The effects of PFT-µ on spermatogonial stem cell viability and Pluripotency.

INTRODUCTION: Spermatogonial stem cells (SSCs) offer remarkable competencies for animal reproduction and overcoming human disease as a result of their differentiation capability. We evaluated the effect of small molecule pifithrin-mu (PFT-µ) as a well-known inhibitor of P53 on SSC biological processes such as viability, apoptosis, and gene expression pattern. METHODS: The SSCs were isolated from the testes of adult NMRI mice and then cultured in DMEM / F12 medium containing 10% FBS. Then, they were characterized by the immunocytochemistry (ICC) technique by high PLZF and low c-Kit expressions. SSCs colony formation assay was carried out and their viability was estimated by MTT (Methylthiazolyldiphenyl-tetrazolium bromide, or 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) assay upon exposure to PFT-µ (0, 0.6, 1.2, 2.5, and 5µM). The apoptosis percentages also were measured using FACS analysis, and finally, Oct4 and Stra8 expression at mRNA levels was assessed using real-time quantitative PCR. RESULTS: The 0.6 and 1.2µM PFT-µ improved the viability of SSC based on MTT assay results; however, 2.5 and 5µM PFT-µ reduced SSC viability compared with the control group. Moreover, PFT-µ at lower concentration enhanced the colony size of SSCs and diminished their apoptosis. As well, as exposure to PFT-µ up-regulated Oct4 expression, while down-regulating the meiotic entry marker, Stra8. CONCLUSION: Based on findings, optimized concentrations of PFT-µ can decrease SSCs apoptosis, and conversely potentiate their pluripotency and self-renewal capacities in vitro.

Generation of Transgenic Cloned Buffalo Embryos Harboring the EGFP Gene in the Y Chromosome Using CRISPR/Cas9-Mediated Targeted Integration.

Sex control technology is of great significance in the production of domestic animals, especially for rapidly breeding water buffalo (bubalus bubalis), which served as a research model in the present study. We have confirmed that a fluorescence protein integrated into the Y chromosome is fit for sexing pre-implantation embryos in the mouse. Firstly, we optimized the efficiency of targeted integration of exogenous gene encoding enhanced green fluorescent protein (eGFP) and mCherry in Neuro-2a cells, mouse embryonic stem cells, mouse embryonic cells (NIH3T3), buffalo fetal fibroblast (BFF) cells. The results showed that a homology arm length of 800 bp on both sides of the target is more efficient that 300 bp or 300 bp/800 bp. Homology-directed repair (HDR)-mediated knock-in in BFF cells was also significantly improved when cells were supplemented with pifithrin-µ, which is a small molecule that inhibits the binding of p53 to mitochondria. Three pulses at 250 V resulted in the most efficient electroporation in BFF cells and 1.5 µg/mL puromycin was found to be the optimal concentration for screening. Moreover, Y-Chr-eGFP transgenic BFF cells and cloned buffalo embryos were successfully generated using CRISPR/Cas9-mediated gene editing combined with the somatic cell nuclear transfer (SCNT) technique. At passage numbers 6-8, the growth rate and cell proliferation rate were significantly lower in Y-Chr-eGFP transgenic than in non-transgenic BFF cells; the expression levels of the methylation-related genes DNMT1 and DNMT3a were similar; however, the expression levels of the acetylation-related genes HDAC1, HDAC2, and HDAC3 were significantly higher (p < 0.05) in Y-Chr-eGFP transgenic BFF cells compared with non-transgenic cells. Y-Chr-eGFP transgenic BFFs were used as donors for SCNT, the results showed that eGFP reporter is suitable for the visualization of the sex of embryos. The blastocyst rates of cloned buffalo embryos were similar; however, the cleavage rates of transgenic cloned embryos were significantly lower compared with control. In summary, we optimized the protocol for generating transgenic BFF cells and successfully generated Y-Chr-eGFP transgenic embryos using these cells as donors.

The p53 inactivators pifithrin-µ and pifithrin-α mitigate TBI-induced neuronal damage through regulation of oxidative stress, neuroinflammation, autophagy and mitophagy.

Traumatic brain injury (TBI) is one of the most common causes of death and disability worldwide. We investigated whether inhibition of p53 using pifithrin (PFT)-α or PFT-µ provides neuroprotective effects via p53 transcriptional dependent or -independent mechanisms, respectively. Sprague Dawley rats were subjected to controlled cortical impact TBI followed by the administration of PFTα or PFT-µ (2 mg/kg, i.v.) at 5 h after TBI. Brain contusion volume, as well as sensory and motor functions were evaluated at 24 h after TBI. TBI-induced impairments were mitigated by both PFT-α and PFT-µ. Fluoro-Jade C staining was used to label degenerating neurons within the TBI-induced cortical contusion region that, together with Annexin V positive neurons, were reduced by PFT-µ. Double immunofluorescence staining similarly demonstrated that PFT-µ significantly increased HO-1 positive neurons and mRNA expression in the cortical contusion region as well as decreased numbers of 4-hydroxynonenal (4HNE)-positive cells. Levels of mRNA encoding for p53, autophagy, mitophagy, anti-oxidant, anti-inflammatory related genes and proteins were measured by RT-qPCR and immunohistochemical staining, respectively. PFT-α, but not PFT-µ, significantly lowered p53 mRNA expression. Both PFT-α and PFT-µ lowered TBI-induced pro-inflammatory cytokines (IL-1ß and IL-6) mRNA levels as well as TBI-induced autophagic marker localization (LC3 and p62). Finally, treatment with PFT-µ mitigated TBI-induced declines in mRNA levels of PINK-1 and SOD2. Our data suggest that both PFT-µ and PFT-α provide neuroprotective actions through regulation of oxidative stress, neuroinflammation, autophagy, and mitophagy mechanisms, and that PFT-µ, in particular, holds promise as a TBI treatment strategy.

Pifithrin-µ modulates microglial activation and promotes histological recovery following spinal cord injury.

BACKGROUND: Treatments immediately after spinal cord injury (SCI) are anticipated to decrease neuronal death, disruption of neuronal connections, demyelination, and inflammation, and to improve repair and functional recovery. Currently, little can be done to modify the acute phase, which extends to the first 48 hours post-injury. Efforts to intervene have focused on the subsequent phases - secondary (days to weeks) and chronic (months to years) - to both promote healing, prevent further damage, and support patients suffering from SCI. METHODS: We used a contusion model of SCI in female mice, and delivered a small molecule reagent during the early phase of injury. Histological and behavioral outcomes were assessed and compared. RESULTS: We find that the reagent Pifithrin-µ (PFT-µ) acts early and directly on microglia in vitro, attenuating their activation. When administered during the acute phase of SCI, PFT-µ resulted in reduced lesion size during the initial inflammatory phase, and reduced the numbers of pro-inflammatory microglia and macrophages. Treatment with PFT-µ during the early stage of injury maintained a stable anti-inflammatory environment. CONCLUSIONS: Our results indicate that a small molecule reagent PFT-µ has sustained immunomodulatory effects following a single dose after injury.

Functional analysis of HSPA1A and HSPA8 in parturition.

Many factors are involved in parturition, such as apoptosis, inflammatory mediators, and hormones. Previous studies indicated that HSP70 directly or indirectly regulates apoptosis, inflammatory immune response and hormone stimulus. To gain new insights into molecular mechanism underlying HSP70 for regulating parturition, we overexpressed and knocked down two representative members of HSP70 (HSPA1A and HSPA8) through transfection of their recombinant plasmid and si-RNA separately in WISH (human amniotic epithelial) cells. The expression changes of several pathways' marker genes were investigated by Western blotting and quantitative real-time PCR (qRT-PCR). Results showed extreme expression changes in the genes of IL-8 and ESR2. HSP70 was found to stimulate estrogen response by regulating ESR2 through ERK1/2 after treating WISH cells with the special phosphorylation inhibitor of ERK1/2 and analyzing the changes of E2 concentration by ELISA. HSP70 was also observed to contribute to preterm birth after administering the special inhibitor of HSP70-PFT-µ with LPS-induced preterm birth mouse model. Overall, HSP70 induces parturition through stimulating immune inflammatory and estrogen response. The balanced HSP70 expression could ensure a smooth parturition, while the imbalanced expression may cause a pathological state like preterm.

2-phenylethynesulphonamide (PFT-µ) enhances the anticancer effect of the novel hsp90 inhibitor NVP-AUY922 in melanoma, by reducing GSH levels.

Heat shock proteins (HSPs), are molecular chaperones that assist the proper folding of nascent proteins. This study aims to evaluate the antitumour effects of the hsp90 inhibitor NVP-AUY922 in melanoma, both in vitro and in vivo. Our results show that NVP-AUY922 inhibits melanoma cell growth in vitro, with down regulation of multiple signalling pathways involved in melanoma progression such as NF-ĸB and MAPK/ERK. However, NVP-AUY922 was unable to limit tumour growth in vivo. Cotreatment of A375M xenografts with NVP-AUY922 and PFT-µ, a dual inhibitor of both hsp70 and autophagy, induced a synergistic increase of cell death in vitro, and delayed tumour formation in A375M xenografts. PFT-µ depleted cells from the reduced form of glutathione (GSH) and increased oxidative stress. The oxidative stress induced by PFT-µ further enhanced NVP-AUY922-induced cytotoxic effects. These data suggest a potential therapeutic role for NVP-AUY922 used in combination with PFT-µ, in melanoma.

Activation of the Mitochondrial Apoptotic Signaling Platform during Rubella Virus Infection.

Mitochondria- as well as p53-based signaling pathways are central for the execution of the intrinsic apoptotic cascade. Their contribution to rubella virus (RV)-induced apoptosis was addressed through time-specific evaluation of characteristic parameters such as permeabilization of the mitochondrial membrane and subsequent release of the pro-apoptotic proteins apoptosis-inducing factor (AIF) and cytochrome c from mitochondria. Additionally, expression and localization pattern of p53 and selected members of the multifunctional and stress-inducible cyclophilin family were examined. The application of pifithrin µ as an inhibitor of p53 shuttling to mitochondria reduced RV-induced cell death to an extent similar to that of the broad spectrum caspase inhibitor z-VAD-fmk (benzyloxycarbonyl-V-A-D-(OMe)-fmk). However, RV progeny generation was not altered. This indicates that, despite an increased survival rate of its cellular host, induction of apoptosis neither supports nor restricts RV replication. Moreover, some of the examined apoptotic markers were affected in a strain-specific manner and differed between the cell culture-adapted strains: Therien and the HPV77 vaccine on the one hand, and a clinical isolate on the other. In summary, the results presented indicate that the transcription-independent mitochondrial p53 program contributes to RV-induced apoptosis.