The PERK Branch of the Unfolded Protein Response Safeguards Protein Homeostasis and Mesendoderm Specification of Human Pluripotent Stem Cells.

Cardiac development involves large-scale rearrangements of the proteome. How the developing cardiac cells maintain the integrity of the proteome during the rapid lineage transition remains unclear. Here it is shown that proteotoxic stress visualized by the misfolded and/or aggregated proteins appears during early cardiac differentiation of human pluripotent stem cells and is resolved by activation of the PERK branch of unfolded protein response (UPR). PERK depletion increases misfolded and/or aggregated protein accumulation, leading to pluripotency exit defect and impaired mesendoderm specification of human pluripotent stem cells. Mechanistically, it is found that PERK safeguards mesendoderm specification through its conserved downstream effector ATF4, which subsequently activates a novel transcriptional target WARS1, to cope with the differentiation-induced proteotoxic stress. The results indicate that protein quality control represents a previously unrecognized core component of the cardiogenic regulatory network. Broadly, these findings provide a framework for understanding how UPR is integrated into the developmental program by activating the PERK-ATF4-WARS1 axis.

Transcriptomics and co-expression network analysis revealing candidate genes for the laccase activity of Trametes gibbosa.

BACKGROUND: Trametes gibbosa, which is a white-rot fungus of the Polyporaceae family found in the cold temperate zone, causes spongy white rot on wood. Laccase can oxidize benzene homologs and is one of the important oxidases for white rot fungi to degrade wood. However, the pathway of laccase synthesis in white rot fungi is unknown. RESULTS: The peak value of laccase activity reached 135.75 U/min/L on the 9th day. For laccase activity and RNA-seq data, gene expression was segmented into 24 modules. Turquoise and blue modules had greater associations with laccase activity (positively 0.94 and negatively -0.86, respectively). For biology function, these genes were concentrated on the cell cycle, citrate cycle, nicotinate, and nicotinamide metabolism, succinate dehydrogenase activity, flavin adenine dinucleotide binding, and oxidoreductase activity which are highly related to the laccase synthetic pathway. Among them, gene_8826 (MW199767), gene_7458 (MW199766), gene_61 (MW199765), gene_1741 (MH257605), and gene_11087 (MK805159) were identified as central genes. CONCLUSION: Laccase activity steadily increased in wood degradation. Laccase oxidation consumes oxygen to produce hydrogen ions and water during the degradation of wood. Some of the hydrogen ions produced can be combined by Flavin adenine dinucleotide (FAD) to form reduced Flavin dinucleotide (FADH2), which can be transmitted. Also, the fungus was starved of oxygen throughout fermentation, and the NADH and FADH2 are unable to transfer hydrogen under hypoxia, resulting in the inability of NAD and FAD to regenerate and inhibit the tricarboxylic acid cycle of cells. These key hub genes related to laccase activity play important roles in the molecular mechanisms of laccase synthesis for exploring industrial excellent strains.

Expression and clinical significance of lncRNA-SChLAP1 in breast cancer.

PURPOSE: The purpose of this study was to investigate the expression of long non-coding RNA (lncRNA)-SChLAP1 in breast cancer (BCa) tissues and its clinical significance in the progression of this cancer. METHODS: 100 pairs of surgically resected BCa tissue samples and normal breast tissues were collected from BCa patients, and SChLAP1 expression in above tissues was detected by quantitative real-time polymerase chain reaction (qRT-PCR). Meanwhile, the receiver operating curve (ROC) was plotted to analyze the efficacy of SChLAP1 in the diagnosis of BCa. In addition, the interplay between SChLAP1 expression and clinical features as well as the pathological features of BCa patients was analyzed. RESULTS: The expression level of SChLAP1 in BCa tissue samples was remarkably higher than that in normal ones. When the area under the ROC curve (AUC)=0.8639 and p<0.001, BCa could be diagnosed by SChLAP1; meanwhile, when the cut-off value was 3.26, the sensitivity was 80% and the specificity was 72%. Moreover, it was uncovered that the SChLAP1 expression was associated with patients' menarche, menopause, age of first pregnancy, or whether breastfeeding is administrated and whether oral contraceptives is taken; in addition, alcohol consumption, body mass index or tumor size and clinical stage were also the factors affecting the expression of SChLAP1. CONCLUSIONS: SChLAP1 is highly expressed in BCa tissues and can serve as a potential biomarker for the diagnosis of this cancer.

Targeted RNA N6 -Methyladenosine Demethylation Controls Cell Fate Transition in Human Pluripotent Stem Cells.

Deficiency of the N6 -methyladenosine (m6 A) methyltransferase complex results in global reduction of m6 A abundance and defective cell development in embryonic stem cells (ESCs). However, it's unclear whether regional m6 A methylation affects cell fate decisions due to the inability to modulate individual m6 A modification in ESCs with precise temporal control. Here, a targeted RNA m6 A erasure (TRME) system is developed to achieve site-specific demethylation of RNAs in human ESCs (hESCs). TRME, in which a stably transfected, doxycycline-inducible dCas13a is fused to the catalytic domain of ALKBH5, can precisely and reversibly demethylate the targeted m6 A site of mRNA and increase mRNA stability with limited off-target effects. It is further demonstrated that temporal m6 A erasure on a single site of SOX2 is sufficient to control the differentiation of hESCs. This study provides a versatile toolbox to reveal the function of individual m6 A modification in hESCs, enabling cell fate control studies at the epitranscriptional level.

Application of transoral endoscopic parathyroidectomy via vestibular approach, endoscopic parathyroidectomy via areola approach for parathyroid adenoma.

BACKGROUND: Transoral endoscopic parathyroidectomy via vestibular approach (TOEPVA) and total endoscopic parathyroidectomy via areola approach (EPA) are commonly used endoscopic parathyroidectomy approaches. This study compares effectiveness of these approaches with conventional open parathyroidectomy (COP) in relation to safety, associated trauma, and feasibility in the treatment of parathyroid adenoma (PTA). METHODS: We examined patients who had undergone TOEPVA (n = 15), EPA (n = 14), and COP (n = 30). All patients had a pathological diagnosis of PTA. We analyzed operative time, intraoperative blood loss, postoperative visual analog scale (VAS) score, postoperative drainage volume, hospital stay and complications such as changes in parathyroid hormone (PTH) and serum calcium before and after surgery. RESULTS: Clinical variables across the three experimental groups were similar except for patient age. TOEPVA and EPA groups had a higher proportion of young patients than COP group. Operation time for endoscopic group was longer than that of open group, and the longest operation time was recorded in TOEPVA group (P = 0.000). Postoperative VAS score: postoperative pain in patients in the endoscopic group was less than that of patients in the open group on the first day (P = 0.001). Postoperative pain in patients of the endoscopic group was significant on the second day (P = 0.044). Pain experienced by patients in the three groups was the same on the third day after surgery (P = 0.312). Postoperative drainage volume in the endoscopic group was more than that in the open group (P = 0.000). There were no significant differences between intraoperative blood loss (P = 0.089), complications (P = 0.407) and hospital stay (P = 0.389) in TOEPVA, EPA and COP groups. PTH and serum calcium levels in the three experimental groups were considerably lower after surgery (P < 0.05). Tumor recurrence was not recorded in the three groups during a follow-up period of between 3 and 36 months. CONCLUSIONS: TOEPVA and EPA are safe treatment options for PTA. The therapeutic effects of TOEPVA and EPA were similar to those of COP in the treatment of PTA.

miR­589­3p sponged by the lncRNA TINCR inhibits the proliferation, migration and invasion and promotes the apoptosis of breast cancer cells by suppressing the Akt pathway via IGF1R.

The long non­coding (lnc)RNA named tissue differentiation inducing non­protein coding RNA (TINCR) is a tumor marker that has not been studied in breast cancer. The present study aimed to investigate the TINCR­targeting micro (mi)RNAs and the regulatory mechanisms of TINCR in breast cancer. Following prediction by TargetScan and confirmation by dual­luciferase reporter assay, TINCR was demonstrated to be a target gene for miR­589­3p. The expression of TINCR and miR­589­3p in breast cancer and adjacent tissues was detected by reverse transcription­quantitative (RT­q)PCR, and the correlation between TINCR and miR­589­3p expression was determined by using Spearman correlation analysis. The 5­years survival was analyzed in patients with breast cancer according to TINCR expression (high or low). The effects of TINCR and miR­589­3p on the proliferation, apoptosis, migratory and invasive abilities of some breast cancer cell lines were detected by MTT assay, flow cytometry, wound healing assay and Transwell assay. The target gene of miR­589­3p was predicted and verified by TargetScan and dual­luciferase reporter assay, and the mechanism of miR­589­3p involvement in breast cancer cells was explored by overexpression or downregulation of miR­589­3p in breast cancer cells. RT­qPCR and western blotting were used to determine the expression of the insulin­like growth factor 1 receptor (IGF1R)/AKT pathway­related genes. The results demonstrated that TINCR expression level was negatively correlated with miR­589­3p expression level in breast cancer tissues and that patients with high expression of TINCR presented with lower survival rates. In addition, TINCR overexpression in cancer cells inhibited miR­589­3p expression, and cell transfection with miR­589­3p mimic partially reversed the effect of TINCR overexpression on the promotion of cancer cell proliferation, migration and invasion, and on the inhibition of cancer cell apoptosis. Furthermore, IGF1R, which is a target gene of miR­589­3p, increased cancer cell proliferation, migration and invasion and inhibited cancer cell apoptosis; however, these effects were partially reversed by miR­589­3p mimic. Furthermore, the results demonstrated that miR­589­3p mimic could downregulate the protein expression of IGF1R and p­AKT. In addition, TINCR overexpression downregulated miR­589­3p expression level. miR­589­3p partially reversed the effects of TINCR overexpression on cancer cell proliferation, migration and invasion, and inhibited cancer cell apoptosis by inhibiting the IGF1R­Akt pathway. The results from the present study demonstrated that TINCR may sponge miR­589­3p in order to inhibit IGF1R­Akt pathway activation in breast cancer cells, promoting therefore cancer cell proliferation, migration and invasion.

PCGF6 regulates stem cell pluripotency as a transcription activator via super-enhancer dependent chromatin interactions.

Polycomb group (PcG) ring finger protein 6 (PCGF6), though known as a member of the transcription-repressing complexes, PcG, also has activation function in regulating pluripotency gene expression. However, the mechanism underlying the activation function of PCGF6 is poorly understood. Here, we found that PCGF6 co-localizes to gene activation regions along with pluripotency factors such as OCT4. In addition, PCGF6 was recruited to a subset of the super-enhancer (SE) regions upstream of cell cycle-associated genes by OCT4, and increased their expression. By combining with promoter capture Hi-C data, we found that PCGF6 activates cell cycle genes by regulating SE-promoter interactions via 3D chromatin. Our findings highlight a novel mechanism of PcG protein in regulating pluripotency, and provide a research basis for the therapeutic application of pluripotent stem cells.

Long non-coding RNA ANRIL promotes carcinogenesis via sponging miR-199a in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is a complex breast cancer subtype characterized by the absence of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (Her2). Long non-coding RNA (lncRNA) antisense non-coding RNA in the INK4 locus (ANRIL) has been verified as oncogenic molecular in series of tumors, however, the role of ANRIL in TNBC carcinogenesis is still unclear. The purpose of present study is to investigate the expression and in-depth regulation of ANRIL on TNBC tumorigenesis. Expression level of ANRIL was up-regulated in TNBC tumor tissue and cell lines compared to noncancerous tissue and non-TNBC cells. Besides, the up-regulated ANRIL expression was closely correlated to poor prognosis. In vitro, loss-of-function experiments showed that ANRIL knockdown interfered by interference oligonucleotide could markedly suppress TNBC cells proliferation and enhance apoptosis. In vivo, ANRIL knockdown inhibited the tumor growth. Bioinformatics analysis and luciferase reporter assay revealed that miR-199a targeted ANRIL at 3'-UTR. Rescue experiments showed that miR-199a inhibitor could reverse the tumor-suppressing role of ANRIL knockdown on TNBC proliferation and apoptosis. Overall, present study demonstrated that ANRIL overexpression modulated TNBC tumorigenesis through acting as molecular 'sponge' for miR-199a, providing a novel insight and therapeutic target for TNBC.

Synthesis of Spiroligomer-Containing Macrocycles.

We demonstrate the synthesis and characterization of the solution conformations of a collection of functionalized spiroligomer-based macrocycles. These macrocycles contain 14 independently controllable stereocenters and four independently controllable functional groups on a highly preorganized scaffold. These molecules are being developed to display complex, preorganized surfaces for binding proteins and to create enzyme-like active sites. In this work, we demonstrate the convergent synthetic approach to this new class of macrocycles and demonstrate that the conformational properties of these molecules can be changed by altering the configuration stereocenters within the backbone.

Proteasome inhibitors with pyrazole scaffolds from structure-based virtual screening.

We performed a virtual screen of ∼340 000 small molecules against the active site of proteasomes followed by in vitro assays and subsequent optimization, yielding a proteasome inhibitor with pyrazole scaffold. The pyrazole-scaffold compound displayed excellent metabolic stability and was highly effective in suppressing solid tumor growth in vivo. Furthermore, the effectiveness of this compound was not negatively impacted by resistance to bortezomib or carfilzomib.

Spiroligozymes for transesterifications: design and relationship of structure to activity.

Transesterification catalysts based on stereochemically defined, modular, functionalized ladder-molecules (named spiroligozymes) were designed, using the "inside-out" design strategy, and mutated synthetically to improve catalysis. A series of stereochemically and regiochemically diverse bifunctional spiroligozymes were first synthesized to identify the best arrangement of a pyridine as a general base catalyst and an alcohol nucleophile to accelerate attack on vinyl trifluoroacetate as an electrophile. The best bifunctional spiroligozyme reacted with vinyl trifluoroacetate to form an acyl-spiroligozyme conjugate 2.7 × 10(3)-fold faster than the background reaction with a benzyl alcohol. Two trifunctional spiroligozymes were then synthesized that combined a urea with the pyridine and alcohol to act as an oxyanion hole and activate the bound acyl-spiroligozyme intermediate to enable acyl-transfer to methanol. The best trifunctional spiroligozyme carries out multiple turnovers and acts as a transesterification catalyst with k(1)/k(uncat) of 2.2 × 10(3) and k(2)/k(uncat) of 1.3 × 10(2). Quantum mechanical calculations identified the four transition states of the catalytic cycle and provided a detailed view of every stage of the transesterification reaction.

Hydrophobic substituent effects on proline catalysis of aldol reactions in water.

Derivatives of 4-hydroxyproline with a series of hydrophobic groups in well-defined orientations have been tested as catalysts for the aldol reactions. All of the modified proline catalysts carry out the intermolecular aldol reaction in water and provide high diastereoselectivity and enantioselectivity. Modified prolines with aromatic groups syn to the carboxylic acid are better catalysts than those with small hydrophobic groups (1a is 43.5 times faster than 1f). Quantum mechanical calculations provide transition structures, TS-1a(water) and TS-1f(water), that support the hypothesis that a stabilizing hydrophobic interaction occurs with 1a.