Miracle-dz/UniFAR-FTUs huggingface dataset:
Regulation of clathrin‐mediated endocytosis by p53

The p53 gene encodes a multi‐functional protein to prevent tumorigenesis. Although there have been many reports of the nuclear functions of p53, little is known about the cytosolic functions of p53. Here, we found that p53 is present in cytosol as well as nuclei under unstressed conditions and binds to clathrin heavy chain (CHC). CHC is known to play a role in receptor‐mediated endocytosis. Based on our findings, we examined the effect of p53 on clathrin‐mediated endocytosis of epidermal growth factor receptor (EGFR). Surprisingly, p53 co‐localized with CHC at the plasma membrane in response to EGF stimulation. In cells with ablated p53 expression by RNAi, EGFR internalization was delayed and intracellular signaling from EGFR was altered. Thus, our findings provide evidence that cytosolic p53 may participate in the regulation of clathrin‐mediated endocytosis to control the correct signaling from EGFR.
Effects of Akt signaling on nuclear reprogramming

Reprogramming of the epigenetic state from differentiated to pluripotent cells can be attained by cell fusion of differentiated somatic cells with embryonic stem (ES) cells or transfer of the nucleus of a differentiated cell into an enucleated oocyte. Activation of Akt signaling is sufficient to maintain pluripotency of ES cells and promotes derivation of embryonic germ (EG) cells from primordial germ cells (PGCs). Here we analyzed the effects of Akt signaling on somatic cell nuclear reprogramming after cell fusion and nuclear transfer. We found that forced activation of Akt signaling stimulated reprogramming after cell fusion of ES cells with thymocytes or mouse embryonic fibroblasts. These hybrid cells showed ES cell characteristics, including in vitro and in vivo differentiation capacity. In contrast, Akt signaling significantly reduced the efficiency of reprogramming with nuclear transfer. Our results demonstrate that Akt signaling plays important roles on the nuclear reprogramming of somatic cells.
N-Myristoylated c-Abl Tyrosine Kinase Localizes to the Endoplasmic Reticulum upon Binding to an Allosteric Inhibitor*

Allosteric kinase inhibitors hold promise for revealing unique features of kinases that may not be apparent using conventional ATP-competitive inhibitors. Here we explore the activity of a previously reported allosteric inhibitor of BCR-Abl kinase, GNF-2, against two cellular isoforms of Abl tyrosine kinase: one that carries a myristate in the N terminus and the other that is deficient in N-myristoylation. Our results show that GNF-2 inhibits the kinase activity of non-myristoylated c-Abl more potently than that of myristoylated c-Abl by binding to the myristate-binding pocket in the C-lobe of the kinase domain. Unexpectedly, indirect immunofluorescence reveals a translocation of myristoylated c-Abl to the endoplasmic reticulum in GNF-2-treated cells, whereas GNF-2 has no detectable effect on the localization of non-myristoylated c-Abl. These results indicate that GNF-2 competes with the NH2-terminal myristate for binding to the c-Abl kinase myristate-binding pocket and that the exposed myristoyl group accounts for the localization to the endoplasmic reticulum. We also demonstrate that GNF-2 can inhibit enzymatic and cellular kinase activity of Arg, a kinase highly homologous to c-Abl, which is also likely to be regulated through intramolecular binding of an NH2-terminal myristate lipid. These results suggest that non-ATP-competitive inhibitors, such as GNF-2, can serve as chemical tools that can discriminate between c-Abl isoform-specific behaviors.
hnRNP K interacts with RNA binding motif protein 42 and functions in the maintenance of cellular ATP level during stress conditions

Heterogeneous nuclear ribonucleoprotein K (hnRNP K) is a conserved RNA‐binding protein that is involved in multiple processes of gene expression, including chromatin remodeling, transcription, RNA splicing, mRNA stability and translation, together with diverse groups of molecular partners. Here we identified a previously uncharacterized protein RNA binding motif protein 42 (RBM42) as hnRNP K‐binding protein. RBM42 directly bound to hnRNP K in vivo and in vitro. RBM42 also directly bound to the 3′ untranslated region of p21 mRNA, one of the target mRNAs for hnRNP K. RBM42 predominantly localized within the nucleus and co‐localized with hnRNP K there. When cells were treated with agents, puromycin, sorbitol or arsenite, which induced the formation of stress granules (SGs), cytoplasmic aggregates of stalled translational pre‐initiation complexes, both hnRNP K and RBM42 localized at SGs. Depletion of hnRNP K by RNA interference decreased cellular ATP level following release from stress conditions. Simultaneous depletion of RBM42 with hnRNP K enhanced the effect of the hnRNP K depletion. Our results indicate that hnRNP K and RBM42 are components of SGs and suggest that hnRNP K and RBM42 have a role in the maintenance of cellular ATP level in the stress conditions possibly through protecting their target mRNAs.
Direct Binding of a Plant LysM Receptor-like Kinase, LysM RLK1/CERK1, to Chitin in Vitro*

Plants induce immune responses against fungal pathogens by recognition of chitin, which is a component of the fungal cell wall. Recent studies have revealed that LysM receptor-like kinase 1/chitin elicitor receptor kinase 1 (LysM RLK1/CERK1) is a critical component for the immune responses to chitin in Arabidopsis thaliana. However, the molecular mechanism of the chitin recognition by LysM RLK1 still remains unknown. Here, we present the first evidence for direct binding of LysM RLK1 to chitin. We expressed LysM RLK1 fused with yeast-enhanced green fluorescent protein (LysM RLK1-yEGFP) in yeast cells. Binding studies using the solubilized LysM RLK1-yEGFP and several insoluble polysaccharides having similar structures showed that LysM RLK1-yEGFP specifically binds to chitin. Subsequently, the fluorescence microscopic observation of the solubilized LysM RLK1-yEGFP binding to chitin beads revealed that the binding was saturable and had a high affinity, with a Kd of ∼82 nm. This binding was competed by the addition of soluble glycol chitin or high concentration of chitin oligosaccharides having 4–8 residues of N-acetyl glucosamine. However, the competition of these chitin oligosaccharides is weaker than that of glycol chitin. These data suggest that LysM RLK1 has a higher affinity for chitin having a longer residue of N-acetyl glucosamine. We also found that LysM RLK1-yEGFP was autophosphorylated in vitro and that chitin does not affect the phosphorylation of LysM RLK1-yEGFP. Our results provide a new dimension to chitin elicitor perception in plants.
Phosphorylation of the Yeast Rpb1 C-terminal Domain at Serines 2, 5, and 7*

The C-terminal domain (CTD) of Rpb1, the largest subunit of RNA polymerase II, acts as a binding platform for various mRNA processing and histone-modifying enzymes that act co-transcriptionally. These factors are targeted to specific phosphorylation states of the CTD that predominate at different stages of transcription. Within the repeating sequence YSPTSPS, serines 2 and 5 are major phosphorylation sites, but serine 7 phosphorylation was recently discovered in mammalian cells. Here we show that CTD serine 7 is also phosphorylated in yeast and that Ser-7(P) chromatin immunoprecipitation patterns resemble those of Ser-5(P). The basal factor TFIIH can phosphorylate Ser-7 in vitro and is necessary for Ser-7(P) in vivo. Interestingly, deletion of the CTD Ser-5(P) phosphatase Rtr1 leads to an increase in Ser-5(P) but not Ser-7(P).
Fission yeast Ku protein is required for recovery from DNA replication stress

The fundamental function of the conserved Ku70–Ku80 heterodimer is to promote the non‐homologous end‐joining (NHEJ) pathway in double‐strand break repair. Although it is thought that Ku plays several roles other than NHEJ in maintaining chromosomal integrity including telomere protection, these precise functions remain unclear. In this study, we describe a novel role of fission yeast Ku proteins encoded by pku70+ and pku80+ genes in dealing with DNA replication stress. In the absence of Rqh1, the fission yeast RecQ helicase, the cells are sensitive to reagents inducing replication stress. pkuΔrqh1Δ double mutant showed synergistic sensitivities to these reagents. However, this synthetic phenotype was not observed when rqh1Δ mutant was coupled with the deletion of lig4+ that encodes a ligase essential for NHEJ, indicating that the role of Ku in replication stress is NHEJ independent. pkuΔrqh1Δ double mutant also showed highly variable copy numbers of rDNA repeats even under unstressed condition. Furthermore, the double mutant exhibited inefficient replication resumption after transient replication stalling. These results suggest the possibility that Ku proteins play an important role in genome integrity recovering replication stress.
RNA‐binding proteins Rbm38 and Rbm24 regulate myogenic differentiation via p21‐dependent and ‐independent regulatory pathways

Skeletal muscle differentiation entails organized sequential events, including cell cycle arrest of proliferating myoblast cells and cell fusion, which lead to the formation of multinucleated myotubes. This process involves both transcriptional and post‐transcriptional regulation of the gene expression of myogenic proteins, as well as cell‐cycle related proteins. RNA‐binding proteins bind to specific sequences of target RNA and regulate gene expression in a post‐transcriptional manner. However, few tissue‐specific RNA binding proteins have been identified. Herein, we report that the RNA binding proteins Rbm24 and Rbm38 were found to be preferentially expressed in muscle during differentiation in vitro. Further, knockdown of either by RNA interference suppressed cell‐cycle arrest and delayed myogenic differentiation in C2C12 cells. In contrast, over‐expression of Rbm24 or Rbm38 induced cell cycle arrest, and then had a positive effect on myogenic differentiation. Immunoprecipitation‐RT‐PCR analysis using tagged Rbm proteins indicated that Rbm38 binds to the p21 transcript in vivo. Consistent with this, differentiation of Rbm38 knockdown cells was rescued by over‐expression of p21. Together, our results suggest that Rbm38 plays a crucial role in cell cycle arrest and myogenic differentiation via its binding to p21.
The Most Prevalent Tooth Shade in a Particular Population: A Survey

Statement of the Problem : Shade matching of a restoration forms a critical final step and is dependent on the knowledge, experience and skill of the dentist. Shade analysis and selection requires accurate and objective evaluation of hue, chroma and value, the three parameters of Albert Munsell’s color system. Objective : 1. To assess the most prevalent tooth shade for anterior and posterior teeth in maxillary and mandibular arches. 2. To evaluate influence of age/gender on shade. 3. To efficiently treat edentulous patients by constructing complete dentures with the most prevalent tooth shade. 4. To purchase dental materials for crown and bridge prosthesis. Materials and Methods : Sample size taken was 400. Four age groups were selected between 15 to 65 years with 100 samples per age group. Subjective method for shade selection was used. Shade selection was done in natural daylight by two operators using the VITA and Ivoclar shade guides. Shade guides were used to examine maxillary and mandibular right and left central incisors and maxillary and mandibular second premolars which were sound, free of any restoration, extrinsic stains and caries free for inclusion in the study. Subjects with tooth abnormalities, intrinsic tooth stains, hypoplasia, fluorosis were excluded from the study. Results : The most prevalent shade in the population of Belgaum is in range of A2 and A3. The most common anterior maxillary shade is A2 cervically and A1 incisally. The mandibular anterior shade is also A2 cervivcally and A1 incisally with more incisal translucency. The posterior shade of prevalence is A3 for maxillary and mandibular teeth. Young people tend to have whiter teeth and when people become older their teeth gradually become yellow in color. Females tend to have whiter teeth compared to males. The shade becomes darker in posteriors; the hue remains same, value decreases.
Transcriptional repressor TIEG1 regulates Bmal1 gene through GC box and controls circadian clockwork

The circadian clock controls daily rhythms in many physiologic processes, and the clock oscillation is regulated by external time cues such as light, temperature, and feeding. In mammals, the transcriptional regulation of clock genes underlies the clock oscillatory mechanism, which is operative even in cultured fibroblasts. We previously demonstrated that glucose treatment of rat‐1 fibroblasts evokes circadian expression of clock genes with a rapid induction of Tieg1 transcript encoding a transcriptional repressor. Here, we found diurnal variation of both Tieg1 mRNA and nuclear TIEG1 protein levels in the mouse liver with their peaks at day/night transition and midnight, respectively. In vitro experiments showed that TIEG1 bound to Bmal1 gene promoter and repressed its transcriptional activity through two juxtaposed GC boxes near the transcription initiation site. The GC box/TIEG1‐mediated repression of Bmal1 promoter was additive to RORE‐dependent repression by REV‐ERBα, a well‐known repressor of Bmal1 gene. In cell‐based real‐time assay, siRNA‐mediated knock‐down of TIEG1 caused period shortening of cellular bioluminescence rhythms driven by Bmal1‐luciferase and Per2‐luciferase reporters. These findings highlight an active role of TIEG1 in the normal clock oscillation and GC box‐mediated regulation of Bmal1 transcription.
Targeted mutagenesis in the sea urchin embryo using zinc‐finger nucleases

We showed that engineered zinc‐finger nucleases (ZFNs), which consist of a zinc‐finger DNA‐binding array and a nuclease domain of the restriction enzyme FokI, can introduce mutations at a specific genomic site in the sea urchin embryo. Using bacterial one‐hybrid screening with zinc‐finger randomized libraries and a single‐strand annealing assay in cultured cells, ZFNs targeting the sea urchin Hemicentrotus pulcherrimus homologue of HesC (HpHesC) were efficiently selected. Consistent with the phenotype observed in embryos injected with an antisense morpholino oligonucleotide against HpHesC, an increase in the primary mesenchyme cell population was observed in embryos injected with a pair of HpHesC ZFN mRNAs. In addition, sequence analysis of the mutations showed that deletions and insertions occurred at the HpHesC target site in the embryos injected with the HpHesC ZFN mRNAs. These results suggest that targeted gene disruption using ZFNs is feasible for the sea urchin embryo.
Hepatic Autophagy Is Suppressed in the Presence of Insulin Resistance and Hyperinsulinemia

Autophagy is essential for maintaining both survival and health of cells. Autophagy is normally suppressed by amino acids and insulin. It is unclear what happens to the autophagy activity in the presence of insulin resistance and hyperinsulinemia. In this study, we examined the autophagy activity in the presence of insulin resistance and hyperinsulinemia and the associated mechanism. Insulin resistance and hyperinsulinemia were induced in mice by a high fat diet, followed by measurements of autophagy markers. Our results show that autophagy was suppressed in the livers of mice with insulin resistance and hyperinsulinemia. Transcript levels of some key autophagy genes were also suppressed in the presence of insulin resistance and hyperinsulinemia. Conversely, autophagy activity was increased in the livers of mice with streptozotocin-induced insulin deficiency. Levels of vps34, atg12, and gabarapl1 transcripts were elevated in the livers of mice with insulin deficiency. To study the mechanism, autophagy was induced by nutrient deprivation or glucagon in cultured hepatocytes in the presence or absence of insulin. Autophagy activity and transcript levels of vps34, atg12, and gabarapl1 genes were reduced by insulin. The effect of insulin was largely prevented by overexpression of the constitutive nuclear form of FoxO1. Importantly, autophagy of mitochondria (mitophagy) in cultured cells was suppressed by insulin in the presence of insulin resistance. Together, our results show that autophagy activity and expression of some key autophagy genes were suppressed in the presence of insulin resistance and hyperinsulinemia. Insulin suppression of autophagy involves FoxO1-mediated transcription of key autophagy genes.
Ultradian rhythm in the intestine of Caenorhabditis elegans is controlled by the C‐terminal region of the FLR‐1 ion channel and the hydrophobic domain of the FLR‐4 protein kinase

Defecation behavior in Caenorhabditis elegans is driven by an endogenous ultradian clock in the intestine. Its periods are positively regulated by FLR‐1, an ion channel of the epithelial sodium channel/degenerin superfamily, and FLR‐4, a protein kinase with a hydrophobic domain at the carboxyl terminus. FLR‐1 has many putative phosphorylation sites in the C‐terminal intracellular region. This structure implies that the periods may be regulated by the phosphorylation of FLR‐1 by FLR‐4, but it remains to be clarified. Here, we show that a truncated FLR‐1 lacking the C‐terminal intracellular region resulted in longer periods, suggesting that this region is involved in the negative regulation of defecation cycle periods. Contrary to our expectation, FLR‐4 was still necessary for the function of the truncated FLR‐1. Furthermore, FLR‐4 containing a kinase‐dead mutation or lacking the whole kinase domain was sufficient for normal defecation cycle periods. FLR‐4 was necessary for the stable expression of FLR‐1::GFP, and its hydrophobic domain was sufficient also for this function. FLR‐1 and FLR‐4 are often colocalized in the plasma membrane. These data showed an unexpected role of FLR‐4: its hydrophobic domain stabilizes the FLR‐1 ion channel, a key regulator of defecation cycle periods in the intestine.
How teachers' perceptions of professional preparedness relate to teacher retention : comparing traditionally and alternatively prepared teachers

Selecting and training potentially effective teachers and retaining those teachers in their work present immense challenges to the teaching profession. This paper examines the relationship between new teachers‘ perceptions of career preparedness and their career intentions, accounting for differences in teachers‘ gender, age, school characteristics, student characteristics, and teacher preparation pathway. This study differs from previous work by employing a nationally representative sample and analyzing three alternative preparation routes—the Teach for America (TFA) program, the New Teacher Project‘s (NTP) various teacher programs, and the Troops-toTeachers (TTT) program. Findings suggest that teachers who report feeling better prepared are more likely to report plans to stay in teaching beyond five years, and those trained by traditional certification programs are more likely to stay in teaching than those certified through alternative programs. Knowing that teachers who feel more prepared are more likely to plan on making teaching a career should guide policy-makers in designing teacher-training programs; fewer vacancies may translate to more competition for open positions, which allow hiring decisions to focus on the most potentially successful applicants. Policy implications include greater scrutiny of teacher education programs, particularly attending to the characteristics of those programs that produce teachers who feel well prepared.
Hematological characteristics and effective screening for compound heterozygosity for Hb constant spring and deletional α+‐thalassemia

Hemoglobin Constant Spring (HbCS) is an unstable α-globin variant causing α-thalassemia phenotypes. The prevalence of compound heterozygosity for HbCS and deletional α + -thalassemia (HbCS/α + -thal) and its characteristics compared with HbCS heterozygosity have not been reported. We performed molecular analysis to detect α + -thal alleles in 75 HbCS heterozygotes and correlated with hematological characteristics. There were 34 HbCS heterozygotes (45.3%) carrying α + -thal. Hematological parameters of HbCS/α + -thal demonstrated lower Hb, MCV, MCH, and MCHC than HbCS heterozygotes, but higher HbCS levels. MCH was the most helpful variable in differentiating these two groups with ROC area under curve (AUC) of 0.927. MCH > 27 pg was able to exclude the presence of α + -thal, while MCH 27 pg was able to exclude the coinheritance with α + -thal, while MCH < 25.5 pg was an effective cut-off for predicting α + -thal with sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of 95.2%, 70.8%, 58.8%, and 97.1%, respectively. Therefore, this could be a cost-effective screening criterion to identify deletional α + -thal in HbCS heterozygotes avoiding expensive molecular testing.
Human ISCA1 Interacts with IOP1/NARFL and Functions in Both Cytosolic and Mitochondrial Iron-Sulfur Protein Biogenesis*

Iron-sulfur proteins play an essential role in many biologic processes. Hence, understanding their assembly is an important goal. In Escherichia coli, the protein IscA is a product of the isc (iron-sulfur cluster) operon and functions in the iron-sulfur cluster assembly pathway in this organism. IscA is conserved in evolution, but its function in mammalian cells is not known. Here, we provide evidence for a role for a human homologue of IscA, named IscA1, in iron-sulfur protein biogenesis. We observe that small interfering RNA knockdown of IscA1 in HeLa cells leads to decreased activity of two mitochondrial iron-sulfur enzymes, succinate dehydrogenase and mitochondrial aconitase, as well as a cytosolic iron-sulfur enzyme, cytosolic aconitase. IscA1 is observed both in cytosolic and mitochondrial fractions. We find that IscA1 interacts with IOP1 (iron-only hydrogenase-like protein 1)/NARFL (nuclear prelamin A recognition factor-like), a cytosolic protein that plays a role in the cytosolic iron-sulfur protein assembly pathway. We therefore propose that human IscA1 plays an important role in both mitochondrial and cytosolic iron-sulfur cluster biogenesis, and a notable component of the latter is the interaction between IscA1 and IOP1.
Mental health service user involvement in research: where have we come from, where are we going?

Purpose – The aim of the research is to assess the extent and value of mental health service user (MHSU) involvement in research in England. Design/methodology/approach – This is a knowledge review, including academic and “grey” literature, and documented testimonial evidence. Findings – The involvement of MHSUs in mental health research has become mainstream. There is clear evidence that involving MHSUs in research adds value. Four gaps in the literature were identified. First, a lack of evidence from non-service-user researchers about their experience of working with MHSUs. Second, a lack of recognition that anyone involved in research may hold more than one role. Third, failure to treat carers as separate from MHSUs, or – often – to include them at all. Fourth, a lack of understanding that MHSUs may have a useful role to play in research on topics other than mental health. Research limitations/implications – The literature would benefit from some evidence about non-service-user researchers’ experiences.