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Recently, Maxim A. Yurkin commented on our paper “Is the nuclear refractive index lower than cytoplasm? Validation of phase measurements and implications for light scattering technologies” as well as on a complementary study “Cell nuclei have lower refractive index and mass density than cytoplasm” from Schürmann et al. In his comment, Yurkin concluded that quantitative phase images of cells with nuclei that are less optically dense than the cytoplasm must exhibit a characteristic concavity, the absence of which is evidence against our conclusion of a less-dense nucleus. In this response, we suggest that Yurkin’s conclusion is reached through an oversimplification of the spatial refractive index distribution within cells, which does not account for high index inclusions such as the nucleolus. We further cite recent studies in 3-dimensional refractive index imaging, in which the preponderance of studies supports our conclusion. Finally, we comment on the current state of knowledge regarding subcellular refractive index distributions in living cells.

Summary Topological cytonuclear discordance is commonly observed in plant phylogenetic and phylogeographic studies, yet few studies have attempted to detect two other forms of cytonuclear discordance (branch length and geographical) and to uncover the causes of the discordance.We used the whole nuclear and chloroplast genome data from 80 individual Asian butternuts to reveal the pattern and processes of cytonuclear discordance.Our findings indicate that the chloroplast genome had substantially deeper divergence (branch‐length discordance) and a steeper cline in the contact zone (geographic discordance) compared with the nuclear genome. After various hypothesis have been tested, the results suggest that incomplete lineage sorting, positive selection and cytonuclear incompatibility are probably insufficient to explain this pattern. However, isolation‐by‐distance analysis and gene flow estimation point to a much higher level of gene flow by pollen compared with by seeds, which may have slowed down lineage divergence and mediated wider contact for nuclear genome compared with the chloroplast genome.Altogether, this study highlights a critical role of sex‐biased dispersal in causing discordance between the nuclear and plastid genome of Asian butternuts. Given its ubiquity among plants, asymmetric gene flow should be given a high priority in future studies of cytonuclear discordance.

Osteoarthritis (OA) is a cartilage degenerative disease commonly observed in the elderly population and is pathologically characterized by the degradation of the cartilage extracellular matrix (ECM). Matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTSs) are critical enzymes involved in the degradation of ECM. Olmesartan is an inhibitor of the angiotensin II receptor developed for the treatment of hypertension, and recent studies show that it exerts anti-inflammatory effects in arthritis. The present study aimed to investigate the mechanism of the protective effect of Olmesartan on cartilage ECM degradation. Interleukin-29 (IL-29) is a novel inflammatory mediator involved in the inflammation and degradation of cartilage in OA, and human T/C-28a2 cells treated with it were the inflammatory model in vitro. We found that the degradation of type 2 collagens and aggrecans was induced by IL-29, accompanied by the upregulation of MMPs and ADAMTSs, but the presence of Olmesartan significantly ameliorated these increases. In addition, Olmesartan abolished IL-29- induced oxidative stress and elevated the expression level of TNF receptor-associated factor 6 (TRAF-6). Mechanistically, we showed that Olmesartan suppressed IL-29- caused inhibitor kappa B α (IκBα) expression and nuclear translocation of nuclear factor kappa-B (NF-κB) p65, indicating it suppressed the activation of the NF-κB pathway. Collectively, our data reveal that Olmesartan exerted a protective function on IL-29- induced type 2 collagen degradation in human chondrocytes.

Accumulating evidence suggests that long non-coding RNAs (lncRNAs) participate in the formation and development of keloids, a benign tumor. In addition, lncRNA H19 has been shown to act on the biological processes of keloids. This study aimed to identify other important mechanisms of the effect of lncRNA H19 on keloid formation. The H19, miR-196b-5p, and SMAD family member 5 (SMAD5) expression levels were detected using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and Western blotting. Subcellular localization of lncRNA H19 was detected using a nuclear–cytoplasmic separation assay. Cell viability and proliferation were measured using counting kit-8 and colony formation assays. Bax and Bcl-2 levels were examined using Western blot analysis. The interaction between H19 and miR-196b-5p or SMAD5 was verified using a dual-luciferase reporter assay. H19 and SMAD5 expression was upregulated in keloid tissue and fibroblasts, whereas miR-196b-5p expression was downregulated. Knockdown of H19, overexpression of miR-196b-5p, or knockdown of SMAD5 inhibited the viability and proliferation of keloid fibroblasts and promoted apoptosis. Overexpression of H19 or SMAD5 and knockdown of miR-196b-5p promoted viability and proliferation and inhibited apoptosis. miR-196b-5p was identified as a H19 sponge, and SMAD5 was identified as a miR-196b-5p target. The combination of lncRNA H19 and miR-196b-5p regulates SMAD5 expression and promotes keloid formation, thus providing a new direction for keloid treatment.

Dexmedetomidine (DEX) has been reported to attenuate cecal ligation perforation (CLP)-stimulated acute lung injury (ALI) by downregulating HMGB1 and RAGE. This study aimed to further investigate the specific mechanisms of RAGE and its potential-related mechanisms of DEX on ALI models in vitro and in vivo. The in vitro and in vivo ALI models were established by lipopolysaccharide treatment in MLE-12 cells and CLP in mice, respectively. The effect of DEX on pathological alteration was investigated by HE staining. Thereafter, the myeloperoxidase (MPO) activity and inflammatory cytokine levels were respectively detected to assess the lung injury of mice using commercial kits. The expression levels of HMGB1, RAGE, NF-κB, and pyroptosis-related molecules were detected by RT-qPCR and Western blot. HE staining showed that lung injury, increased inflammatory cell infiltration, and lung permeability was found in the ALI mice, and DEX treatment significantly attenuated lung tissue damage induced by CLP. The MPO activity and inflammatory cytokines (TNF-α, IL-1β, and NLRP3) levels were also significantly reduced after DEX treatment compared with those in the ALI mice. Moreover, DEX activated the HMGB1/RAGE/NF-κB pathway and upregulated the pyroptosis-related proteins. However, the protective DEX effect was impaired by RAGE overexpression in ALI mice and MLE-12 cells. Additionally, DEX treatment significantly suppressed HMGB1 translocation from the nucleus region to the cytoplasm, and this effect was reversed by RAGE overexpression. These findings suggested that DEX may be a useful ALI treatment, and the protective effects on ALI mice may be through the inhibition of HMGB1/RAGE/NF-κB pathway and cell pyroptosis., Graphical abstractDexmedetomidine (DEX) has protective effects on acute lung injury (ALI) in vitro and in vivo. The possible mechanisms may be closely associated with inflammatory response, caspase-1-mediated cell pyroptosis, and high-mobility group protein 1 (HMGB1)/receptor for advanced glycation end products (RAGE)/nuclear factor-κB pathway. Moreover, DEX could promote the HMGB1 translocation from the cytoplasm to the nucleus in lipopolysaccharide-activated MLE-12 cells, whereas the action of RAGE overexpression was opposite.

Curcumin suppressed ultraviolet (UV) induced skin carcinogenesis and activated the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway. However, whether curcumin protects skin injury caused by UV is still unknown. A vitro model was established and curcumin effects on Hacat cells were detected. Nrf2 was knocked down in Hacat cells to verify the Nrf2 role in the protective effect of curcumin. Results indicated that ultraviolet A (UVA) (or ultraviolet B (UVB)) irradiation would lead to decreased cell proliferation, increased cell apoptosis, decreased catalase, heme oxygenase 1, and superoxide dismutase expression, and increased levels of protein carbonylation and malondialdehyde (p

Subcellular localization of transcripts is highly associated with regulation of gene expression, synthesis of protein, and also the development of the human brain cortex. Although many mechanisms are prevalent in the occurrence of neuroinflammation, the mechanisms based on differences in subcellular localization of transcripts have not been explored. To characterize the dynamic profile of nuclear and cytoplasmic transcripts during the progress of haemorrhage-induced neuroinflammation, we isolated nucleo-cytoplasmic RNA fractions of oxyhaemoglobin (oxy-Hb) treated microglia cells and sequenced both fractions. We discovered that cytoplasmic retained genes were the major forces to maintain the neuroinflammatory microenvironment with 10 hub genes and 40 conserved genes were identified. Moreover, antisense RNA Gm44096 and lincRNA Gm47270, which co-expressed with a crowd of inflammatory genes in the cytoplasm, were discovered as regulatory strategies for sustaining the neuroinflammatory microenvironment. Thus, our study provides a new perspective on understanding haemorrhage-induced neuroinflammation and also reveals a mechanism of lncRNA responsible for maintaining the neuroinflammatory microenvironment.

Expansion microscopy (ExM) has been successfully used to improve the spatial resolution when imaging tissues by optical microscopy. In ExM, proteins of a fixed sample are crosslinked to a swellable acrylamide gel, which expands when incubated in water. Therefore, ExM allows enlarged subcellular structures to be resolved that would otherwise be hidden to standard confocal microscopy. Herein, we aim to validate ExM for the study of peroxisomes, mitochondria, nuclei and the plasma membrane. Upon comparison of the expansion factors of these cellular compartments in HEK293 cells within the same gel, we found significant differences, of a factor of above 2, in expansion factors. For peroxisomes, the expansion factor differed even between peroxisomal membrane and matrix marker; this underlines the need for a thorough validation of expansion factors of this powerful technique. We further give an overview of possible quantification methods for the determination of expansion factors of intracellular organelles, and we highlight some potentials and challenges., In Expansion Microscopy (ExM) subcellular structures are imaged in isotropically expanded fixed samples, consequently allowing to resolve enlarged subcellular structures, otherwise hidden to standard microscopy. Upon comparison of the expansion factors of different cellular compartments in cells within the same gel, we found significant differences in expansion factors of a factor of above 2.

Super-resolution microscopy revealed that the plant nuclear lamina proteins NMCP1 and NMCP2 organize into filamentous networks with filament bundles., Plant genomes lack genes encoding intermediate filament proteins, including lamins; however, functional lamin analogues are presumed to exist in plants. Plant-specific coiled-coil proteins, that is, nuclear matrix constituent proteins (NMCPs), are the most likely candidates as the structural elements of the nuclear lamina because they exhibit a lamin-like domain arrangement. They are exclusively localized at the nuclear periphery and have functions that are analogous to those of lamins. However, their assembly into filamentous polymers has not yet been confirmed. In this study, we examined the higher-order structure of NMCP1 and NMCP2 in Apium graveolens cells by using stimulated emission depletion microscopy combined with immunofluorescence cell labelling. Our analyses revealed that NMCP1 and NMCP2 form intricate filamentous networks, which include thick segments consisting of filament bundles, forming a dense filamentous layer extending across the nuclear periphery. Furthermore, the outermost chromatin distribution was found to be in the nucleoplasm-facing region of the nuclear lamina. Recombinant Daucus carota NMCP1 with a His-tag produced in Escherichia coli refolded into dimers and self-assembled into filaments and filament bundles. These results suggest that NMCP1 and NMCP2 organize into the nuclear lamina by forming a filamentous network with filament bundles that localize at the nuclear periphery.

The vitamin D receptor (VDR) and aryl hydrocarbon receptor (AHR) are two nuclear receptors that exert their effects by binding with ligands and forming a molecular complex. These complexes translocate to the nucleus and activate the expression of a series of genes which have a response element to VDR or AHR. Both receptors have been identified in the pathogenesis of endometriosis, a common disease characterized by the formation of endometrium-like tissue in ectopic zones. Despite numerous therapies, there is no definitive cure for endometriosis at the pharmacological level. Our study aims to describe the location and the expression of VDR and AHR at the protein level. For this purpose, an evaluation was performed using tissue from the three normal phases of the endometrium (proliferative, early, and late secretory) and in endometriosis by immunohistochemistry, using anti-VDR and anti-AHR antibodies. We demonstrate that in the nuclei of glandular cells in endometriosis, the expression of VDR and AHR is mutually exclusive—when the expression of one receptor is high, the other one is low—suggesting a possible target in the treatment of endometriosis. We also identify a significant change in the expression of glandular cytoplasmic AHR between the proliferative and late secretory endometrium. Supplementary Information The online version contains supplementary material available at 10.1007/s00418-021-02005-9.

The occurrence of hypoxia-ischemia (HI) in the developing brain is closely associated with neuronal injury and even death. However, the underlying molecular mechanism is not fully understood. This study was designed to investigate phosphatase and tensin homolog (PTEN) nuclear translocation and its possible role in rat cortical neuronal damage following oxygen-glucose deprivation (OGD) in vitro. An in vitro OGD model was established using primary cortical neurons dissected from newborn Sprague-Dawley rats to mimic HI conditions. The PTENK13R mutant plasmid, which contains a lysine-to-arginine mutation at the lysine 13 residue, was constructed. The nuclei and cytoplasm of neurons were separated. Neuronal injury following OGD was evidenced by increased lactate dehydrogenase (LDH) release and apoptotic cell counts. In addition, PTEN expression was increased and the phosphorylation of extracellular signal-regulated kinase 1/2 (p-ERK1/2) and activation of nuclear factor kappa B (NF-κB) were decreased following OGD. PTENK13R transfection prevented PTEN nuclear translocation; attenuated the effect of OGD on nuclear p-ERK1/2 and NF-κB, apoptosis, and LDH release; and increased the expression of several anti-apoptotic proteins. We conclude that PTEN nuclear translocation plays an essential role in neuronal injury following OGD via modulation of the p-ERK1/2 and NF-κB pathways. Prevention of PTEN nuclear translocation might be a candidate strategy for preventing brain injury following HI.

Cataract is the leading cause of visual impairment globally. Racemization of lens proteins may contribute to cataract formation in aging individuals. As a special type of age-related cataract (ARC), diabetic cataract (DC) is characterized by the early onset of cortical opacification and finally developed into a mixed type of cortical and nuclear opacification. We compared racemization of Asp 58 residue, a hotspot position in αA-crystallin, from the cortex and nucleus of diabetic and age-matched senile cataractous lenses, by identifying L-Asp/L-isoAsp/D-Asp/D-isoAsp by mass spectrometry. Compared to nondiabetic cataractous lenses, DC lenses showed a significantly increased cortex/nucleus ratio of D-Asp 58, which originated primarily from an increased percentage of D-Asp 58 in the lens cortex of DC. Moreover, patients diagnosed with diabetes for over 10 years showed a lower cortex/nucleus ratio of D-isoAsp 58 in the lens compared with those who had a shorter duration of diabetes, which originated mainly from an increased percentage of D-isoAsp 58 in the lens nucleus of DC with increasing time of hyperglycemia. Further analysis confirmed decreased protein solubility in diabetic cataractous lenses. The different racemization pattern in DC may be distinguished from ARC and influence its phenotype over the protracted duration of diabetes.

cDNA microarray data conducted by our group revealed overexpression of CXCL2 and CXCL8 in ovarian cancer (OC) microenvironment. Herein, we have proven that the chemokine receptor, CXCR2, is a pivotal molecule in re-sensitizing OC to cisplatin, and its inhibition decreases cell proliferation, viability, tumor size in cisplatin-resistant cells, as well as reversed the overexpression of mesenchymal epithelium transition markers. Altogether, our study indicates a central effect of CXCR2 in preventing tumor progression, due to acquisition of cisplatin chemoresistant phenotype by tumor cells, and patients’ high lethality rate. We found that the overexpression of CXCR2 by OC cells is persistent and anomalously confined to the cellular nuclei, thus pointing to an urge in developing highly lipophilic molecules that promptly permeate cells, bind to and inhibit nuclear CXCR2 to fight OC, instead of relying on the high-cost genetic engineered cells.

Suppression of type I interferon (IFN) response is one pathological outcome of the infection of highly pathogenic human coronaviruses. To effect this, severe acute respiratory syndrome coronavirus (SARS-CoV) and SARS-CoV-2 encode multiple IFN antagonists. In this study, we reported on the IFN antagonism of SARS-CoV-2 main protease NSP5. NSP5 proteins of both SARS-CoV and SARS-CoV-2 counteracted Sendai virus-induced IFN production. NSP5 variants G15S and K90R commonly seen in circulating strains of SARS-CoV-2 retained the IFN-antagonizing property. The suppressive effect of NSP5 on IFN-β gene transcription induced by RIG-I, MAVS, TBK1 and IKKϵ suggested that NSP5 likely acts at a step downstream of IRF3 phosphorylation in the cytoplasm. NSP5 did not influence steady-state expression or phosphorylation of IRF3, suggesting that IRF3, regardless of its phosphorylation state, might not be the substrate of NSP5 protease. However, nuclear translocation of phosphorylated IRF3 was severely compromised in NSP5-expressing cells. Taken together, our work revealed a new mechanism by which NSP5 proteins encoded by SARS-CoV and SARS-CoV-2 antagonize IFN production by retaining phosphorylated IRF3 in the cytoplasm. Our findings have implications in rational design and development of antiviral agents against SARS-CoV-2.

Cellular senescence is the irreversible cell cycle arrest in response to DNA damage. Because senescent cells accumulate with age and contribute to chronic inflammation, they are promising therapeutic targets for healthspan extension. The senescent phenotype can vary depending on cell type and on the specific insults that induce senescence. This variability is also reflected in the extensive remodeling of the genome organization within the nucleus of senescent cells. Here, we give an overview of the nuclear changes that occur in different forms of senescence, including changes to chromatin state and composition and to the three-dimensional organization of the genome, as well as alterations to the nuclear envelope and to the accessibility of repetitive genomic regions. Many of these changes are shared across all forms of senescence, implicating nuclear organization as a fundamental driver of the senescent state and of how senescent cells interact with the surrounding tissue.

Coxsackievirus B3 (CVB3) is a prevalent etiological agent for viral myocarditis and neurological disorders, particularly in infants and young children. Virus-encoded proteinases have emerged as cytopathic factors that contribute to disease pathogenesis in part through targeting the cellular recycling machinery of autophagy. Although it is appreciated that CVB3 can usurp cellular macroautophagy/autophagy for pro-viral functions, the precise mechanisms by which viral proteinases disrupt autophagy remain incompletely understood. Here we identified TFEB (transcription factor EB), a master regulator of autophagy and lysosome biogenesis, as a novel target of CVB3 proteinase 3 C. Time-course infections uncovered a significant loss of full-length TFEB and the emergence of a lower-molecular mass (~63 kDa) fragment. Cellular and in vitro cleavage assays revealed the involvement of viral proteinase 3 C in the proteolytic processing of TFEB, while site-directed mutagenesis identified the site of cleavage after glutamine 60. Assessment of TFEB transcriptional activity using a reporter construct discovered a loss of function of the cleavage fragment despite nuclear localization and retaining of the ability of DNA and protein binding. Furthermore, we showed that CVB3 infection was also able to trigger cleavage-independent nuclear translocation of TFEB that relied on the serine-threonine phosphatase PPP3/calcineurin. Finally, we demonstrated that both TFEB and TFEB [Δ60] serve roles in viral egress albeit through differing mechanisms. Collectively, this study reveals that CVB3 targets TFEB for proteolytic processing to disrupt host lysosomal function and enhance viral infection. Abbreviations:ACTB: actin beta; CLEAR: coordinated lysosomal enhancement and regulation; CVB3: coxsackievirus B3; DAPI: 4′,6-diamidino-2-phenylindole; GFP: green fluorescent protein; LAMP1: lysosomal associated membrane protein 1; LTR: LysoTracker Red; PPP3/calcineurin: protein phosphatase 3; PPP3CA: protein phosphatase 3 catalytic subunit A; p-TFEB: phospho-Ser211 TFEB; si-CON: scramble control siRNA; TFEB: transcription factor EB; TFEB [Δ60]: TFEB cleavage fragment that lacks the first 60 amino acids; VP1: viral capsid protein 1

Tumor metastasis is the leading cause of death in patients with colorectal cancer (CRC). Circular RNAs (circRNAs) have been shown to be involved in cancer progression. However, the regulatory mechanisms of circRNAs involved in CRC tumor metastasis are currently unknown. Methods: High-throughput sequencing was performed on 6 pairs of CRC and adjacent normal tissues to identify the expression profiles of mRNA and circRNA. circ1662 was assessed by RNA-ISH and IHC of a tissue chip. The function of circ1662 in CRC was evaluated by knocking down or overexpressing circ1662. MeRIP-qPCR, RIP-qPCR, and RNA pull-down were performed to determine the relationship between METTL3, circ1662, and YAP1. Results: A novel circRNA, circ1662, exhibited significantly higher expression in CRC tissues than paired normal tissues. High circ1662 expression was correlated with poor prognosis and tumor depth in patients with CRC. Functionally, circ1662 promoted CRC cell invasion and migration by controlling EMT in vitro and in vivo. Mechanistically, circ1662 directly bound to YAP1 and accelerated its nuclear accumulation to regulate the SMAD3 pathway. Additionally, circ1662 enhanced CRC invasion and migration depending on YAP1 and SMAD3. Interestingly, METTL3 induced circ1662 expression by binding its flanking sequences and installing m6A modifications. Clinically, circ1662 expression strongly correlated with METTL3 and YAP1 protein expression. Moreover, YAP1 expression was negatively correlated with SMAD3 expression. Conclusions: METTL3-induced circ1662 promoted CRC cell invasion and migration by accelerating YAP1 nuclear transport. This result implies that circ1662 is a new prognostic and therapeutic marker for CRC metastasis.

During their different life stages, parasites undergo remarkable morphological, physiological, and behavioral “metamorphoses” to meet the needs of their changing habitats. This is even true for ectoparasites, such as the monogeneans, which typically have a free-swimming larval stage (oncomiracidium) that seeks out and attaches to the external surfaces of fish where they mature. Before any obvious changes occur, there are ultrastructural differences in the oncomiracidium’s outer surface that prepare it for a parasitic existence. The present findings suggest a distinct variation in timing of the switch from oncomiracidia epidermis to the syncytial structure of the adult tegument and so, to date, there are three such categories within the Monogenea: (1) Nuclei of both ciliated cells and interciliary cytoplasm are shed from the surface layer and the epidermis becomes a syncytial layer during the later stages of embryogenesis; (2) nuclei of both ciliated cells and interciliary syncytium remain distinct and the switch occurs later after the oncomiracidia hatch (as in the present study); and (3) the nuclei remain distinct in the ciliated epidermis but those of the interciliary epidermis are lost during embryonic development. Here we describe how the epidermis of the oncomiracidium of Discocotyle sagittata is differentiated into two regions, a ciliated cell layer and an interciliary, syncytial cytoplasm, both of which are nucleated. The interciliary syncytium extends in-between and underneath the ciliated cells and sometimes covers part of their apical surfaces, possibly the start of their shedding process. The presence of membranous whorls and pyknotic nuclei over the surface are indicative of membrane turnover suggesting that the switch in epidermis morphology is already initiated at this stage. The body tegument and associated putative sensory receptors of subadult and adult D. sagittata are similar to those in other monogeneans.

A large portion of the human genome is transcribed into long noncoding RNAs that can range from 200 nucleotides to several kilobases in length. The number of identified lncRNAs is still growing, but only a handful of them have been functionally characterized. However, it is known that the functions of lncRNAs are closely related to their subcellular localization. Cytoplasmic lncRNAs can regulate mRNA stability, affect translation and act as miRNA sponges, while nuclear-retained long noncoding RNAs have been reported to be involved in transcriptional control, chromosome scaffolding, modulation of alternative splicing and chromatin remodelling. Through these processes, lncRNAs have diverse regulatory roles in cell biology and diseases. OIP5-AS1 (also known as Cyrano), a poorly characterized lncRNA expressed antisense to the OIP5 oncogene, is deregulated in multiple cancers. We showed that one of the OIP5-AS1 splicing forms (ENST00000501665.2) is retained in the cell nucleus where it associates with chromatin, thus narrowing down the spectrum of its possible mechanisms of action. Its knockdown with antisense LNA gapmeRs led to inhibited expression of a sense partner, OIP5, strongly suggesting a functional coupling between OIP5 and ENST00000501665.2. A subsequent bioinformatics analysis followed by RAP-MS and RNA Immunoprecipitation experiments suggested its possible mode of action; in particular, we found that ENST00000501665.2 directly binds to a number of nuclear proteins, including SMARCA4, a component of the SWI/SNF chromatin remodelling complex, whose binding motif is located in the promoter of the OIP5 oncogene.

Rationale: Annexin-A1 (ANXA1) has previously been proposed to play a crucial role in neuronal apoptosis during ischemic stroke injury. Our recent study demonstrated that ANXA1 was modified by SUMOylation, and that this modification was greatly weakened after cerebral ischemia, but its effect on neuronal death and the underlying mechanism have not been fully elucidated. Methods: Mice subjected to middle cerebral artery occlusion were established as the animal model and primary cultured neurons treated with oxygen-glucose deprivation and reperfusion was established as the cell model of ischemic stroke. The Ni2+-NTA agarose affinity pull-down assay was carried out to determine the SUMOylation level of ANXA1. Co-immunoprecipitation assays was utilized to explore the protein interaction. Immunoblot analysis, quantitative real-time PCR, Luciferase reporter assay were performed to identify the regulatory mechanism. LDH release and TUNEL staining was performed to investigate the neuronal cytotoxicity and apoptosis, respectively. Results: In this study, we identified the deSUMOylating enzyme sentrin/SUMO-specific protease 6 (SENP6) as a negative regulator of ANXA1 SUMOylation. Notably, we found that SENP6-mediated deSUMOylation of ANXA1 induced its nuclear translocation and triggered neuronal apoptosis during cerebral ischemic injury. A mechanistic study demonstrated that SENP6-mediated deSUMOylation of ANXA1 promoted TRPM7- and PKC-dependent phosphorylation of ANXA1. Furthermore, blocking the deSUMOylation of ANXA1 mediated by SENP6 inhibited the transcriptional activity of p53, decreased Bid expression, suppressed caspase-3 pathway activation and reduced the apoptosis of primary neurons subjected to oxygen-glucose deprivation and reperfusion. More importantly, SENP6 inhibition by overexpression of a SENP6 catalytic mutant in neurons resulted in significant improvement in neurological function in the mouse model of ischemic stroke. Conclusions: Taken together, the results of this study identified a previously unidentified function of SENP6 in neuronal apoptosis and strongly indicated that SENP6 inhibition may provide therapeutic benefits for cerebral ischemia.

YTH domain family 2 (YTHDF2) is an N6-methyladenosine (m6A) binding protein promoting mRNA degradation in various biological processes. Despite its essential roles, the role of YTHDF2 in determining cell fates has not been fully elucidated. Notch signaling plays a vital role in determining cell fates, such as proliferation, differentiation, and apoptosis. We investigated the effect of YTHDF2 on Notch signaling. Our results show that YTHDF2 inhibits Notch signaling by downregulating the Notch1, HES1, and HES5 mRNA levels. Analyzing YTHDF2 deletion mutants indicates that the YTH domain is critical in regulating the Notch signal by directly binding m6A of Notch1 mRNA. Recently, YTHDF2 nuclear translocation was reported under heat shock conditions, but its physiological function is unknown. In our study, the YTH domain is required for YTHDF2 nuclear translocation. In addition, under heat shock stress, the Notch signal was significantly restored due to the increased expression of the Notch1 targets. These results suggest that YTHDF2 in the cytoplasm may act as an intrinsic suppressor in Notch signaling by promoting Notch1 mRNA degradation under normal cellular conditions. Conversely, upon the extracellular stress such as heat shock, YTHDF2 nuclear translocation resulting in reduced Notch1 mRNA decay may contribute to the increasing of Notch intracellular domain (NICD) regulating the survival-related target genes.

Neonatal infectious pneumonia (NIP) is a common infectious disease that develops in the neonatal period. The purpose of our study was to explore the potential roles of 25(OH)-Vitamin D (25-OH-VD) and its anti-inflammatory mechanism in NIP. The results showed that serum 25-OH-VD level was negatively correlated with the severity of NIP, whereas Spearman’s correlation analysis showed a significant positive correlation between the severity of NIP and the levels of pneumonia markers procalcitonin (PCT) and interleukin-6 (IL-6). The expression of vitamin D receptor (VDR) was down-regulated, while the transforming growth factor β (TGFβ), nuclear YAP, and TAZ were up-regulated in the peripheral blood mononuclear cells (PBMCs) of neonates with severe pneumonia. Neonates with 25-OH-VD deficiency were associated with an increased risk of NIP. In BEAS-2B cells, down-regulation of nuclear YAP and TAZ was found in the lipopolysaccharide (LPS) + VD group relative to the LPS-induced group. Additionally, positive rate of nuclear YAP, as detected by immunocytochemistry (ICC), and the nuclear translocation of nuclear YAP/TAZ by IFA in the LPS+VD group showed an intermediate level between that of the control and LPS-induced groups. Furthermore, the expressions of VDR and CYP27B1 were significantly increased in the LPS+VD group as compared to those in the LPS-induced group. The anti-inflammatory mechanism in NIP was achieved due to the 25-OH-VD mediating TGFβ/YAP/TAZ pathway, which suggested that using 25-OH-VD might be a potential strategy for NIP treatment.

Nuclear localization of cytoplasmic RNA virus proteins mediated by intrinsic nuclear localization signal (NLS) plays essential roles in successful virus replication. We previously reported that NLS mutation in the matrix (M) protein obviously attenuates the replication and pathogenicity of Newcastle disease virus (NDV), but the attenuated replication mechanism remains unclear. In this study, we showed that M/NLS mutation not only disrupted M’s nucleocytoplasmic trafficking characteristic but also impaired viral RNA synthesis and transcription. Using TMT-based quantitative proteomics analysis of BSR-T7/5 cells infected with the parental NDV rSS1GFP and the mutant NDV rSS1GFP-M/NLSm harboring M/NLS mutation, we found that rSS1GFP infection stimulated much greater quantities and more expression changes of differentially expressed proteins involved in host cell transcription, ribosomal structure, posttranslational modification, and intracellular trafficking than rSS1GFP-M/NLSm infection. Further in-depth analysis revealed that the dominant nuclear accumulation of M protein inhibited host cell transcription, RNA processing and modification, protein synthesis, posttranscriptional modification and transport; and this kind of inhibition could be weakened when most of M protein was confined outside the nucleus. More importantly, we found that the function of M protein in the cytoplasm effected the inhibition of TIFA expression in a dose-dependent manner, and promoted NDV replication by down-regulating TIFA/TRAF6/NF-κB-mediated production of cytokines. It was the first report about the involvement of M protein in NDV immune evasion. Taken together, our findings demonstrate that NDV replication is closely related to the nucleocytoplasmic trafficking of M protein, which accelerates our understanding of the molecular functions of NDV M protein.

ACE2 was observed as the cell surface receptor of the SARS-CoV-2 virus. Interestingly, we also found ACE2 positivity inside the cell nucleus. The ACE2 levels changed during cell differentiation and aging and varied in distinct cell types. We observed ACE2 depletion in the aortas of aging female mice, similarly, the aging caused ACE2 decrease in the kidneys. Compared with that in the heart, brain and kidneys, the ACE2 level was the lowest in the mouse lungs. In mice exposed to nicotine, ACE2 was not changed in olfactory bulbs but in the lungs, ACE2 was upregulated in females and downregulated in males. These observations indicate the distinct gender-dependent properties of ACE2. Differentiation into enterocytes, and cardiomyocytes, caused ACE2 depletion. The cardiomyogenesis was accompanied by renin upregulation, delayed in HDAC1-depleted cells. In contrast, vitamin D2 decreased the renin level while ACE2 was upregulated. Together, the ACE2 level is high in non-differentiated cells. This protein is more abundant in the tissues of mouse embryos and young mice in comparison with older animals. Mostly, downregulation of ACE2 is accompanied by renin upregulation. Thus, the pathophysiology of COVID-19 disease should be further studied not only by considering the ACE2 level but also the whole renin-angiotensin system.

BRCA1-associated protein-1(BAP1) expression is commonly lost in several tumors including malignant pleural mesothelioma (MPM). Presence or absence of immunohistochemical BAP1 nuclear staining in tumor cells is currently used for differential diagnosis of MPM. In this study, a large cohort of 596 MPM tumors with available clinical data was analyzed to examine associations of BAP1 staining pattern with clinical and molecular features that may reflect the impact ofBAP1mutation on MPM biology. Cases were classified according to the BAP1 staining pattern of tumor cells. Exome and RNA-sequencing data were available for subsets of cases. Levels of mRNA encoding claudin 15 (CLDN15) and vimentin (VIM) were determined using RT-qPCR on 483 cases to estimate the relative proportions of epithelial-like and mesenchymal-like components in each tumor. Four BAP1 staining patterns were observed: single-pattern nuclear staining (36%), single-pattern cytoplasmic staining (25%), single-pattern absent staining (12%), and combinations of these staining patterns (27%). This study confirmed prior reports that nuclear BAP1 is more frequently associated with wild-typeBAP1and sarcomatoid histology. However, no associations between BAP1 staining pattern(s) and mutations in specific protein domains and/or mutation type were observed. BAP1 staining patterns were significantly associated (p < 0.001) withBAP1gene expression, MPM histologic subtypes, molecular clusters, and markers of epithelial-to-mesenchymal transition. Frequent observation of combinations of BAP1 staining patterns in MPM tumors indicated intra-tumoral heterogeneity ofBAP1status. Cytoplasmic BAP1 staining was identified as a putative indicator of favorable prognosis in non-epithelioid MPM. In conclusion, novel significant associations among different BAP1 staining patterns and subgroups of MPM tumors were observed, suggesting that the role ofBAP1in tumor progression may be more complex than its presumed tumor suppressor function. Cytoplasmic staining was identified as a putative indicator of favorable prognosis in non-epithelioid MPM, potentially addressing a critical need in clinical decision-making in this disease. (c) 2020 The Authors.The Journal of Pathologypublished by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland. National Cancer InstituteUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USANIH National Cancer Institute (NCI) [NCI 2 R01 CA120528-11A1]; International Mesothelioma Program at Brigham and Women's Hospital; NCI Cancer Center Support GrantUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USANIH National Cancer Institute (NCI) [NIH 5 P30 CA06516] This work was supported by grants to RB from the National Cancer Institute (NCI 2 R01 CA120528-11A1) and the International Mesothelioma Program at Brigham and Women's Hospital. The study sponsors played no role in the study design, collection, analysis, interpretation of data, writing of the report, or decision to submit the paper for publication. We thank the Dana-Farber/Harvard Cancer Center in Boston, MA, for the use of the Specialized Histopathology Core, which provided histology and immunohistochemistry service. Dana-Farber/Harvard Cancer Center is supported in part by an NCI Cancer Center Support Grant #NIH 5 P30 CA06516.

New findings What is the central question of this study? Type 2 diabetes mellitus (T2DM) causes skeletal muscle atrophy; does it affect resistance training (RT)-mediated molecular adaptations and subsequent muscle hypertrophy? What is the main finding and its importance? Although skeletal muscle mass and regulation were not preserved under conditions of T2DM, the response of RT-induced skeletal muscle hypertrophy was not impaired in T2DM rat skeletal muscle. These findings suggest that the capacity of RT-mediated muscle mass gain is not diminished in the T2DM condition. Abstract Type 2 diabetes mellitus (T2DM) is known to cause skeletal muscle atrophy. However, it is not known whether T2DM affects resistance training (RT)-mediated molecular adaptations and subsequent muscle hypertrophy. Therefore, we investigated the effect of T2DM on response of skeletal muscle hypertrophy to chronic RT using a rat resistance exercise mimetic model. T2DM and healthy control rats were subjected to 18 bouts (3 times per week) of chronic RT on unilateral lower legs. RT significantly increased gastrocnemius muscle mass and myonuclei in both T2DM and healthy control rats to the same extent, even though T2DM caused muscle atrophy in the resting condition. Further, T2DM significantly reduced mechanistic target of rapamycin complex 1 (mTORC1) activity (phosphorylation of p70S6KThr389 and 4E-BP1Thr37/46 ) to insulin stimulation and the number of myonuclei in the untrained basal condition, but RT-mediated adaptations were not affected by T2DM. These findings suggested that although the skeletal muscle mass and regulation were not preserved under basal conditions of T2DM, the response of RT-induced skeletal muscle hypertrophy was not impaired in T2DM rat skeletal muscle.

Aberrant activation of β‐catenin has been implicated in a variety of human diseases, including cancer. In spite of significant progress, the regulation of active Wnt/β‐catenin‐signaling pathways is still poorly understood. In this study, we show that F‐box protein 16 (FBXO16) is a putative tumor suppressor. It is a component of the SCF (SKP1‐Cullin1‐F‐box protein) complex, which targets the nuclear β‐catenin protein to facilitate proteasomal degradation through the 26S proteasome. FBXO16 interacts physically with the C‐terminal domain of β‐catenin and promotes its lysine 48‐linked polyubiquitination. In addition, it inhibits epithelial‐to‐mesenchymal transition (EMT) by attenuating the level of β‐catenin. Therefore, depletion of FBXO16 leads to increased levels of β‐catenin, which then promotes cell invasion, tumor growth, and EMT of cancer cells. Furthermore, FBXO16 and β‐catenin share an inverse correlation of cellular expression in clinical breast cancer patient samples. In summary, we propose that FBXO16 functions as a putative tumor suppressor by forming an SCFFBXO16 complex that targets nuclear β‐catenin in a unique manner for ubiquitination and subsequent proteasomal degradation to prevent malignancy. This work suggests a novel therapeutic strategy against human cancers related to aberrant β‐catenin activation. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

The relationship between the radiation dose delivered to a tumor and its effect is not completely predictable. Uncertainty in the estimation of the boron concentration in a tumor, variation in the radiation sensitivity of the tumor cells, and the complexity of the interactions between the four types of radiation comprising the boron neutron capture therapy (BNCT) dose contribute to this uncertainty. We reanalyzed the data of our previous papers to investigate the variation in radiosensitivity of tumor cells to the 10B(n,α)7Li dose: the dose generated by the reaction of thermal neutrons and 10B, hereafter the ‘boron-neutron dose’. The radiosensitivities of five tumors (EL4, SAS/neo, SAS/mp53, SCCVII and B16-BL6 melanoma) were examined. For the combination of p-boron-L-phenylalanine (BPA: C9H12BNO4) with neutron irradiation, D0, the cell survival curve for the boron-neutron dose was the smallest for the SAS/neo, followed by the EL4, SAS/mp53, SCCVII and B16-BL6 melanoma, in that order. For the combination of mercaptoundecahydrododecaborate (BSH: Na2B12H11SH) with neutron irradiation, D0 was the smallest for the EL4, followed by the SAS/neo, B16–BL6melanoma, SAS/mp53 and SCCVII, in that order. The relationships between these D0 values and the nucleocytoplasmic ratios (Xncs) or cell size indices (Xcs) obtained by histopathological microslide image were as follows: (D0 = 0.1341Xnc–1.586, R2 = 0.9721) for all tumor types with BPA-BNCT, and D0 = 0.0122Xcs–0.1319 (R2 = 0.9795) for four tumor types (all except the B16-BL6 melanoma) with BSH-BNCT. Based on these results, we proposed a new biologically equivalent effectiveness factor: the absolute biological effectiveness (ABE) factor. The ABE factor is Gy/D0. Thus, the ABE dose is the physical dose multiplied by the ABE factor, and refers to the dose needed to decrease the cell survival rate to e–ABE dose/Gy.

RFWD3 is an E3 ubiquitin ligase that plays important roles in DNA damage response and DNA replication. We have previously demonstrated that the stabilization of RFWD3 by PCNA at the replication fork enables ubiquitination of the single-stranded binding protein, RPA and its subsequent degradation for replication progression. Here, we report that RFWD3 associates with the Origin Recognition Complex (ORC) and ORC-Associated (ORCA/LRWD1), components of the pre-replicative complex required for the initiation of DNA replication. Overexpression of ORC/ORCA leads to the stabilization of RFWD3. Interestingly, RFWD3 seems to stabilize ORC/ORCA in cells expressing wild type p53, as the depletion of RFWD3 reduces the levels of ORC/ORCA. Further, the catalytic activity of RFWD3 is required for the stabilization of ORC. Our results indicate that the RFWD3 promotes the stability of ORC, enabling efficient pre-RC assembly.

The BMP/TGFβ-Smad, Notch and VEGF signaling guides formation of endothelial tip and stalk cells. However, the crosstalk of bone morphogenetic proteins (BMPs) and vascular endothelial growth factor receptor 2 (VEGFR2) signaling has remained largely unknown. We demonstrate that BMP family members regulate VEGFR2 and Notch signaling, and act via TAZ-Hippo signaling pathway. BMPs were found to be regulated after VEGF gene transfer in C57/Bl6 mice and in a porcine myocardial ischemia model. BMPs 2/4/6 were identified as endothelium-specific targets of VEGF. BMP2 modulated VEGF-mediated endothelial sprouting via Delta like Canonical Notch Ligand 4 (DLL4). BMP6 modulated VEGF signaling by regulating VEGFR2 expression and acted via Hippo signaling effector TAZ, known to regulate cell survival/proliferation, and to be dysregulated in cancer. In a matrigel plug assay in nude mice BMP6 was further demonstrated to induce angiogenesis. BMP6 is the first member of BMP family found to directly regulate both Hippo signaling and neovessel formation. It may thus serve as a target in pro/anti-angiogenic therapies. Electronic supplementary material The online version of this article (10.1007/s10456-020-09748-4) contains supplementary material, which is available to authorized users.

Nuclear movement and positioning play a role in developmental processes throughout life. Nuclear movement and positioning are mediated primarily by linker of nucleoskeleton and cytoskeleton (LINC) complexes. LINC complexes are comprised of the inner nuclear membrane SUN proteins and the outer nuclear membrane (ONM) KASH proteins. In Arabidopsis pollen tubes, the vegetative nucleus (VN) maintains a fixed distance from the pollen tube tip during growth, and the VN precedes the sperm cells (SCs). In pollen tubes of wit12 and wifi, mutants deficient in the ONM component of a plant LINC complex, the SCs precede the VN during pollen tube growth and the fixed VN distance from the tip is lost. Subsequently, pollen tubes frequently fail to burst upon reception. In this study, we sought to determine if the pollen tube reception defect observed in wit12 and wifi is due to decreased sensitivity to reactive oxygen species (ROS). Here, we show that wit12 and wifi are hyposensitive to exogenous H2O2, and that this hyposensitivity is correlated with decreased proximity of the VN to the pollen tube tip. Additionally, we report the first instance of nuclear Ca2+ peaks in growing pollen tubes, which are disrupted in the wit12 mutant. In the wit12 mutant, nuclear Ca2+ peaks are reduced in response to exogenous ROS, but these peaks are not correlated with pollen tube burst. This study finds that VN proximity to the pollen tube tip is required for both response to exogenous ROS, as well as internal nuclear Ca2+ fluctuations.

Osteoporosis (OP) is an age-related osteolytic disease and characterized by low bone mass and more prone to fracture due to active osteoclasts. Proliferating cell nuclear antigen (PCNA) has been long identified as a nuclear protein playing critical roles in the regulation of DNA replication and repair. Recently, a few studies have demonstrated the cytoplasmic localization of PCNA and its function associated with apoptosis in neutrophil and neuroblastoma cells. However, the involvement of PCNA, including the cytoplasmic PCNA, in the osteoclast differentiation remains unclear. In the present study, we show that PCNA is translocated from nucleus to cytoplasm during the RANKL-induced osteoclast differentiation, and localized in the actin belt of mature osteoclast. Knockdown of PCNA significantly affected the integrity of actin belt, the formation of multinucleated osteoclasts, the expression of osteoclast-specific genes, and the in vitro bone resorption. Interactomic study has revealed β-actin as the major interacting partner of the cytoplasmic PCNA, suggesting that cytoplasmic PCNA might play a critical role in the differentiation of osteoclast through regulation of actin-cytoskeleton remodeling. Taken together, our results demonstrate the critical role of cytoplasmic PCNA during the process of osteoclast differentiation, and provided a potential therapeutic target for treatment of osteoclast-related bone diseases.

Thyroid tumors are the most common types of endocrine malignancies and are commonly treated with radioactive iodine (RAI) to destroy remaining cancer cells following surgical intervention. We previously reported that the expression levels of double-stranded DNA-dependent protein kinase catalytic subunit (DNA-PKcs), which plays a key role in non-homologous end joining, are correlated with the radiosensitivity of cancer cells. Specifically, cells expressing high levels of DNA-PKcs exhibited radiation resistance, whereas cells expressing low levels were sensitive to radiation treatment. In this study, we observed full-length native DNA-PKcs (460 kDa) in radiation-resistant FRO and KTC-2 cells through western blot analysis using an antibody against the C-terminus of DNA-PKcs. In contrast, cleaved DNA-PKcs (175 kDa) were observed in radiation-sensitive TPC-1 and KTC-1 cells. Almost equal amounts of DNA-PKcs were observed in moderately radiation-sensitive WRO cells. We also describe a simple method for the prediction of radiation therapy efficacy in individual cases of thyroid cancers based on staining for DNA-PKcs in human cancer cell lines. Immunofluorescent staining showed that native DNA-PKcs was localized largely in the cytoplasm and only rarely localized in the nuclei of radiation-resistant thyroid cancer cells, whereas in radiation-sensitive cancer cells a 175-kDa cleaved C-terminal fragment of DNA-PKcs was localized mainly inside the nuclei. Therefore, DNA-PKcs moved to the nucleus after γ-ray irradiation. Our results suggest a new method for classifying human thyroid tumors based on their cellular distribution patterns of DNA-PKcs in combination with their radiosensitivity., Journal of radiation research, 61(4), pp.511-516; 2020

Inflammatory osteolysis is a common osteolytic specificity that occurs during infectious orthopaedic surgery and is characterized by an imbalance in bone homeostasis due to excessive osteoclast bone resorption activity. Epothilone B (Epo B) induced α-tubulin polymerization and enhanced microtubule stability, which also played an essential role in anti-inflammatory effect on the regulation of many diseases. However, its effects on skeletal system have rarely been investigated. Our study demonstrated that Epo B inhibited osteoclastogenesis in vitro and prevented inflammatory osteolysis in vivo. Further analysis showed that Epo B also markedly induced mature osteoclasts apoptosis during osteoclastogenesis. Mechanistically, Epo B directly suppressed osteoclastogenesis by the inhibitory regulation of the phosphorylation and activation of PI3K/Akt/STAT3 signaling directly, and the suppressive regulation of the CD9/gp130/STAT3 signaling pathway indirectly. The negative regulatory effect on STAT3 signaling further restrained the translocation of NF-κB p65 and NFATc1 from the cytosol to the nuclei during RANKL stimulation. Additionally, the expression of osteoclast specific genes was also significantly attenuated during osteoclast fusion and differentiation. Taken together, these findings illustrated that Epo B protected against LPS-induced bone destruction through inhibiting osteoclastogenesis via regulating the STAT3 dependent signaling pathway.

Altered expression of family with sequence similarity 84, member B (FAM84B) has been found in various human cancers. However, the expression and function of FAM84B in pancreatic ductal adenocarcinoma (PDAC) has not been studied. Here, by analyzing The Cancer Genome Atlas cohort, we found that FAM84B amplification was observed in 11% of 141 PDAC patients, and FAM84B amplification was correlated with higher mRNA expression of FAM84B. FAM84B amplification and overexpression was significantly correlated with poor overall survival. Moreover, knockdown of FAM84B in PDAC cell lines suppressed cell proliferation and induced apoptosis. FAM84B knockdown also suppressed mitochondrial function and glycolysis of PDAC cells. Interestingly, knockdown of FAM84B decreased the nuclear accumulation of β-catenin, and the expression of c-Myc and lactate dehydrogenase A, but enhanced the expression of Survivin. On the contrary, FAM84B overexpression displayed reversed effects in cell proliferation, apoptosis, mitochondrial function, and glycolysis, which was blocked by the Wnt/β-catenin pathway inhibitor (XAV939). In addition, PDAC cells with lower expression of FAM84B were more sensitive to gemcitabine-induced cell proliferation inhibition both in vitro and in vivo. In conclusion, FAM84B plays an important role in aerobic glycolysis and tumorigenesis in PDAC and Wnt/β-catenin may be involved in this process.

Macroautophagy/autophagy is an important catabolic process for maintaining cellular homeostasis by adapting to various stress conditions. Autophagy is mediated by a double-membrane autophagosome, which sequesters a portion of cytoplasmic components for delivery to the vacuole. Several autophagy-related (ATG) genes play crucial roles in autophagosome formation. The induction of ATG genes must be tightly regulated to maintain a proper autophagic activity, but their regulatory mechanisms are still largely unknown. Here, we report that the trehalose-6-phosphate phosphatase Tps2 functions as a positive regulator of autophagy in Saccharomyces cerevisiae. Cellular trehalose levels do not affect autophagy regulation by Tps2. Loss of Tps2 leads to impaired autophagic flux and reduced ATG8 expre/ssion under nitrogen starvation. In tps2Δ cells, Ume6 is predominantly dephosphorylated and represses ATG8 transcription by binding to its promoter region. Tps2 regulates nuclear translocation and activation of Rim15 kinase, a negative regulator of Ume6, by causing the dissociation of Rim15 from the 14-3-3 proteins Bmh1/2 under nitrogen starvation, suggesting that Rim15 mediates the function of Tps2 as a positive regulator of ATG8 induction. Furthermore, Tps2 plays a crucial role in the dephosphorylation of Ser1061 and Thr1075 residues of Rim15, which is important for controlling the dissociation of Rim15 from Bmh1/2 under nitrogen starvation. Together, our results reveal the role of Tps2 as a positive regulator of autophagy and provide new insight into the regulatory mechanisms of ATG gene expression. Abbreviations: ATG: autophagy-related; ChIP: chromatin immunoprecipitation; Co-IP: co-immunoprecipitation; DAPI: 4ʹ,6-diamidino-2-phenylindole; GFP: green fluorescent protein; PKA: protein kinase A; PtdIns3K: phosphatidylinositol 3-kinase; Rim15KI: kinase-inactive Rim15; Rim15-2A: Rim15S1061A,T1075A; TEM: transmission electron microscopy; TORC1: target of rapamycin complex 1

As an indispensable structure protein, the herpes simplex virus 1 (HSV-1) UL6 has been described to exert numerous roles in viral proliferation. However, its exact subcellular localization and subcellular transport mechanism is not well known. In the present study, by utilizing confocal fluorescent microscopy, UL6 was shown to mainly locate in the nucleus in enhanced yellow fluorescent protein or Flag tag fused expression plasmid-transfected cells or HSV-1-infected cells, whereas its predicted nuclear localization signal was nonfunctional. In addition, by exploiting dominant negative mutant and inhibitor of different nuclear import receptors, as well as co-immunoprecipitation and RNA interference assays, UL6 was established to interact with importin α1, importin α7 and transportin-1 to mediate its nuclear translocation under the help of Ran-mediated GTP hydrolysis. Accordingly, these results will advance the knowledge of UL6-mediated biological significances in HSV-1 infection cycle.

Background: Inhibitors of the CDK family of proteins have been approved for the treatment of a variety of tumours; however, the use of this type of drug alone is often ineffective and elicits adverse side effects. The development of new drugs administered in combination with CDK inhibitors is expected to improve the therapeutic effect. Methods: We identified the function of suppressor of cytokine signalling 1 (SOCS1) in hepatocellular carcinoma (HCC) cell models and the xenograft mouse model. The molecular mechanism of SOCS1 regulating cell proliferation was proved through transcriptome sequencing, western blot and Co-IP. Findings: When SOCS1 expression was artificially upregulated, HCC cell lines were arrested at the G1-S transition in the cell cycle. Interestingly, during this process, total CyclinD1 protein increased, but the effective proportion decreased. We found that the deficiency of CyclinD1 in the nucleus is probably due to the decrease in the stability of nuclear CyclinD1 caused by the ubiquitin-based degradation of P21 and P27, thus inhibiting the progression of the cell cycle to S phase. After P21 expression was increased, the levels of the component that inactivates CyclinD1 decreased as expected. It showed that P21 has a partial promoting effect on cancer. Interpretation: As a tumour inhibitory factor in HCC, SOCS1 is a good indicator of prognosis, tumour size and long-term survival after resection. SOCS1 is expected to become a drug target in combined with CDK family inhibitors. Funding Statement: Innovative Research Groups of National Natural Science Foundation of China (No. 81721091), Major program of National Natural Science Foundation of China (No.91542205), National S&T Major Project (No. 2017ZX10203205), Zhejiang International Science and Technology Cooperation Project (No.2016C04003), National Natural Science Foundation of China (No. 81773070). Declaration of Interests: The authors declare that they have no competing interests. Ethics Approval Statement: The study protocol was approved by the ethics committee of the First Affiliated Hospital of Zhejiang University. All experiments containing animals according to protocols approved by Animal Care and Use Committee of Zhejiang University.

Herpes simplex virus 1 (HSV-1) is a representative alphaherpesvirus that can provoke a series of severe diseases to human being, but its exact pathogenesis is not perfectly understood. UL2, a uracil-DNA glycosylase involved in the process of HSV-1 DNA replication, has been shown to be predominantly targeted to the nuclei in our previous study, yet little is established regarding the subcellular localization signal or its related function of UL2 during HSV-1 propagation. Here, by creating a number of UL2 variants merged with enhanced yellow fluorescent protein, an authentic nuclear localization signal (NLS) of UL2 was, for the first time, identified and profiled to amino acids (aa) 1 to 17 (MKRACSRSPSPRRRPSS), and 12RRR14 was indispensable for its nuclear accumulation. Besides, the predicted nuclear export signal (aa 225 to 240) of UL2 was determined to be nonfunctional. Based on the HSV-1 bacterial artificial chromosome and homologous recombination technique, three recombinant viruses with mutations of the identified NLS, deletion and revertant of UL2 were constructed to assess the effect of UL2 nuclear targeting on HSV-1 replication. Compared to the wild type HSV-1, UL2 deletion remarkably restrained viral production, and mutation of NLS targeting UL2 to cytoplasm (pan-cellular distribution) in recombinant virus-infected cells showed a certain degree of deficiency in HSV-1 proliferation. Moreover, recombinant virus with UL2 deletion exhibited serious damages of viral DNA synthesis and mRNA expression, and these processes were partially disrupted in the recombinant virus with UL2 NLS mutation. Collectively, we had established a functional NLS in UL2 and showed that the NLS-mediated nuclear translocation of UL2 was important for efficient production of HSV-1. These data were of significance for further clarifying the biological function of UL2 during HSV-1 infection.

Triple negative breast cancer (TNBC) is a heterogeneous disease, which lacks expression of the estrogen receptor (ER), progesterone receptor (PR) and the human epidermal growth factor 2 receptor (HER2). This subtype of breast cancer has the poorest prognosis with limited therapies currently available, and hence additional options are needed. CAPER is a coactivator of the activator protein-1 (AP-1) (interacting specifically with the c-Jun component) and the ER and is known to be involved in human breast cancer pathogenesis. Recent published data have demonstrated a role for CAPER in TNBC and, as such, disrupting the function of CAPER with c-Jun could be a novel approach to treat TNBC patients. The data presented here shows the development and in vitro testing of CAPER-derived peptides that inhibit the coactivator activity of CAPER with c-Jun. These CAPER peptides result in a decrease in cell number and an increase in apoptosis in two TNBC cell lines, MDA-MB-231 and BT-549, while having no effect on the non-tumorigenic cell line MCF 10A. Additionally, two modes of action were demonstrated which appear to be cell line dependent: 1) a modulation of phosphorylated c-Jun leading to a decrease in Bcl-2 in MDA-MB-231 cells and a decrease in p21 in BT-549 cells and 2) a decrease in DNA repair proteins, leading to impaired DNA repair function in MDA-MB-231 cells. The data presented here supports further development of CAPER-derived peptides for the treatment of TNBC.

Differentiating neutrophils undergo large-scale changes in nuclear morphology. How such alterations in structure are established and modulated upon exposure to microbial agents is largely unknown. Here, we found that prior to encounter with bacteria, an armamentarium of inflammatory genes was positioned in a transcriptionally passive environment suppressing premature transcriptional activation. Upon microbial exposure, however, human neutrophils rapidly (

Herpes simplex virus (HSV) type 1 (HSV-1) infection exhibited high heterogeneity at individual cells level, including the different gene expression patterns and varying amounts of progeny virus. However, the underlying mechanism of such variability remains obscure. The importance of host long noncoding RNAs (lncRNAs) in virus infection had been recognized, while the contribution of lncRNAs to the heterogeneous infection remains unknown. Herein, a prior single-cell RNA sequencing data using HSV-1 reporter strain expressing ICP4-YFP was re-analyzed to obtain the differentially expressed lncRNA between the successfully initiated viral gene expression (ICP4-YFP+) cells and the aborted infection cells (ICP4-YFP-). The ICP4-YFP+ population show a higher abundance of MAMDC2 antisense 1 (MAMDC2-AS1) lncRNA than ICP4-YFP- population. MAMDC2-AS1 silencing reduces the expression of HSV-1 immediate early (IE) genes and limit HSV-1 infection in human host cells. Consistently, ectopic expression of MAMDC2-AS1 enhances HSV-1 IE genes transcription and facilitates the formation of HSV-1-induced plaques. Mechanically, both RNA-pull down and RNA immunoprecipitation assays show that MAMDC2-AS1 interacts with the RNA binding protein heat shock protein 90α (Hsp90α), a molecular chaperone involving in the nuclear import of HSV-1. The MAMDC2-AS1-Hsp90α interaction facilitates the nuclear transport of viral tegument protein VP16, the core factor initiating the expression of HSV-1 IE genes. The transcription factor YY1 mediates the induction of MAMDC2-AS1 upon HSV-1 infection. Our study elucidates the contribution of lncRNA to HSV-1 infection susceptibility in human cells and the role of Hsp90α RNA binding activity in HSV-1 infection.

In budding yeast, the Rho-family GTPase Cdc42 has several functions that depend on its subcellular localization and the cell cycle stage. During bud formation, Cdc42 localizes to the plasma membrane at the bud tip and bud neck where it carries out functions in actin polymerization, spindle positioning, and exocytosis to ensure proper polarity development. Recent live-cell imaging analysis revealed a novel localization of Cdc42 to a discrete intracellular focus associated with the vacuole and nuclear envelope. The discovery of this novel Cdc42 localization led to the identification of a new function in ESCRT-mediated nuclear envelope sealing. However, other aspects of this intracellular localization and its functional implications were not explored. Here, we further characterize the Cdc42 focus and present several novel observations that suggest possible additional Cdc42 functions at the nucleus, including nucleus-vacuole junction formation, nuclear envelope tethering, nuclear migration, and nucleopodia formation.