Cell Protein Research Articles

Carnosol alleviates cisplatin–induced acute kidney injury by regulating apoptosis and pyroptosis

AbstractThe use of the common anticancer drug cisplatin (CP) in clinical practice often leads to acute kidney injury (AKI); however, no protective therapy is available. Therefore, new drugs that reduce the nephrotoxicity induced by CP are urgently needed. Carnosol (CA) is an antioxidant found. We investigated the renoprotective effects of CA on CP‐induced AKI in male C57BL/6 mice and HK2 cells. CA mitigated renal dysfunction, histopathological changes and tubular injury in vivo, as indicated by the expression of NGAL, KIM1 and HMGB1. Moreover, the numbers of apoptotic cells and the expression of apoptotic proteins were dramatically reduced after CA treatment in mouse kidneys and HK2 cells. CA significantly ameliorated CP‐induced inflammation and decreased TNF‐α and IL‐1β levels in vivo and in vitro and macrophage infiltration in the mouse kidney. CA decreased the expression levels of p‐p65/p65, NLRP3 and ASC, which indicates that CA suppressed the activation of the NF‐κB/NLRP3 signaling axis induced by CP in vivo and in vitro. In addition, CA decreased the levels of certain protein in pyroptotic cells, as indicated by the expression of cleaved caspase‐1, GSDMD, and mature IL‐1β and IL‐18 in vivo and in vitro. Finally, CA reduced the level of cleaved caspase‐1, but those of GSDMD and NLRP3 protein were not significantly different after treatment with the NLRP3 inhibitor MCC950 and were elevated by the NLRP3 activator nigericin. In conclusion, this study revealed that CA protects against CP‐induced AKI by decreasing apoptosis and NF‐κB/NLRP3/GSDMD‐mediated pyroptosis, which provides new insight into the prevention of AKI.

Upregulation of ENAH by a PI3K/AKT/β-catenin cascade promotes oral cancer cell migration and growth via an ITGB5/Src axis

BackgroundOral cancer accounts for 2% of cancer-related deaths globally, with over 90% of cases being oral cavity squamous cell carcinomas (OSCCs). Approximately 50% of patients with OSCC succumb to the disease within 5 years, primarily due to the advanced stage at which it is typically diagnosed. This underscores an urgent need to identify proteins related to OSCC progression to develop effective diagnostic and therapeutic strategies.MethodsTo identify OSCC progression-related proteins, we conducted integrated proteome and transcriptome analyses on cancer tissues from patients and patient-derived xenograft (PDX) model mice. We investigated the role of protein-enabled homolog (ENAH), identified as an OSCC progression-associated protein, through proliferation, transwell migration, and invasion assays in OSCC cells. The mechanisms underlying ENAH-mediated functions were elucidated using gene knockdown and ectopic expression techniques in OSCC cells.ResultsENAH was identified as a candidate associated with OSCC progression based on integrated analyses, which showed increased ENAH levels in primary OSCC tissues compared with adjacent noncancerous counterparts, and sustained overexpression in the cancer tissues of PDX models. We confirmed that level of ENAH is increased in OSCC tissues and that its elevated expression correlates with poorer survival rates in patients with OSCC. Furthermore, the upregulation of ENAH in OSCC cells results from the activation of the GSK3β/β-catenin axis by the EGFR/PI3K/AKT cascade. ENAH expression enhances cell proliferation and mobility by upregulating integrin β5 in oral cancer cells.ConclusionsThe upregulation of ENAH through a PI3K/AKT/β-catenin signaling cascade enhances oral cancer cell migration and growth via the ITGB5/Src axis. These findings offer a new interpretation of the ENAH function in the OSCC progression and provide crucial information for developing new OSCC treatment strategies.

Spontaneous high clonal expansion of Wilms’ tumor gene 1-specific cytotoxic T-lymphocytes in patients with Wilms’ tumor gene 1-expressing solid tumor

Wilms’ tumor protein 1 (WT1)-targeted immunotherapy has been used in patients with leukemia and solid tumors. However, the spontaneous WT1-specific immune response before WT1 peptide vaccination in patients with WT1-expressing tumors (PTs) remains unclear. Therefore, we investigated whether WT1-specific cytotoxic CD8+ T-lymphocytes (CTLs) are clonally expanded in the peripheral blood outside of tumor sites. Clonal expansion of WT1126 peptide (a.a.126–134)-specific CTLs (WT1126-CTLs) was compared between seven PTs and five healthy volunteers (HVs), and their T-cell receptors (TCRs) were analyzed at the single-cell level. Overall, 433 and 351 TCR β-chains of WT1126-CTLs were detected from PTs and HVs, respectively, and complementarity-determining region 3 was sequenced for clonality analysis. The frequencies of WT1126-CTLs were higher in human leukocyte antigen (HLA)-A*02:01+ PTs than in HLA-A*02:01+ HVs, although the difference was not statistically significant. WT1126-CTLs of differentiated types, including memory and effector, were higher in PTs than in HVs; whereas, those of the naïve type were higher in HVs than in PTs. WT1126-CTL clonality was significantly higher in PTs than in HVs. Furthermore, the frequency of effector WT1126-CTLs positively correlated with WT1126-CTL clonality in PTs; whereas, the frequency of naïve phenotype WT1126-CTLs tended to be negatively correlated with clonality. In conclusion, these results suggest that the WT1 protein in tumor cells is highly immunogenic, thereby stimulating endogenous naïve-type WT1126-CTLs and enabling them to clonally expand and differentiate into effector-type WT1126-CTLs.

LncRNA MALAT1 silencing represses CXCL12-induced proliferation, invasion, and homing behavior in multiple myeloma by inhibiting CXCR4

ABSTRACT Background: Long non-coding RNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) has been identified in multiple myeloma (MM); its influence on the homing behavior in MM cells is unclear. This study elucidates the impact and mechanism by which MALAT1 affects the homing behavior in MM cells. Methods: Bone marrow was obtained from patients with MM. MALAT1 and C-X-C motif chemokine receptor 4 (CXCR4) expression in cluster of differentiation 138-purified (CD138) plasma cells were detected by qRT-PCR and Western blotting. MALAT1, CXCR4, and C-X-C motif chemokine ligand 12 (CXCL12) influences on MM cell viability, proliferation, and invasion was monitored by CCK-8, 5-Ethynyl-2'-deoxyuridine, and Transwell assays. qRT-PCR, Western blotting, and ELISA were utilized to detect homing effect-related proteins in MM cells, including intercellular adhesion molecule 1 (ICAM-1) and very late antigen-4 (VLA-4). Results: MALAT1 and CXCR4 were overexpressed in CD138-purified plasma cells from bone marrow of MM patients, associating with bone damage. MALAT1 upregulated CXCR4 in MM cells. MALAT1 overexpression enhanced MM cell viability, proliferation, and invasion, whereas CXCR4 silencing reversed them. CXCR4 silencing attenuated MALAT1 induction on ICAM-1 and VLA-4 expression in MM cells. CXCL12 upregulation intensified MM cell proliferation and invasion and ICAM-1 and VLA-4 expression in MM cells. MALAT1 silencing counteracted the impact of CXCL12. Conclusion: MALAT1 silencing may repress CXCL12 induction on MM cell proliferation, invasion, and homing behavior by inhibiting CXCR4. MALAT1 may be a promising target for MM treatment.

Ionomic and proteomic changes highlight the effect of silicon supply on the nodules functioning of Trifolium incarnatum L.

IntroductionNumerous studies have reported the beneficial effects of silicon (Si) in alleviating biotic or abiotic stresses in many plant species. However, the role of Si in Fabaceae facing environmental stress is poorly documented. The aim of this study is to investigate the effect of Si on physiological traits and nodulation efficiency in Trifolium incarnatum L.MethodsSi was supplied (1.7 mM in the form of Na2SiO3) plants inoculated with Rhizobium leguminosarum bv trifolii and plant physiological traits and nodule ionomic and molecular traits were monitored over 25 days.ResultsSi supply promoted shoot biomass, the quantity of both Si and N in roots and shoots, and the number, biomass and density of nodules and their nitrogenase abundance which contribute to better dinitrogen (N2) fixation. Ionomic analysis of nodules revealed that Si supply increased the amount of several macroelements (potassium, phosphorus and sulfur) and microelements (copper, zinc and molybdenum) known to improve nodulation efficiency and N2 fixation. Finally, comparative proteomic analysis (+Si versus -Si) of nodules highlighted that Si modulated the proteome of both symbionts with 989 and 212 differentially accumulated proteins (DAPs) in the infected host root cells and their symbiont bacteria, respectively.DiscussionAmong the DAPs, the roles of those involved in nodulation and N2 fixation are discussed. For the first time, this study provides new insights into the effects of Si on both nodular partners and paves the way for a better understanding of the impact of Si on improving nodule function, and more specifically, on the nodules’ N2-fixing capacity.

USP2 Mitigates Reactive Oxygen Species-Induced Mitochondrial Damage via UCP2 Expression in Myoblasts

Ubiquitin-specific protease 2 (USP2) maintains mitochondrial integrity in culture myoblasts. In this study, we investigated the molecular mechanisms underlying the protective role of USP2 in mitochondria. The knockout (KO) of the Usp2 gene or the chemical inhibition of USP2 induced a robust accumulation of mitochondrial reactive oxygen species (ROS), accompanied by defects in mitochondrial membrane potential, in C2C12 myoblasts. ROS removal by N-acetyl-L-cysteine restored the mitochondrial dysfunction induced by USP2 deficiency. Comprehensive RT-qPCR screening and following protein analysis indicated that both the genetic and chemical inhibition of USP2 elicited a decrease in uncoupling protein 2 (UCP2) at mRNA and protein levels. Accordingly, the introduction of a Ucp2-expressing construct effectively recovered the mitochondrial membrane potential, entailing an increment in the intracellular ATP level in Usp2KO C2C12 cells. In contrast, USP2 deficiency also decreased peroxisome proliferator-activated receptor γ coactivator 1α (PGC1α) protein in C2C12 cells, while it upregulated Ppargc1a mRNA. Overexpression studies indicated that USP2 potentially stabilizes PGC1α in an isopeptidase-dependent manner. Given that PGC1α is an inducer of UCP2 in C2C12 cells, USP2 might ameliorate mitochondrial ROS by maintaining the PGC1α–UCP2 axis in myoblasts.

Establishment of liquid biopsy procedure for the analysis of circulating cell free DNA, exosomes, RNA and proteins in colorectal cancer and adenoma patients.

Liquid biopsy has an underexplored diagnostic potential in colorectal cancer (CRC). Sufficient quantity and quality of its elements (circulating cell-free DNA (ccfDNA), exosomes and exosomal RNA) are essential for accurate results. The present study aims to establish the optimal protocol for handling liquid biopsy samples. Samples were obtained by collecting peripheral blood from colorectal adenoma patients in CellSave tubes. Plasma was separated within six hours using differential centrifugation and aliquots stored at - 20/- 80°C until further processing. Three methods for isolation of ccfDNA, and two combinations of kits for isolation of exosomes and exosomal RNA were tested. The quality and quantity of ccfDNA isolates were evaluated. Exosomes were characterised by determining size, concentration, and total and specific protein content. Expression of chosen microRNAs, miR-19a-3p and miR-92-3p, which have been implicated in CRC progression, were determined. The vacuum-column-based kit showed the highest quantities of isolated ccfDNA (P-value < 0.001). Kits for exosome isolation significantly differed in size (P-value = 0.016), concentration (P-value = 0.016) and protein content (P-value = 0.016). There was no significant difference in expressions of miR-19a-3p (P-value = 0.219) and miR-92a-3p (P-value = 0.094) between the two isolation kits. The new, adapted protocol described, enables simultaneous analysis of multiple elements when investigating potential biomarkers of CRC.

Cellular NONO protein binds to the flavivirus replication complex and promotes positive-strand RNA synthesis.

A cellular protein, non-POU-domain-containing octamer binding protein (NONO), bound to the replication complex of Japanese encephalitis virus (JEV) by directly interacting with the viral 3' UTR RNA and NS3 protein. These interactions were also identified in West Nile virus (WNV) and Zika virus (ZIKV). The infection of JEV or the expression of JEV NS3 protein in cells could induce relocation of NONO protein from the nucleus to the cytoplasm. In JEV-infected cells, the NS3, NS5, and viral RNA could be concurrently detected in the immunoprecipitation by the NONO-specific antibody, suggesting that NONO could integrate into the replication complex of JEV. Further results of co-immunoprecipitation assays showed that NONO protein interacted with NS3 helicase domains 1 and 2 by its two RNA recognize motifs (RRMs). The knockdown and knockout of NONO in cells could significantly reduce the replication of JEV and ZIKV but had no effect on the replication of vesicular stomatitis virus (VSV). The effect of NONO protein on JEV proliferation occurred during the replication stage, rather than the attachment and entry stages. The level of viral positive-strand RNA in NONO knockout cells was significantly reduced than that in wild-type cells at 12-48 h post-JEV infection. However, the level of negative-strand virus RNA had no difference between NONO knockout and wild-type cells at 12-24 h post-infection. In summary, our study identified a cellular protein that bound to the replication complex of flavivirus and facilitated the synthesis of positive-strand RNA.IMPORTANCEOver half of the world's population is at risk of flaviviruses infection, posing a serious global health concern. To date, there are no antiviral drugs or treatments for the severe symptoms caused by the infection of flaviviruses. Some cellular proteins could participate in the replication of virus, and these cellular proteins were also ideal targets in antiviral strategy. Here, we identified cellular NONO protein was recruited by flavivirus NS3 protein to the cytoplasm, serving as a "scaffold" for viral replication complex. Our findings also revealed that NONO protein was critical for flavivirus positive-strand RNA synthesis. Specific areas where NONO interacted with flavivirus NS3 proteins and viral UTRs have also been identified. These results propose a new mechanism for cellular protein to participate in flavivirus replication and also raise a new potential anti-flavivirus strategy.

Pulsed electric field at resonance frequency combat Klebsiella pneumoniabiofilms.

Healtcare-associated infections have increased due to the development of antimicrobial resistance (AMR) of Gram-negative pathogens (GNPs) and the development of outbreacks over the past two decades. In this work, we investigated how exposure to positive electric pulses affects the growth characteristics of Klebsiella pneumonia (K. pneumonia), a common cause of pneumonia. We explored the impact of varying exposure frequencies (0.2-2Hz) and time (15-90min, at resonance frequency) on bioelectric signals produced during cell division, biofilm formation, and bacterial antibiotic susceptibility. Our research found that an extremely low-frequency pulsed electric field (ELF-PEF) significantly inhibited K. pneumonia growth. Specifically, exposure to 0.8Hz for one hour increased the antibiotic susceptibility of K. pneumonia to inhibitors of cell wall formation, proteins, β-lactamase, DNA, and other substances. We also noticed a notable decrease in K. pneumonia biofilm development exposed to ELF-PEF. Our results suggest that the interaction of K. pneumonia cells with ELF-PEF at the specified frequency and time alters cellular activity and bacterial structure. This technique may be used in the future to treat K. pneumonia infections both in vitro and in vivo.

Mechanical confinement matters: Unveiling the effect of two-photon polymerized 2.5D and 3D microarchitectures on neuronal YAP expression and neurite outgrowth

The effect of mechanical cues on cellular behaviour has been reported in multiple studies so far, and a specific aspect of interest is the role of mechanotransductive proteins in neuronal development. Among these, yes-associated protein (YAP) is responsible for multiple functions in neuronal development such as neuronal progenitor cells migration and differentiation while myocardin-related transcription factor A (MRTFA) facilitates neurite outgrowth and axonal pathfinding. Both proteins have indirectly intertwined fates via their signalling pathways. There is little literature investigating the roles of YAP and MRTFA in vitro concerning neurite outgrowth in mechanically confined microenvironments. Moreover, our understanding of their relationship in immature neurons cultured within engineered confined microenvironments is still lacking. In this study, we fabricated, via two-photon polymerization (2PP), 2.5D microgrooves and 3D polymeric microchannels, with a diameter range from 5 to 30 μm. We cultured SH-SY5Y cells and differentiated them into immature neuron-like cells on both 2.5D and 3D microstructures to investigate the effect of mechanical confinement on cell morphology and protein expression. In 2.5D microgrooves, both YAP and MRTFA nuclear/cytoplasmic (N/C) ratios exhibited maxima in the 10 μm grooves indicating a strong relation with mechanical-stress-inducing confinement. In 3D microchannels, both proteins’ N/C ratio exhibited minima in presence of 5 or 10 μm channels, a behaviour that was opposite to the ones observed in the 2.5D microgrooves and that indicates how the geometry and mechanical confinement of 3D microenvironments are unique compared to 2.5D ones due to focal adhesion, actin, and nuclear polarization. Further, especially in presence of 2.5D microgrooves, cells featured an inversely proportional relationship between YAP N/C ratio and the average neurite length. Finally, we also cultured human induced pluripotent stem cells (hiPSCs) and differentiated them into cortical neurons on the microstructures for up to 2 weeks. Interestingly, YAP and MRTFA N/C ratios also showed a maximum around the 10 μm 2.5D microgrooves, indicating the physiological relevance of our study. Our results elucidate the possible differences induced by 2.5D and 3D confining microenvironments in neuronal development and paves the way for understanding the intricate interplay between mechanotransductive proteins and their effect on neural cell fate within engineered cell microenvironments.

An Electrochemical Biosensor Analysis of the Interaction of a Two-Vector Phospholipid Composition of Doxorubicin with dsDNA and Breast Cancer Cell Models In Vitro

Objectives: The main aim of our experiments was to demonstrate the suitability of cell-based biosensors for searching for new anticancer medicinal preparations. Methods: The effect of the substance doxorubicin, doxorubicin embedded in phospholipid nanoparticles, and doxorubicin with phospholipid nanoparticles modified by targeting vectors (cRGD and folic acid) on dsDNA and breast cancer cell lines (MCF-7, MDA-MB-231) was studied. Results: In the obtained doxorubicin nanoforms, the particle size was less than 60 nm. Our study of the percentage of doxorubicin inclusion showed the almost complete embeddability of the substance into nanoparticles for all samples, with an average of 95.4 ± 4.6%. The calculation of the toxicity index of the studied doxorubicin samples showed that all substances were moderately toxic drugs in terms of adenine and guanine. The biosensor analysis using electrodes modified with carbon nanotubes showed an intercalation interaction between doxorubicin and its derivatives and dsDNA, except for the composition of doxorubicin with folic acid with a linker length of 2000 (NPh-Dox-Fol(2.0)). The results of the electroanalysis were normalized to the total cell protein (mg) and cell concentration. The highest intensity of the electrochemical signals was observed in intact control cells of the MCF-7 and MDA-MB-231 cell lines. Conclusions: The proposed electrochemical approach is useful for the analysis of cell line responses to the medicinal preparations.

From Waste to Protein: Optimizing Rice Husk Utilization for Sustainable Single Cell Protein Production

The increasing global demand for protein has prompted a search for sustainable and cost-effective alternatives to traditional animal and dairy sources. Single-cell protein (SCP) produced from microorganism’s offers a promising solution due to its high protein content and rapid growth rates. This study addresses the challenge of utilizing rice husk, an abundant yet underutilized agricultural waste in Nigeria, as a substrate for SCP production. Through optimization of the production of SCP using Bacillus sp. AT-b3 and Bacillus sp. CMF 12 as model organisms. Molecular identification through 16S rRNA sequencing confirmed Bacillus sp. AT-b3 and Bacillus sp. CMF 12 as the most potent isolates for SCP production. Optimization studies were conducted using the One-Factor-at-a-Time (OFAT) method to determine the ideal fermentation conditions. The results showed that the optimal temperature for SCP production was 40 °C, with Bacillus sp. AT-b3 demonstrating superior production efficiency. pH optimization revealed that neutral pH (pH 7) was ideal for maximizing SCP production, with Bacillus spp. AT-b3 outperforming Bacillus sp. CMF 12 at this pH. Substrate concentration studies indicated that 2.0% was optimal for SCP production, and incubation time optimization indicated 48 hours as the optimal period for maximum yield. Amino acid profiling of the SCP produced showed significant variations between the two isolates. Bacillus sp. CMF 12 was richer in essential amino acids like Arginine and Methionine, while Bacillus sp. AT-b3 had a higher Glycine content at (p&lt;0.05). The findings of this study suggest that both strains have potential applications in nutritional supplements, with Bacillus sp. AT-b3 being particularly suited for industrial-scale SCP production. This study concludes that Bacillus sp. AT-b3 is an efficient SCP producer under optimal conditions of neutral pH, moderate temperature, and appropriate substrate concentration. Further research is recommended to explore pilot-scale production, alternative substrates, and comprehensive safety assessments.

An optimized ROP6 mRNA construct successfully expressed immunogenic Toxoplasma gondii ROP6 protein in cell culture

Toxoplasma gondii is an apicomplexan parasite infecting all mammals including humans and causes toxoplasmosis. There is no vaccine available for humans and thus vaccine development efforts continue using novel antigens and/or vaccine platforms. Since our previous microarray screening study showed that ROP6 is a suitable antigen to be used in vaccine studies, in this study, we aimed to design an optimized mRNA construct encoding the ROP6 protein and then demonstrate its efficiency and immunogenicity using in vitro methods. For this, we constructed a pT7CFE1-Chis/ROP6 vector encoding optimized ROP6 mRNA containing EMCV 5′UTR with IRES and a 20 nucleotides fragment from alpha globin 3′ UTR. Then, we generated the optimized ROP6 mRNAs with anti-reverse cap analogue (ARCA) and approximately 150 nucleotide long poly-A tail. Next, HEK293T cells were transfected with the optimized ROP6 mRNAs to show recombinant ROP6 protein expression capability. Moreover, we expressed in vitro recombinant ROP6 protein in HeLa cell lysate using the pT7CFE1-Chis/ROP6 vector to reveal the immunogenicity of recombinant ROP6 protein using sera samples collected from mice infected with PRU strain of T. gondii. The IFA and Western blot results showed that the optimized ROP6 mRNAs successfully expressed the recombinant ROP6 protein in HEK293T cells. Moreover the recombinant ROP6 protein expressed in HeLa cell lysate strongly reacted with sera samples collected from mice. The absorbance difference detected among positive and negative mice serum samples analyzed was statistically significant, indicating that the recombinant ROP6 protein was immunogenic (P = 0.0003). In conclusion, this study demonstrated that the optimized ROP6 mRNAs can be used in the development of mRNA vaccines against toxoplasmosis.

An enzyme-fused phycobiliprotein synthesis system developed for visual whole-cell biosensors for the detection of cadmium during wastewater treatment

Transcription factor-based whole-cell biosensors are simple and inexpensive tools for monitoring heavy metals during environmental processes. However, most biosensors are depend on instrumentation or have low sensitivity. Here, all the enzymes in the phycobiliprotein synthesis system were fused with the phycocyanin apoprotein to create a system to synthesize enzyme-fused phycobiliproteins with comparable fluorescence. We replaced the biliprotein synthesis system T7 promoter with cadmium-sensing genetic elements to create two visual cadmium biosensors (Epcad-cbsA and Epcad-cbsS). Cd2+ was detected at nanomolar concentrations by measuring the fluorescence intensity and evaluating the degree of color change of the cell pellets. Moreover, good linear correlations were observed with Cd2+ concentrations up to 250nM. Compared with cellular fluorescence, our cadmium biosensor is more sensitive when cell color is used as the output signal, with detection limits of 7.6nM for Epcad-cbsA and 3.2nM for Epcad-cbsS. This visual platform was validated by assessing bioavailable cadmium during wastewater treatment. By immobilizing large amounts of phycobilin with the proteins in cells, our designed biosensors do not require pigment extraction or specialized light sources or detection devices. This work demonstrates that a biliprotein synthesis system can be a novel platform for the development of simple, low-cost, and highly sensitive visual biosensors for real-world applications.

Polyethylene microplastic modulates the toxicity of pentachlorophenol to the microalgae Isochrysis galbana, clone t-ISO

Pentachlorophenol (PCP) and polyethylene microplastic (PE-MP) have been designated as emerging and persistent pollutants, respectively. The combined effects of those pollutants are still unknown, especially to organisms like phytoplankton that may adsorb to their surface. Therefore, the purpose of this study was to investigate for the first time the effects of PE-MP alone and in combination with PCP on the microalgae Isochrysis galbana, clone t-ISO following 72 h of exposure. Photosynthetic pigments amounts, carotenoid, protein, carbohydrate and fatty acids have been assessed. Acute toxicity test showed that the 72 h median inhibition concentration (72 h-EC50) was 148.2, 0.66 and 087 mg L−1 for PE-MP, PCP and their mixture. The utmost effects in growth inhibition rates were noted with 0.5 and 1.25 mg L−1 PCP (23% and 85%, respectively), and 100 and 300 mg L−1 PE-MP (49% and 64%, respectively). Moreover, it was found that those concentrations had a major impact on the photosynthetic pigments, protein, carbohydrate, and fatty acids amounts in algal cells. Furthermore, levels of H2O2 and Malondialdehyde (MDA), as well as the activities of catalase (CAT), superoxide dismutase (SOD), and ascorbate peroxidase (APX), indicated the induction of an oxidative stress in algal cells. It appears that adding PE-MP at a no-effect concentration (25 mg L−1) reduces the toxicity caused by PCP due to its adsorption to polyethylene microplastics.

Ac34deoGlcNAz: A Selective Probe for Identifying O-GlcNAc-Modified Proteins in Prostate Cancer.

O-GlcNAcylation is a prevalent protein modification in eukaryotic cellsandincreasing evidences indicated that over-expressed O-GlcNAcylation are intimately linked to the development and prognosis of prostate cancer.Thus, exploring this modification in the context of prostate cancer is vital for understanding the underlying mechanisms and hopefully used forfuture targeted therapies. In this paper, we use our previously established metabolic probes to comprehensively compare the labeling efficiency ofO-GlcNAc modified proteins in PC3 cells. Our results demonstrated that all the tested probes were non-toxic to PC3 cells and only Ac4GlcNAz, Ac4GalNAzand Ac34deoGlcNAzexhibited robust labeling signals amongst the probes. Further investigations by western blot and flow cytometry analysis revealed that Ac34deoGlcNAzwas a specific and efficient probe for intracelluar protein labeling with negligible S-glyco-modification signal. In addition, proteomic analysis further confirmed that 94% of the proteins identified byAc34deoGlcNAzwere in the form of O-linked GlcNAc, these enriched O-GlcNAcylated proteins were mainly involved in the regulated processes of prostate cancer. Our results here together prove Ac34deoGlcNAzis a safe and reliable probefor metabolic labeling O-GlcNAc modified proteins in prostate cancer, providing a mean to fully exploitthe regulatory mechanism of O-GlcNAcylation in the process of prostate cancer.

The stability of the Opi1p repressor for phospholipid biosynthetic gene expression in Saccharomyces cerevisiae is dependent on its interactions with Scs2p and Ino2p

The yeast Saccharomyces cerevisiae Opi1p negatively regulates phospholipid biosynthetic genes. Under derepressing conditions, Opi1p binds to the endoplasmic reticulum/nuclear membrane with the aid of the membrane protein Scs2p and phosphatidic acids under derepressing conditions. Under repressing conditions, it enters the nucleus to inhibit the positive transcription factors Ino2p and Ino4p. While the spatial regulation of Opi1p is understood, the regulation of its abundance remains unclear. We investigated the role of Scs2p and Ino2p in Opi1p stability by overexpressing these proteins in yeast cells. Opi1p was stable in the presence of Scs2p, but mutations in residues required for interaction with Scs2p caused Opi1p unstable. Even in the absence of Scs2p, Opi1p remained stable in the strain having a mutation to increase phosphatidic acid levels. Conversely, overproduction of Ino2p reduced Opi1p stability, whereas a mutant Ino2p that cannot interact with Opi1p did not. Additionally, Opi1p was stable in strains lacking Ino2p or with a mutated Ino2p-binding domain. These findings suggest that regulation, adding another layer to the regulation of phospholipid biosynthetic gene expression by Opi1p.

Essential magnetosome proteins MamI and MamL from magnetotactic bacteria interact in mammalian cells

To detect cellular activities deep within the body using magnetic resonance platforms, magnetosomes are the ideal model of genetically-encoded nanoparticles. These membrane-bound iron biominerals produced by magnetotactic bacteria are highly regulated by approximately 30 genes; however, the number of magnetosome genes that are essential and/or constitute the root structure upon which biominerals form is largely undefined. To examine the possibility that key magnetosome genes may interact in a foreign environment, we expressed mamI and mamL as fluorescent fusion proteins in mammalian cells. Localization and potential protein-protein interaction(s) were investigated using confocal microscopy and fluorescence correlation spectroscopy (FCS). Enhanced green fluorescent protein (EGFP)-MamI and the red fluorescent Tomato-MamL displayed distinct intracellular localization, with net-like and punctate fluorescence, respectively. Remarkably, co-expression revealed co-localization of both fluorescent fusion proteins in the same punctate pattern. An interaction between MamI and MamL was confirmed by co-immunoprecipitation. In addition, changes in EGFP-MamI distribution were accompanied by acquisition of intracellular mobility which all Tomato-MamL structures displayed. Analysis of extracts from these cells by FCS was consistent with an interaction between fluorescent fusion proteins, including an increase in particle radius. Co-localization and interaction of MamI and MamL demonstrate that select magnetosome proteins may associate in mammalian cells.

PiRNA array analysis provide insight into the mechanism of DEHP-induced testicular toxicology in pubertal male rats

Di-(2-ethylhexyl) phthalate (DEHP), a widely used plasticizer, could cause male reproductive toxicity by disrupting spermatogenesis. Piwi-interacting RNAs (piRNAs) are a small non-coding RNAs specifically highly expressed in the germline and interact with PIWI proteins to regulate spermatogenesis. Accumulating studies have confirmed that environmental poisons could induce male reproductive injury via altering piRNA expression. However, it remains unclear whether DEHP causes male reproductive dysfunction by perturbing piRNA levels. In this study, we conducted piRNA microarray expression analyses on testes of DEHP-exposed and control male rats and performed some in vitro and in vivo studies to explore the role of piRNA on DEHP-induced male reprouctive toxicity. Our results showed that DEHP exposure leaded to changed expression profile of piRNAs in pubertal male rat testes. And bioinformatics analyses revealed that down-regulated piR-rno-26751 probably targeted Insr mRNA expression regulation. Results from gene and protein expression tests demonstrated that DEHP caused decreased expression of INSR mainly in spermatogonia. Moreover, MEHP, the main metabolite of DEHP resulted in cell apoptosis and down-regulation of INSR and its downstream p-IRS1, p-PI3K, p-AKT and p-FOXO1 in GC-1spg cells. Conversely, overexpression of INSR restored cell apoptosis the down-regulation of the above proteins in GC-1spg cells. In conclusion, these findings suggest that DEHP-induced down-regulation of piR-rno-26751 targets the suppression of INSR, leading to apoptosis of spermatogonia in pubertal male rats.

Integrated mRNA-seq and miRNA-seq analysis reveals key transcription factors of HNF4α and KLF4 in ADPKD

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is the most prevalent genetic disorder affecting the kidneys. Understanding epigenetic regulatory mechanisms and the role of microRNAs (miRNAs) is crucial for developing therapeutic interventions. Two mRNA datasets (GSE7869 and GSE35831) and miRNA expression data (GSE133530) from ADPKD patients were used to find differentially expressed genes (DEGs) and differentially expressed miRNAs (DEMs), with a focus on genes regulated by hub transcription factors (TFs) and their target genes. The expression of hub TFs was validated in human kidneys and animal models through Western Blot (WB) and RT-PCR analysis. The location of the hub TF proteins in kidney cells was observed by a laser confocal microscope. A total of 2037 DEGs were identified. DEM analysis resulted in 59 up-regulated and 107 down-regulated miRNAs. Predicted target DEGs of DEMs indicated two top dysregulated TFs: hepatocyte nuclear factor 4 alpha (HNF4α) and Kruppel-like factor 4 (KLF4). RT-PCR, WB, and immunochemistry results showed that mRNA and protein levels of HNF4α were significantly decreased while KLF4 levels were significantly up-regulated in human ADPKD kidneys and Pkd1 conditional knockout mice compared with normal controls. Laser confocal microscopy revealed that KLF4 was mainly located in the cytoplasm while HNF4α was in the nucleus. Functional enrichment analysis indicated that genes regulated by HNF4α were mainly associated with metabolic pathways, while KLF4-regulated genes were linked to kidney development. Drug response prediction analysis revealed potential drug candidates for ADPKD treatment, including BI-2536, Sepantronium, and AZD5582. This integrated analysis provides new epigenetic insights into the complex miRNA-TF-mRNA network in ADPKD and identifies HNF4α and KLF4 as key TFs. These findings offer valuable resources for further research and potential drug development for ADPKD.