Myc signaling pathway in human osteosarcoma cells

Background Diosmetin is a bioflavonoid compound naturally abundant in citrus fruits. It is found to perform a variety of activities, while its antitumor property in osteosarcoma, a malignant tumor with unmet clinical treatment, remained unknown. Methods Colony formation assay, cell cycle analysis and apoptosis analysis were conducted respectively to observe the effect of diosmetin on cell proliferation and apoptosis in human osteosarcoma cells. Western blot and immunoprecipitation were used to detect the expression of apoptotic molecules and activation of STAT3/c-Myc pathway in Saos-2 and U2SO cells. Results Diosmetin significantly inhibited cell proliferation, induced cell cycle arrest at G2/M phase and promoted cell apoptosis in both Saos-2 and U2SO cells. Moreover, Diosmetin downregulated the expression of anti-apoptotic protein Bcl-xL while upregulated the levels of pro-apoptotic proteins including cleaved Caspase-3, cleaved-PARP and Bax. Furthermore, diosmetin dose-dependently inhibited STAT3 phosphorylation, reduced the expression of its downstream protein c-Myc and impeded the interaction between STAT3 molecules. Conclusions These results suggest that diosmetin exerts anti-osteosarcoma effects by suppressing cell proliferation and inducing apoptosis via inhibiting the activation of STAT3/c-Myc signaling pathway, which provide the possibility for diosmetin to be a chemotherapeutic candidate for osteosarcoma.

Increased susceptibility of cystic fibrosis airway epithelial cells to ferroptosis

Background Defective chloride transport in airway epithelial cells (AECs) and the associated lung disease are the main causes of morbidity and early mortality in cystic fibrosis (CF). Abnormal airway iron homeostasis and the presence of lipid peroxidation products, indicative of oxidative stress, are features of CF lung disease. Results Here, we report that CF AECs (IB3-1) are susceptible to ferroptosis, a type of cell death associated with iron accumulation and lipid peroxidation. Compared to isogenic CFTR corrected cells (C38), the IB3-1 cells showed increased susceptibility to cell death upon exposure to iron in the form of ferric ammonium citrate (FAC) and the ferroptosis inducer, erastin. This phenotype was accompanied by accumulation of intracellular ferrous iron and lipid peroxides and the extracellular release of malondialdehyde, all indicative of redox stress, and increased levels of lactate dehydrogenase in the culture supernatant, indicating enhanced cell injury. The ferric iron chelator deferoxamine (DFO) and the lipophilic antioxidant ferrostatin-1 inhibited FAC and erastin induced ferroptosis in IB3-1 cells. Glutathione peroxidase 4 (GPX4) expression was decreased in IB3-1 cells treated with FAC and erastin, but was unchanged in C38 AECs. Necroptosis appeared to be involved in the enhanced susceptibility of IB3-1 AECs to ferroptosis, as evidenced by partial cell death rescue with necroptosis inhibitors and enhanced mixed lineage kinase domain-like (MLKL) localisation to the plasma membrane. Conclusion These studies suggest that the increased susceptibility of CF AECs to ferroptosis is linked to abnormal intracellular ferrous iron accumulation and reduced antioxidant defences. In addition, the process of ferroptotic cell death in CF AECs does not appear to be a single entity and for the first time we describe necroptosis as a potential contributory factor. Iron chelation and antioxidant treatments may be promising therapeutic interventions in cystic fibrosis. Graphical Abstract

Dual effects of entomopathogenic fungi on control of the pest Lobesia botrana and the pathogenic fungus Eutypella microtheca on grapevine

Background Entomopathogenic fungi (EPF) are the natural enemies of insect pests. Nevertheless, research on the use of EPF for simultaneous prevention of pest and disease agents on the same crop is limited. In this study, we explored the potential dual effects of three strains of the EPF Metarhizium anisopliae on the control of detrimental agents of Vitis vinifera L., including different developmental stages (larvae, pupae, and adult) of the insect pest Lobesia botrana and the phytopathogenic fungus Eutypella microtheca. Methods Laboratory pathogenicity trials were performed to examine the effects of the three M. anisopliae strains on the mortality rate of L. botrana. In addition, field trials were conducted to assess the biocontrol potential of one selected M. anisopliae strain on the larval stage of L. botrana. Moreover, inhibitory effects of the three EPF strains on E. microtheca growth were examined in vitro. Results All the M. anisopliae strains were highly effective, killing all stages of L. botrana as well as inhibiting the growth of E. microtheca. The in vitro mortality of larvae treated with the strains was over 75%, whereas that of treated pupae and adults was over 85%. The three EPF strains showed similar efficacy against larvae and adult stages; nevertheless, pupal mortality was observed to be strain dependent. Mortality of L. botrana larvae ranged from 64 to 91% at field conditions. Inhibition of E. microtheca growth reached 50% in comparison to the control. Conclusions Our study showed that M. anisopliae strains were highly effective in ensuring control of two different detrimental agents of V. vinifera L., providing new evidence to support the dual effects of entomopathogenic fungi.

Non-coding RNAs and chromatin: key epigenetic factors from spermatogenesis to transgenerational inheritance

Cellular fate and gene expression patterns are modulated by different epigenetic factors including non-coding RNAs (ncRNAs) and chromatin organization. Both factors are dynamic throughout male germ cell differentiation on the seminiferous tubule, despite the transcriptional inactivation in the last stages of spermatogenesis. Sperm maturation during the caput-to-cauda transit on the epididymis involves changes in chromatin organization and the soma-to-germ line transference of ncRNAs that are essential to obtain a functional sperm for fertilization and embryo development. Here, the male environment (diseases, drugs, mental stress) is crucial to modulate these epigenetic factors throughout sperm maturation, affecting the corresponding offspring. Paternal transgenerational inheritance has been directly related to sperm epigenetic changes, most of them associated with variations in the ncRNA content and chromatin marks. Our aim is to give an overview about how epigenetics, focused on ncRNAs and chromatin, is pivotal to understand spermatogenesis and sperm maturation, and how the male environment impacts the sperm epigenome modulating the offspring gene expression pattern.

Mild poikilocapnic hypoxia increases very low frequency haemoglobin oxygenation oscillations in prefrontal cortex

Background The aim of the study was to investigate the effect of mild cerebral hypoxia on haemoglobin oxygenation (HbO2), cerebrospinal fluid dynamics and cardiovascular physiology. To achieve this goal, four signals were recorded simultaneously: blood pressure, heart rate / electrocardiogram, HbO2 from right hemisphere and changes of subarachnoid space (SAS) width from left hemisphere. Signals were registered from 30 healthy, young participants (2 females and 28 males, body mass index = 24.5 ± 2.3 kg/m2, age 30.8 ± 13.4 years). Results We analysed the recorded signals using wavelet transform and phase coherence. We demonstrated for the first time that in healthy subjects exposed to mild poikilokapnic hypoxia there were increases in very low frequency HbO2 oscillations (< 0.052 Hz) in prefrontal cortex. Additionally, SAS fluctuation diminished in the whole frequency range which could be explained by brain oedema. Conclusions Consequently the study provides insight into mechanisms governing brain response to a mild hypoxic challenge. Our study supports the notion that HbO2 and SAS width monitoring might be beneficial for patients with acute lung disease.

The therapeutic potential of mesenchymal stem cells in treating osteoporosis

Osteoporosis (OP), a common systemic metabolic bone disease, is characterized by low bone mass, increasing bone fragility and a high risk of fracture. At present, the clinical treatment of OP mainly involves anti-bone resorption drugs and anabolic agents for bone, but their long-term use can cause serious side effects. The development of stem cell therapy and regenerative medicine has provided a new approach to the clinical treatment of various diseases, even with a hope for cure. Recently, the therapeutic advantages of the therapy have been shown for a variety of orthopedic diseases. However, these stem cell-based researches are currently limited to animal models; the uncertainty regarding the post-transplantation fate of stem cells and their safety in recipients has largely restricted the development of human clinical trials. Nevertheless, the feasibility of mesenchymal stem cells to treat osteoporotic mice has drawn a growing amount of intriguing attention from clinicians to its potential of applying the stem cell-based therapy as a new therapeutic approach to OP in the future clinic. In the current review, therefore, we explored the potential use of mesenchymal stem cells in human OP treatment.

Effects of enriched-potassium diet on cardiorespiratory outcomes in experimental non-ischemic chronic heart failure

Background Chronic heart failure (CHF) is a global health problem. Increased sympathetic outflow, cardiac arrhythmogenesis and irregular breathing patterns have all been associated with poor outcomes in CHF. Several studies showed that activation of the renin-angiotensin system (RAS) play a key role in CHF pathophysiology. Interestingly, potassium (K+) supplemented diets showed promising results in normalizing RAS axis and autonomic dysfunction in vascular diseases, lowering cardiovascular risk. Whether subtle increases in dietary K+ consumption may exert similar effects in CHF has not been previously tested. Accordingly, we aimed to evaluate the effects of dietary K+ supplementation on cardiorespiratory alterations in rats with CHF. Methods Adult male Sprague–Dawley rats underwent volume overload to induce non-ischemic CHF. Animals were randomly allocated to normal chow diet (CHF group) or supplemented K+ diet (CHF+K+ group) for 6 weeks. Cardiac arrhythmogenesis, sympathetic outflow, baroreflex sensitivity, breathing disorders, chemoreflex function, respiratory–cardiovascular coupling and cardiac function were evaluated. Results Compared to normal chow diet, K+ supplemented diet in CHF significantly reduced arrhythmia incidence (67.8 ± 15.1 vs. 31.0 ± 3.7 events/hour, CHF vs. CHF+K+), decreased cardiac sympathetic tone (ΔHR to propranolol: − 97.4 ± 9.4 vs. − 60.8 ± 8.3 bpm, CHF vs. CHF+K+), restored baroreflex function and attenuated irregular breathing patterns. Additionally, supplementation of the diet with K+ restores normal central respiratory chemoreflex drive and abrogates pathological cardio-respiratory coupling in CHF rats being the outcome an improved cardiac function. Conclusion Our findings support that dietary K+ supplementation in non-ischemic CHF alleviate cardiorespiratory dysfunction.

The role of vulpinic acid as a natural compound in the regulation of breast cancer-associated miRNAs

Background Breast cancer is the most frequently diagnosed cancer, and no effective treatment solution has yet been found. The number of studies based on the research of novel natural compounds in the treatment of breast cancer has been increasing in recent years. The anticancer properties of natural compounds are related to the regulation of microRNA (miRNA) expression. Therefore, changing the profile of miRNAs with the use of natural products is very important in cancer treatment. However, the role of vulpinic acid and related miRNAs in breast cancer progression remains unknown. Vulpinic acid, methyl (as2E)-2-(3-hydroxy-5-oxo-4-phenylfuran-2-ylidene)-2 phenylacetate, is a natural product extracted from the lichen species and shows an anticancer effect on different cancer cells. Methods This study examines the effects of vulpinic acid on the miRNA levels of breast cancer (MCF-7) cells and its relationship with cell proliferation and apoptosis levels. The antiproliferative effect of vulpinic acid was screened against MCF-7 breast cancer cells and MCF-12A breast epithelial cells using the xCELLigence real-time cell analysis system. We analyzed the altered miRNA expression profile in MCF-7 breast cancer cells versus MCF-12A cells following their response to vulpinic acid through microarray analysis. The microarray analysis results were confirmed through quantitative real-time PCR and bioinformatics analysis. Results The results of the miRNA array and bioinformatic analyses demonstrated that 12 miRNAs were specifically responsive to vulpinic acid in MCF-7 breast cancer cells. This is the first study to reveal that vulpinic acid inhibits the expression of 12 miRNAs and suppresses breast cancer cell proliferation. The study also revealed that vulpinic acid may downregulate the expression of 12 miRNAs by repressing the FOXO-3 gene. The miRNA targets were mainly found to play a role in the apoptosis, cell cycle and MAPK pathways. Moreover, Bcl-2, Bax, procaspase-3 and procaspase-9 protein levels were assessed by western blot analysis for validation of apoptosis at the protein level. Conclusion This study revealed the molecular mechanisms of vulpinic acid on breast cancer and showed that vulpinic acid regulates apoptosis signaling pathways by decreasing the expression of miRNAs. The miRNA expression patterns illuminate the underlying effect of vulpinic acid in breast cancer treatment. Graphical Abstract