Cytoplasmic Activity Research Articles

Yap1-Scribble polarization is required for hematopoietic stem cell division and fate

AbstractYap1 and its paralogue Taz largely control epithelial tissue growth. We have identified that hematopoietic stem cell (HSC) fitness response to stress depends on Yap1 and Taz. Deletion of Yap1 and Taz induces a loss of HSC quiescence, symmetric self-renewal ability, and renders HSC more vulnerable to serial myeloablative 5-fluorouracil treatment. This effect depends on the predominant cytosolic polarization of Yap1 through a PDZ domain-mediated interaction with the scaffold Scribble. Scribble and Yap1 coordinate to control cytoplasmic Cdc42 activity and HSC fate determination in vivo. Deletion of Scribble disrupts Yap1 copolarization with Cdc42 and decreases Cdc42 activity, resulting in increased self-renewing HSC with competitive reconstitution advantages. These data suggest that Scribble/Yap1 copolarization is indispensable for Cdc42-dependent activity on HSC asymmetric division and fate. The combined loss of Scribble, Yap1, and Taz results in transcriptional upregulation of Rac-specific guanine nucleotide exchange factors, Rac activation, and HSC fitness restoration. Scribble links Cdc42 and the cytosolic functions of the Hippo signaling cascade in HSC fate determination.

Analyzing structural differences between insulin receptor (IR) and IGF1R for designing small molecule allosteric inhibitors of IGF1R as novel anti-cancer agents

IR and insulin-like growth factor-1 receptor (IGF-1R) share high degree of sequence and structural similarity that hinders the development of anticancer drugs targeting IGF1R, which is dysregulated in many cancers. Although IR and IGF1R mediate their activities through similar signalling pathways, yet they show different physiological effects. The exact molecular mechanism(s) how IR and IGF1R exert their distinct functions remain largely unknown. Here, we performed in silico analysis and generated GFP-fusion proteins of wild type IR and its K1079R mutant to analyze their subcellular localization, cytoplasmic and nuclear activities in comparison to IGF1R and its K1055R mutant. We showed that, like K1055R mutation in IGF1R, K1079R mutation does not impede the subcellular localization and nuclear activities of IR. Although K1079R mutation significantly decreases the kinase activity of IR but not as much as K1055R mutation, which was seen to drastically reduce the kinase activity of IGF1R. Moreover, K1079 residue in IR is seen to be sitting in a pocket which is different than the allosteric inhibitor binding pocket present in its homologue (IGF1R). This is for the first time such a study has been conducted to identify structural differences between these receptors that could be exploited for designing small molecule allosteric inhibitor(s) of IGF1R as novel anti-cancer drugs

Hydrogen promotes the activation of Cu, Zn superoxide dismutase in a rat corneal alkali-burn model.

To investigate the effects of hydrogen (H2) on Cu, Zn superoxide dismutase (SOD1) activation in a rat model of corneal alkali burn. In each rat, one cornea was subjected to alkali exposure. Physiological saline (saline group) or H2-dissolved saline (H2 group) was instilled continuously on the cornea for 5min before and after alkali exposure. Inflammatory cells, neovascularization, and cytoplasmic SOD1 levels were evaluated immunohistochemically in enucleated eyes from both groups. Three-dimensional ultrastructural tissue changes in the eyes were analyzed using low-vacuum scanning electron microscopy. The numbers of both inflammatory and vascular endothelial cells were significantly reduced in the corneas of the H2 group (P<0.01). Furthermore, H2 treatment increased both cytoplasmic SOD1 levels (P<0.01) and activity in corneal epithelial cells (P<0.01). Notably, the SOD1 activity level in the H2 group was approximately 2.5-fold greater than that in the saline group. H2 treatment suppresses inflammation and neovascularization in the injured cornea and indirectly suppresses oxidative insult to the cornea by upregulating the SOD1 enzyme protein level and activity.

APC2CDH1 negatively regulates agrin signaling by promoting the ubiquitination and proteolytic degradation of DOK7.

Neuromuscular junctions (NMJs) are peripheral synapses between motoneurons and skeletal muscle fibers that are critical for the control of muscle contraction. Dysfunction of these synapses has been implicated in congenital myasthenic syndrome (CMS). In vertebrates, agrin-LRP4-MuSK signaling plays a critical role in acetylcholine receptor (AChR) clustering and NMJ formation. The adaptor protein DOK7 is the downstream substrate of MuSK and also a cytoplasmic activator of MuSK. The role of DOK7 in the promotion of AChR clustering and the mechanisms involved have been well studied; however, the negative regulation of DOK7 after MuSK activation remains unknown. Anaphase-promoting complex 2 (APC2), the core subunit of APC/C E3 ligase complex, was originally believed to regulate cell-cycle transitions. Here, we show that APC2 is enriched at post-synapse of NMJs in postmitotic myotubes. In response to agrin stimulation, APC2 negatively regulates AChR clustering by promoting the ubiquitination of DOK7 at lysine 243 for its proteolytic degradation, which relies on MuSK kinase activity and the phosphorylation of tyrosine 106 in DOK7. Thus, this study provides a mechanism whereby agrin signaling is negatively regulated as part of vertebrate NMJ homeostasis.

Conjugates of Ciprofloxacin and Levofloxacin with Cell-Penetrating Peptide Exhibit Antifungal Activity and Mammalian Cytotoxicity.

Seven conjugates composed of well-known fluoroquinolone antibacterial agents, ciprofloxacin (CIP) or levofloxacin (LVX), and a cell-penetrating peptide transportan 10 (TP10-NH2) were synthesised. The drugs were covalently bound to the peptide via an amide bond, methylenecarbonyl moiety, or a disulfide bridge. Conjugation of fluoroquinolones to TP10-NH2 resulted in congeners demonstrating antifungal in vitro activity against human pathogenic yeasts of the Candida genus (MICs in the 6.25–100 µM range), whereas the components were poorly active. The antibacterial in vitro activity of most of the conjugates was lower than the activity of CIP or LVX, but the antibacterial effect of CIP-S-S-TP10-NH2 was similar to the mother fluoroquinolone. Additionally, for two representative CIP and LVX conjugates, a rapid bactericidal effect was shown. Compared to fluoroquinolones, TP10-NH2 and the majority of its conjugates generated a relatively low level of reactive oxygen species (ROS) in human embryonic kidney cells (HEK293) and human myeloid leukemia cells (HL-60). The conjugates exhibited cytotoxicity against three cell lines, HEK293, HepG2 (human liver cancer cell line), and LLC-PK1 (old male pig kidney cells), with IC50 values in the 10–100 µM range and hemolytic activity. The mammalian toxicity was due to the intrinsic cytoplasmic membrane disruption activity of TP10-NH2 since fluoroquinolones themselves were not cytotoxic. Nevertheless, the selectivity index values of the conjugates, both for the bacteria and human pathogenic yeasts, remained favourable.

Biomimetic nanoparticle loading obatoclax mesylate for the treatment of non-small-cell lung cancer (NSCLC) through suppressing Bcl-2 signaling

Lung cancer still remains a leading cause of cancer mortality in the world. Obatoclax mesylate (OM), a B cell chronic lymphocytic leukemia/lymphoma 2 (Bcl-2) family antagonist, is a potential antitumor drug. However, its poor aqueous solubility restricts its clinical application. Although these inherent defects, nanotechnology can be used to improve the solubility and tumor target of OM, promoting its antitumor efficiency. In the present study, the poly(lactic-coglycolic acid) (PLGA) was used and combined with red blood-cell membrane (RBCm) to explore if OM-loaded RBCm nanoparticles could improve the antitumor efficacy of OM for the treatment of lung cancer with relatively lower side effects compared with the free OM. The good physicochemical stability of the prepared RBCm-OM/PLGA nanoparticles was confirmed, and the optimal size of 153 nm was screened out, along with sustained drug release behavior. We found that RBCm-OM/PLGA nanoparticles effectively reduced the proliferation of lung cancer cells. Additionally, RBCm-OM/PLGA nanoparticles considerably induced apoptosis in lung cancer cells by reducing Bcl-2 expression levels, accompanied with the improved Cyto-c releases in cytoplasm and Caspase-3 activation. Mitochondrial membrane potential was also obviously impaired in lung cancer cells incubated with RBCm-OM/PLGA nanoparticles. Compared with free OM, RBCm-OM/PLGA nanoparticles could greatly prolong the drug circulation time in vivo and upgraded the drug concentration accumulated in tumor tissue. Furthermore, RBCm-OM/PLGA nanoparticles exerted stronger antitumor efficacy in vivo against lung cancer progression with superior safety. Therefore, RBCm-OM/PLGA nanoparticles provided new potential for lung cancer therapy with the improved safety and therapeutic effect.

Nuclear Functions of TOR: Impact on Transcription and the Epigenome.

The target of rapamycin (TOR) protein kinase is at the core of growth factor- and nutrient-dependent signaling pathways that are well-known for their regulation of metabolism, growth, and proliferation. However, TOR is also involved in the regulation of gene expression, genomic and epigenomic stability. TOR affects nuclear functions indirectly through its activity in the cytoplasm, but also directly through active nuclear TOR pools. The mechanisms by which TOR regulates its nuclear functions are less well-understood compared with its cytoplasmic activities. TOR is an important pharmacological target for several diseases, including cancer, metabolic and neurological disorders. Thus, studies of the nuclear functions of TOR are important for our understanding of basic biological processes, as well as for clinical implications.

SO025DCR2 ACCELERATES RENAL FIBROSIS AND TUBULAR CELL SENESCENCE BY PHOSPHORYLATING TYR 194 OF PRDX1 IN DIABETIC NEPHROPATHY

Abstract Background and Aims Cell senescence of renal tubular epithelial cells (RTECs), which is involved in renal tubulointerstitial fibrosis (TIF), is a key event in the progression of diabetic nephropathy (DN). However, the underlying mechanism remains unclear. This study aims to investigate the role and mechanism of decoy receptor 2 (DcR2) in TIF and cell senescence of RTECs. Method Streptozotocin (STZ)-induced DN mice model and high glucose (HG)-induced cell senescent model were constructed. The expression of DcR2 were regulated by ultrasound-mediated gene transfer of DcR2-siRNA or DcR2-overexpression plasmid in vivo. The co-expression of DcR2 with senescent markers (p16, p21 and SA-β-gal) and fibrotic markers (FN, collagen I and α-SMA). The interaction of DcR2 and its ligand TRAIL and antagonistic receptor DR5 were detected by laser confocal and Co-IP. The DcR2-interaction proteins were screened in renal tissue of DN patients and HG-induced cells by Co-IP combining with LC-MS/MS. The interaction of DcR2 and PRDX1 was validated by Co-IP, pull down assay and immunofluorescence co-staining. Peroxidase activity of PRDX1 was assessed by the kits of reactive oxygen species (ROS) and specific 2-cys peroxidase activity. The level of PRDX1 phosphorylation was measured through WB and the phosphorylated site were studied by wild-type PRDX1 and mutant Tyr194 PRDX1. Results DcR2 was co-expressed with senescent markers and co-localization with fibrotic markers in DN patients and STZ-DN mice. Knockdown of DcR2 effectively decreased renal fibrosis and tubular cell senescence in STZ-induced DN mice. However, DcR2 overexpression showed the opposite effects. DcR2 didn’t interacted with TRAIL or DR5 in vivo and vitro. The following study indicated that PRDX1 is a novel DcR2-interacting protein using quantitative proteomics, which have peroxidase and antioxidant activity in cytoplasm. The interaction of DcR2-PRDX1 mediated RTEC senescence in vitro. Knockdown of DcR2 inhibited the level of ROS and promoted the peroxidase activity of PRDX1, resulting in oxidative stress and cell senescence, and DcR2 overexpression presented the contrary results. DcR2 regulated the level of PRDX1 phosphorylation by interacting Tyr194 of PRDX1, and the mutant Tyr194 of PRDX1 can’t be phosphorylated by DcR2. Conclusion DcR2 mediates renal fibrosis and RTEC senescence by phosphorylating with PRDX1, suggesting DcR2 could serve as a potential therapeutic target for the amelioration of DN progression.

Tracking intracellular forces and mechanical property changes in mouse one-cell embryo development.

Cells comprise mechanically active matter that governs their functionality, but intracellular mechanics are difficult to study directly and are poorly understood. However, injected nanodevices open up opportunities to analyse intracellular mechanobiology. Here, we identify a programme of forces and changes to the cytoplasmic mechanical properties required for mouse embryo development from fertilization to the first cell division. Injected, fully internalized nanodevices responded to sperm decondensation and recondensation, and subsequent device behaviour suggested a model for pronuclear convergence based on a gradient of effective cytoplasmic stiffness. The nanodevices reported reduced cytoplasmic mechanical activity during chromosome alignment and indicated that cytoplasmic stiffening occurred during embryo elongation, followed by rapid cytoplasmic softening during cytokinesis (cell division). Forces greater than those inside muscle cells were detected within embryos. These results suggest that intracellular forces are part of a concerted programme that is necessary for development at the origin of a new embryonic life.

Increased nuclear but not cytoplasmic activities of CELF1 protein leads to muscle wasting.

mRNA processing is highly regulated during development through changes in RNA-binding protein (RBP) activities. CUG-BP, Elav-like family member 1 (CELF1, also called CUGBP1) is an RBP, the expression of which decreases in skeletal muscle soon after birth. CELF1 regulates multiple nuclear and cytoplasmic RNA processing events. In the nucleus, CELF1 regulates networks of postnatal alternative splicing (AS) transitions, while in the cytoplasm, CELF1 regulates mRNA stability and translation. Stabilization and misregulation of CELF1 has been implicated in human diseases including myotonic dystrophy type 1, Alzheimer's disease and multiple cancers. To understand the contribution of nuclear and cytoplasmic CELF1 activity to normal and pathogenic skeletal muscle biology, we generated transgenic mice for doxycycline-inducible and skeletal muscle-specific expression of active CELF1 mutants engineered to be localized predominantly to either the nucleus or the cytoplasm. Adult mice expressing nuclear, but not cytoplasmic, CELF1 are characterized by strong histopathological defects, muscle loss within 10days and changes in AS. In contrast, mice expressing cytoplasmic CELF1 display changes in protein levels of targets known to be regulated at the level of translation by CELF1, with minimal changes in AS. These changes are in the absence of overt histopathological changes or muscle loss. RNA-sequencing revealed extensive gene expression and AS changes in mice overexpressing nuclear and naturally localized CELF1 protein, with affected genes involved in cytoskeleton dynamics, membrane dynamics, RNA processing and zinc ion binding. These results support a stronger role for nuclear CELF1 functions as compared to cytoplasmic CELF1 functions in skeletal muscle wasting.

MON-919 Primary Neuroendocrine Tumor of the Central Nervous Sistem, a Case Report and Literature Review

IntroductionNeuroendocrine Neoplasms are rare, with an incidence of 5 to 100,000 inhabitants, constituting 1% of all malignancies, presenting high survival rates in general, even in metastatic diseases. However, in those poorly differentiated, as in the following case, survival is around 4% in 5 years. We will describe a case of primary neuroendocrine tumor in the brain, of which is uncommon in the literature.Clinical caseA 26 years women was referred to the ER of Santa Casa de São Paulo, in January 2019, to be evaluated by neurosurgery, due to progressive left hemiparesis and headache for 3 months, which got worse in 4 days. On CT scan, there was a 6 x 6 cm solid-cystic, expansive, lesion in the right frontal lobe, with perilesional edema and contralateral midline 1.3cm deviation and subfalcine herniation.Thus, the tumor was resected soon, with anatomopathological analysis showing poorly differentiated tumor of cells with scarce cytoplasm, hyperchromatic nuclei and high mitotic activity.Immunohistochemical analysis finds 50% Ki67, with focal p53, TTF1, CD99, CD 56 and synaptophysin positivity. The main hypotheses, then, consisted of Neuroendocrine Carcinoma.Four months after surgery, the patient reported worsening deficit, headache, pain, weight loss, being referred to the Emergency Room, once more. In RM an expansive lesion was found 6.6 x 4.4 cm, in the right frontoparietal surgical cavity, edema, compression and 0,4 cm midline deviation.The patient was once again submitted to emergency neurosurgery, with microsurgical resection. The pathology was identical to the previous one.We proceed with hormonal evaluation, regarding to Medular Thyroid carcinoma, Gastrinoma, Insulinoma, Pheochromocytoma, Carcinoid tumor and others.Imaging exams were also performed to investigate other primary sites: no changes in CT scan of the chest and abdomen and PET CT FDG. However, this one showed recurrence of the intracranial lesion, with three sites of involvement, all hypermetabolic: one of 4.1 x 2.9 cm (SUV 4.9) and another of 3.9 x 3.3 cm (SUV 8, 4) in the right frontoparietal region and medial nodule to the right thalamus of 1.2 cm (SUV 6.1).Patient currently maintain left hemiparesis, frequent pain, taking carbamazepine due to epileptic seizures, and considerable weight loss. She has an important limitation of daily activities and basic self-care, with 50% Karnofsky scale. Due to relapse, palliative radiotherapy was initiated in the region of the tumors.ConclusionThe patient had a poor outcome in relation to cancer, with little possibility of treatment due to poor tumor differentiation and poor performance status.

Effect of Cameraria ohridella on accumulation of proteins, peroxidase activity and composition in Aesculus hippocastanum leaves

This study analyzed the dynamics of leaf damage, the content of easily soluble proteins, benzidine-peroxidase activity and the composition of acid enzyme isoforms in the leaves of Aesculus hippocastanum L. under the influence of the chestnut miner Cameraria ohridella Deschka et Dimić (Lepidoptera, Gracillariidae) in plantations in one of the Ukraine’s largest industrial cities Dnipro, located in the steppe zone of Ukraine. During July-August, the destruction of leaves by the pest in relatively stable horse chestnut trees increased to 13.3%, while in unstable trees it increased to 97.5%. The maximum amount of protein was found in horse chestnut leaves in July, which decreased significantly in August and correlated with the level of leaf damage by the pest. The horse chestnut leaves were characterized by a more than twofold increase in cytoplasmic peroxidase activity in cases of high level of damage to the leaves by the phytophage. These observations indicate that consumption of the leaves by C. ohridella causes oxidative stress, which leads to the activation of enzyme. The horse chestnut trees have been shown to respond specifically to the attack of C. ohridella due to changes in the activity of individual peroxidase isozymes, the expression of which varies across the spectrum. In the leaves of the horse chestnut trees with a high level of damage by miner, the expression of enzyme isoforms in the pH range of 4.08–4.15 is significantly activated, which can be considered as a reliable biochemical marker of plant sensitivity of Ae. hippocastanum to phytophagous attack by C. ohridella. However, increase in the degree of phytophage invasion does not change the activity of isoperoxidase 4.21, and isoperoxidase with pI 4.25, 4.42 and 4.58 correspond to a decrease in activity compared with relatively resistant Ae. hippocastanum plants. We noted that the high total activity of peroxidase, as well as active adjustments in the spectrum of the peroxidase system, obviously, contribute to maintaining the functional integrity of the photosynthetic system of leaves of Ae. hippocastanum under the influence of the chestnut miner C. ohridella by neutralizing reactive oxygen species.

Loss of Functional Non‐Coding RNA Diversity in Diabetic Cardiac Mitochondria

Individuals with diabetes mellitus are predisposed to cardiovascular complications, but no effective predictive or therapeutic measure is in place to prevent these morbidities. Maintenance of cardiac function is heavily dependent on mitochondrial bioenergetics, making the regulation of mitochondria crucial in understanding cardiac pathologies. Both declining cardiovascular health as well as diminished mitochondrial efficiency have been separately correlated with disruption of non‐coding RNA (ncRNA) presence and activity. The recent detection of ncRNA within mitochondria by our lab and others has led us to investigate whether or not certain disease states could dysregulate ncRNA distribution and function inside the organelle. The objective of this study was to identify ncRNAs impacted within the mitochondrion by type 2 diabetes mellitus in human patients and evaluate the downstream effects caused by their dysregulation. Cytoplasm and mitochondria from human atrial appendages in type 2 diabetic (n=7) and non‐diabetic (n=7) patients were isolated and RNA purified for high‐throughput sequencing. In silico analyses of differentially expressed ncRNA revealed potential functions relevant to mitochondrial bioenergetics and cardiac dysfunction, primarily through targeting of mitochondrial and cytoplasmic mRNA but additionally for interactions between ncRNAs themselves. Approximately 20 tRNAs, 6 rRNAs, 447 lncRNAs, and 154 miRNAs were identified through mitochondrial sequencing, using a mean count value greater than 50 as a cutoff for baseline expression. Of these, all 6 rRNAs as well as 41 lncRNAs and 140 miRNAs were significantly downregulated in expression in type 2 diabetics compared to non‐diabetics. In contrast, 37 lncRNAs were significantly increased relative to non‐diabetic patients. The majority of lncRNAs and miRNAs were determined to have RNA‐binding properties, either for mRNAs or other non‐coding RNAs, based on predictive software and databases such as LncBase, NPInter, and miRcode. The mRNAs identified as targets for dysregulated miRNAs were correlated with regulation of proteins such as insulin, Akt, ERK1/2, and similarly metabolically‐crucial factors according to Ingenuity Pathway Analysis, implying cytoplasmic activity despite being found in the mitochondrion. Additionally, various cellular pathways influencing growth and viability were identified to be potentially impacted by miRNA dysregulation. Overall this study reveals a loss of non‐coding RNA diversity within type 2 diabetic cardiac mitochondria, which could contribute to mitochondrial dysfunction and ultimately cardiovascular morbidities associated with type 2 diabetes mellitus. These findings may also support ncRNA as therapeutic and diagnostic targets for type 2 diabetic cardiac complications.Support or Funding InformationThis work was supported by: The National Heart, Lung, and Blood Institute [R01 HL‐128485] (JMH), American Heart Association [AHA‐17PRE33660333] (QAH), West Virginia IDeA Network of Biomedical Research WV‐INBRE support by National Institute of Health Grant [P20GM103434], and the Community Foundation for the Ohio Valley Whipkey Trust.

Lactate dehydrogenase: An important molecule involved in acetamizuril action against Eimeria tenella

Lactate dehydrogenase (LDH), a vital enzyme in anaerobic glycolysis, is closely associated with the survival of parasites. Previous studies of some parasites have shown that LDH exhibits unique physicochemical properties and molecular structures and may be an ideal potential target for diagnosis and drug screening. In this study, we aimed to investigate the effects of acetamizuril, a novel anticoccidial compound, on LDH in the second-generation merozoites of Eimeria tenella (mz-LDH). Quantitative real-time PCR, Western blot, immunofluorescence and enzyme activity assays were each applied to detect the changes of mz-LDH. Our results indicated that the mRNA and protein levels of mz-LDH were reduced upon acetamizuril treatment. Immunolocalization of mz-LDH demonstrated that considerable amount of mz-LDH was distributed around or in the nuclei of second-generation merozoites within the untreated group; in contrast, the acetamizuril-treated group had very low level of mz-LDH. Meanwhile, LDH enzyme activity assay suggested that a decreased LDH enzyme activity in both cytoplasm and nucleus of merozoites in the acetamizuril-treated group. Moreover, the induced apoptosis in second-generation merozoites by the acetamizuril was evaluated by detecting caspase 3 activity, and acetamizuril was found to significantly increase caspase 3 activity. The above findings show that LDH may play an important role in the mediating the activity of acetamizuril against coccidiosis, and further investigation into this aspect might contribute to new light on the pathogenesis of E. tenella during its interaction with acetamizuril.

Evaluation of sodium alginate for encapsulation-vitrification of testicular Leydig cells

This study reports encapsulation-vitrification of Leydig cells. The Leydig cells were encapsulated in sodium alginate beads of different sizes and cryopreserved by vitrification or slow freezing. Physico-chemical characterization of beads was done by Fourier Transform Infrared Spectroscopy (FTIR), X-Ray Diffraction (XRD), Fluorescence Recovery after Photobleaching (FRAP) and in vitro biodegradation study. Surface morphology of cryopreserved cell-encapsulated beads was evaluated by Environmental Scanning Electron Microscopy (E-SEM), encapsulation efficiency and viability of cells were assessed by Trypan blue assay, mitochondrial activity (MTT assay) and cytoplasmic esterase enzyme activity (FDA assay), respectively. Results showed that vitrification gives better results than slow freezing with respect to surface morphology as well as cell viability of the cell-encapsulated beads (86.94 ± 2.20% vs. 67.94 ± 2.30%; p < 0.05). Encapsulation of cells in small diameter beads (1.8 mm) gave a better cell proliferation rate than large (2.1 mm and 2.7 mm). There was a significant difference in the population doubling time (47.9 ± 1.7 h vs. 67.1 ± 2.5 h) and cell proliferation rate (0.50 ± 0.24 vs. 0.36 ± 0.24 per day) of vitrified-warmed cell encapsulated beads with different diameter (p < 0.05). Encapsualtion in sodium alginate beads is a promising method for cryopreservation of Leydig cells by slow freezing as well as vitrification.

Mislocalized cytoplasmic p27 activates PAK1-mediated metastasis and is a prognostic factor in osteosarcoma.

The development of pulmonary metastasis is the leading cause of death in osteosarcoma (OS), which is the most common malignant bone tumor in children. We have previously reported that the tumor suppressor p27 (KIP1, CDKN1B) is frequently mislocalized to the cytoplasm of OS. However, its prognostic significance and metastatic mechanism are still elusive. Here, we show that cytoplasmic p27 significantly correlated with a higher metastatic status and poorer survival of OS patients (n = 136, P < 0.05), highlighting the clinical significance of p27 mislocalization in OS. Mechanistically, cytoplasmic p27 is co‐immunoprecipitated with p21‐activated kinase 1 (PAK1), which resulted in higher PAK1 phosphorylations, actin polymerization, and cell motility in p27‐mislocalized OS cells. Silencing PAK1 expression in different p27‐mislocalized OS cell lines decreased the migratory and adhesion abilities in vitro, as well as the development of pulmonary metastases in vivo. Similar PAK1‐dependent motility was also observed in other p27‐mislocalized cancer cell lines. In summary, our study suggests that cytoplasmic p27‐mediated PAK1 activation is crucial for OS metastasis. A biomarker‐guided targeted therapeutic approach for metastatic OS and other cancers harboring p27 mislocalization can be developed, where cytoplasmic p27 is used for risk stratification and PAK1 can be exploited as a potential therapeutic target.

Cholesterol derivatives induce dephosphorylation of the histone deacetylases Rpd3/HDAC1 to upregulate autophagy

ABSTRACT Histone deacetylases (HDACs) are important for global gene expression and contribute to numerous physiological events. Deacetylase Rpd3 in yeast and its conserved homolog HDAC1 in mammals oppositely regulate autophagy; however, how Rpd3/HDAC1 is regulated to mediate autophagy remains unclear. Here, we showed autophagy occurrence in silkworm (Bombyx mori) required BmRpd3, wherein steroid hormone 20-hydroxyecdysone (20E) signaling regulated its protein level and nuclear localization negatively. Inhibition of MTOR led to dephosphorylation and nucleo-cytoplasmic translocation of BmRpd3/HsHDAC1. Besides, cholesterol, 20E, and 27-hydroxycholesterol could all induce massive dephosphorylation and cytoplasmic localization of BmRpd3/HsHDAC1, and thus autophagy by affecting MTORC1 activity. In addition, three phosphorylation sites (Ser392, Ser421, and Ser423) identified in BmRpd3 were conserved in HsHDAC1. Single or triple phosphorylation-site mutation attenuated the phosphorylation levels of BmRpd3/HsHDAC1, leading to their cytoplasmic localization and autophagy activation. In general, cholesterol derivatives, especially hydroxylated cholesterol, caused dephosphorylation and nucleo-cytoplasmic shuttling of BmRpd3/HsHDAC1 through inhibition of MTOR signaling to facilitate autophagy in B. mori and mammals. These findings improve our understandings of BmRpd3/HsHDAC1-mediated autophagy induced by cholesterol derivatives and shed light on their potential as a therapeutic target for neurodegenerative diseases and autophagy-related studies. Abbreviations: 20E: 20-hydroxyecdysone; 27-OH: 27-hydroxycholesterol; ACTB: actin beta; AMPK: AMP-activated protein kinase; Atg: autophagy-related; BmSqstm1: Bombyx sequestosome 1; CQ: chloroquine; HDAC: histone deacetylase; LMNB: Lamin B1; MTOR: mechanistic target of rapamycin kinase; PE: phosphatidylethanolamine; SQSTM1/p62: sequestosome 1; TUBA1A: tubulin alpha 1a.

Mammalian RNA Decay Pathways Are Highly Specialized and Widely Linked to Translation.

SummaryRNA decay is crucial for mRNA turnover and surveillance and misregulated in many diseases. This complex system is challenging to study, particularly in mammals, where it remains unclear whether decay pathways perform specialized versus redundant roles. Cytoplasmic pathways and links to translation are particularly enigmatic. By directly profiling decay factor targets and normal versus aberrant translation in mouse embryonic stem cells (mESCs), we uncovered extensive decay pathway specialization and crosstalk with translation. XRN1 (5′-3′) mediates cytoplasmic bulk mRNA turnover whereas SKIV2L (3′-5′) is universally recruited by ribosomes, tackling aberrant translation and sometimes modulating mRNA abundance. Further exploring translation surveillance revealed AVEN and FOCAD as SKIV2L interactors. AVEN prevents ribosome stalls at structured regions, which otherwise require SKIV2L for clearance. This pathway is crucial for histone translation, upstream open reading frame (uORF) regulation, and counteracting ribosome arrest on small ORFs. In summary, we uncovered key targets, components, and functions of mammalian RNA decay pathways and extensive coupling to translation.

Antibacterial Peptide BSN-37 Kills Extra- and Intra-Cellular Salmonella enterica Serovar Typhimurium by a Nonlytic Mode of Action.

The increasing rates of resistance to traditional anti-Salmonella agents have made the treatment of invasive salmonellosis more problematic, which necessitates the search for new antimicrobial compounds. In this study, the action mode of BSN-37, a novel antibacterial peptide (AMP) from bovine spleen neutrophils, was investigated against Salmonella enterica serovar Typhimurium (S. Typhimurium). Minimum inhibitory concentrations (MICs) and time-kill kinetics of BSN-37 were determined. The cell membrane changes of S. Typhimurium CVCC541 (ST) treated with BSN-37 were investigated by testing the fluorescence intensity of membrane probes and the release of cytoplasmic β-galactosidase activity. Likewise, cell morphological and ultrastructural changes were also observed using scanning and transmission electron microscopes. Furthermore, the cytotoxicity of BSN-37 was detected by a CCK-8 kit and real-time cell assay. The proliferation inhibition of BSN-37 against intracellular S. Typhimurium was performed in Madin-Darby canine kidney (MDCK) cells. The results demonstrated that BSN-37 exhibited strong antibacterial activity against ST (MICs, 16.67 μg/ml), which was not remarkably affected by the serum salts at a physiological concentration. However, the presence of CaCl2 led to an increase in MIC of BSN-37 by about 4-fold compared to that of ST. BSN-37 at the concentration of 100 μg/ml could completely kill ST after co-incubation for 6 h. Likewise, BSN-37 at different concentrations (50, 100, and 200 μg/ml) could increase the outer membrane permeability of ST but not impair its inner membrane integrity. Moreover, no broken and ruptured cells were found in the figures of scanning and transmission electron microscopes. These results demonstrate that BSN-37 exerts its antibacterial activity against S. Typhimurium by a non-lytic mode of action. Importantly, BSN-37 had no toxicity to the tested eukaryotic cells, even at a concentration of 800 μg/ml. BSN-37 could significantly inhibit the proliferation of intracellular S. Typhimurium.

Not Fade Away: Mechanisms of Neuronal ATP Homeostasis

In this issue of Neuron, Ashrafi etal. (2020) identify a feedforward signaling mechanism that couples neuronal activity to the homeostatic maintenance of axonal and synaptic ATP production. This mechanism is achieved via changes in cytoplasmic calcium and activation of brain-specific, mitochondrial MICU3.