PKM2 is a potential prognostic biomarker and related to immune infiltration in lung cancer.

Pyruvate kinase M2 (PKM2), a subtype of pyruvate kinase, plays a crucial role as a key enzyme in the final step of glycolysis. It is involved in regulating the tumor microenvironment and accelerating tumor progression. However, the relationship between PKM2 expression and the prognosis and immune infiltration remains unclear in lung cancer. In this study, we analyzed PKM2 expression in pan-cancer, and investigated its association with prognosis and immune cell infiltration of lung cancer by using multiple online databases, including Gent2, Tumor Immune Estimation Resource (TIMER), Gene Expression Profiling Interactive Analysis (GEPIA), PrognoScan, Kaplan-Meier plotter, and The Human Protein Atlas (HPA). The results showed that PKM2 expression is elevated in tumor tissues compared with the adjacent normal tissues of most cancers, including lung cancer. Prognostic analysis indicated that high expression of PKM2 was associated with poorer prognosis in overall lung cancer patients, especially in lung adenocarcinoma (LUAD). Notably, PKM2 exhibited a strong correlation with B cells and CD4+ T cells in LUAD; and with B cells, CD8+ T cells, CD4+ cells, and macrophages in lung squamous cell carcinoma (LUSC). Furthermore, PKM2 expression displayed a significant negative correlation with the expression of immune cell markers in both LUAD and LUSC. These findings suggested that PKM2 could serve as a promising prognostic biomarker for lung cancer and provided insights into its essential role in modulating the immune cell infiltration.

Functional characterization of a rare pathogenic variant c.875G > A, p.(Cys292Tyr) in COMP.

BACKGROUND: The protein encoded by the cartilage oligomeric matrix protein (COMP) gene is a noncollagenous extracellular matrix (ECM) protein that is important for chondrocyte formation and growth. Variations in the COMP gene cause pseudoachondroplasia (PSACH), which is mainly characterized by short-limbed dwarfing in the clinic. AIMS: To characterize the function of a rare pathogenic variant in the COMP gene (c.875G > A, p.Cys292Tyr). MATERIALS & METHODS: We performed 3D structural analysis, in vitro expression analysis, and immunofluorescence to characterize the effects of the variant on protein structure, expression, and cellular localization respectively. RESULTS: Variation modeling showed that the interactions between amino acids were changed after the variation, and there were 31 changes in the secondary structure of mutant COMP (MT-COMP). Western blot showed that the intracellular quantity of MT-COMP was higher than the wild-type COMP (WT-COMP). Cellular immunofluorescence results showed that WT-COMP was less abundant and homogenously distributed in cells, while the MT-COMP accumulated in the cytoplasm. DISCUSSION: Herein, we report a variant of COMP in a Chinese family with PSACH. We have shown that the rare missense variant, COMP c.875G > A, previously reported in ClinVar and identified in our patient, results in excessive accumulation of mutant protein in the cytoplasm, and is therefore pathogenic. CONCLUSION: Through in silico and experimental analyses, we provide evidence that COMP c.875G > A is the likely cause of PSACH in a Chinese family.

Total Synthesis of (+)-Shearilicine.

Herein we report the first total synthesis of the indole diterpenoid natural product shearilicine by an 11-step sequence via a generalizable precursor to the highly oxidized subclass of indole diterpenoids. A native chiral auxiliary strategy was employed to access the target molecule in an enantiospecific fashion. The formation of the key carbazole substructure was achieved through a mild intramolecular Heck cyclization, wherein a computational study revealed noncovalent substrate-ligand and ligand-ligand interactions that promoted migratory insertion.

A novel NPHS2 mutation (c.865A > G) identified in a Chinese family with steroid-resistant nephrotic syndrome alters subcellular localization of nephrin.

BACKGROUND: NPHS2 is the causative gene of nephrotic syndrome type 2 (MIM 600995) which often clinically manifests as steroid-resistant nephrotic syndrome (SRNS). The NPHS2 gene encodes a slit diaphragm (SD) associated protein podocin. OBJECTIVE: This study reported a novel disease-causing mutation of NPHS2 in a Chinese family with SRNS. We also investigated the pathogenic mechanism of the variants in this family. METHOD: A Chinese family with SRNS was recruited. Whole exome sequencing was performed to screen for disease-causing mutation. Sanger sequencing was used to confirm the results. In vitro functional experiments including immunoblotting, co-immunoprecipitation and double immunofluorescence staining were performed to explore the pathogenic mechanisms of mutations. RESULTS: In this family, compound heterozygous mutations of NPHS2 (c.467dupT and c.865A > G) were identified and segregated with the disease. The maternal c.865A > G was a novel variant, leading to amino acid substitution (p.K289E). In vitro functional assays indicated that c.467dupT (p.L156FfsX11) mutant lost interaction with nephrin. Both K289E and L156FfsX11 mutants showed sharply diminished plasma membrane localization. Furthermore, abnormal distribution of podocin mutants also altered the cell membrane localization of nephrin. CONCLUSION: We reported a family with SRNS caused by compound heterozygous mutations of NPHS2 (c.467dupT and c.865A > G). c.865A > G (p.K289E) in NPHS2 was a novel causative variant associated with SRNS. Both variants in this family not only affected the normal cell membrane localization of podocin, but also altered the cell membrane localization of nephrin which is the major architectural protein of SD.

Compound heterozygous KCNV2 variants contribute to cone dystrophy with supernormal rod responses in a Chinese family.

BACKGROUND: Cone dystrophy with supernormal rod response (CDSRR) is an autosomal recessive retinal disorder characterized by myopia, dyschromatopsia, nyctalopia, photophobia, and nystagmus. CDSRR is caused by mutations in KCNV2, the gene encoding for an electrically silent Kv subunit (Kvs) named Kv8.2. METHODS: A Chinese CDSRR family was recruited. Complete ophthalmology clinical examinations were performed to clarify the phenotype. Genetic examination was underwent using whole exome sequencing (WES). In addition, a candidate gene was validated by Sanger sequencing. Expression analysis in vitro including immunoblotting, quantitative real-time PCR (qRT-PCR), and co-immunoprecipitation experiments was performed to investigate the pathogenic mechanism of the identified gene variants. RESULTS: WES identified two KCNV2 heterozygous mutations from the proband. Sanger sequencing validated that the patient's parents had, respectively, carried those two mutations. Further in vitro functional experiments indicated that the mutated alleles had led the Kv8.2 proteins to fail in expressing and interacting with the Kv2.1 protein, respectively. CONCLUSIONS: This study expanded the KCNV2 mutation spectrum. It can also be deduced that CDSRR has a broad heterogeneity. It is further confirmed that the inability expression of Kv8.2 proteins and the failure of Kv8.2 proteins to interact with Kv2.1 may have accounted for the etiology of CDSRR based on previous studies and this study.

Identification of two rare mutations c.1318G>A and c.6438+2T>G in a Chinese DMD family as genetic markers.

BACKGROUND: Duchenne muscular dystrophy (DMD) is a fatal X-linked recessive disorder with no effective treatment, which underscores the importance of avoiding the birth of children with DMD by identifying pathogenic mutations and obtaining an accurate prenatal diagnosis. OBJECTIVE: The objective of this study was to analyze the genetic defect of a Chinese family where all male patients have died of DMD. METHODS: Multiplex ligation dependent probe analysis (MLPA) and next-generation sequencing (NGS) were employed to detect DMD mutations. The candidate mutations were then validated by Sanger sequencing. In vitro splicing assay was further conducted to examine the potential effect of the novel DMD splice site mutation on splicing. RESULTS: We found that two rare DMD mutations c.1318G>A and c.6438+2T>G passed from generation to generation among female carriers and they may be used as genetic markers in the Chinese DMD family. In vitro splicing assay further revealed that the novel classical splice site mutation c.6438+2T>G gave rise to a new donor splice site, which resulted in a frame shift of the transcripts and a premature termination at position 2159 in exon 45 (p.Y2144Nfs*16). CONCLUSION: We found that two co-inherited mutations passed from generation to generation in female carriers and they may be used as genetic markers in the Chinese DMD family. Our findings not only expanded the DMD mutation spectrum, but also provided an important basis for identifying of female carriers and avoiding the birth of affected male children in this DMD family.

PIWIL2 stabilizes ß-catenin to promote cell cycle and proliferation in tumor cells.

PIWIL2 belongs to the PIWI protein subfamily and is widely expressed in a variety of tumors. Previous studies have shown that PIWIL2 has the characteristics of oncogene. Recently we reported that PIWIL2 suppresses GSK3ß activity to regulate circadian rhythms through SRC-PI3K-AKT pathway. As GSK3ß is a key part of the ß-catenin destruction complex, it plays a vital role in regulating the degradation of ß-catenin. Besides, the activated ß-catenin/CyclinD1 pathway is involved in the proliferation of tumor cells. It is intriguing to investigate whether PIWIL2 regulates ß-catenin and downstream pathway. In this study, we found that PIWIL2 suppressed GSK3ß induced phosphorylation and ubiquitination of ß-catenin, and thus increased ß-catenin accumulation in the nucleus. By up-regulating ß-catenin and CyclinD1, PIWIL2 can promote cell cycle and proliferation in tumor cells. Taken together, our results revealed a novel function of PIWIL2 in regulating ß-catenin/CyclinD1 pathway in tumor cells, providing a new perspective for PIWIL2 as an oncogene.

PIWIL1 suppresses circadian rhythms through GSK3ß-induced phosphorylation and degradation of CLOCK and BMAL1 in cancer cells.

Circadian rhythms are maintained by series of circadian clock proteins, and post-translation modifications of clock proteins significantly contribute to regulating circadian clock. However, the underlying upstream mechanism of circadian genes that are responsible for circadian rhythms in cancer cells remains unknown. PIWIL1 participates in many physiological processes and current discoveries have shown that PIWIL1 is involved in tumorigenesis in various cancers. Here we report that PIWIL1 can suppress circadian rhythms in cancer cells. Mechanistically, by promoting SRC interacting with PI3K, PIWIL1 can activate PI3K-AKT signalling pathway to phosphorylate and inactivate GSK3ß, repressing GSK3ß-induced phosphorylation and ubiquitination of CLOCK and BMAL1. Simultaneously, together with CLOCK/BMAL1 complex, PIWIL1 can bind with E-BOX region to suppress transcriptional activities of clock-controlled genes promoters. Collectively, our findings first demonstrate that PIWIL1 negatively regulates circadian rhythms via two pathways, providing molecular connection between dysfunction of circadian rhythms and tumorigenesis.

Vinylogous acyl triflates as an entry point to α,ß-disubstituted cyclic enones via Suzuki-Miyaura cross-coupling.

An alternative protocol for the B-alkyl Suzuki-Miyaura reaction to produce cyclic α,ß-disubstituted enones is reported. The use of ß-triflyl enones as coupling partners in lieu of their halogenated analogs provides enhanced substrate stability to light and chromatography without adversely affecting reactivity. This protocol allows efficient access to the synthetically challenging α,ß-disubstituted enone motif under mild conditions.

A novel CRX variant (p.R98X) is identified in a Chinese family of Retinitis pigmentosa with atypical and mild manifestations.

BACKGROUND: Retinitis pigmentosa (RP) is the most common form of hereditary retinal degeneration that can cause inherited blindness. RP has extreme genetic and clinical heterogeneity, which brings a major obstacle to obtaining an accurate molecular diagnosis. OBJECTIVE: To analyze the genetic defect in a Chinese family of RP with a few atypical manifestations. METHODS: Whole-exome sequencing (WES) was applied to identify the disease-associated genes. Sanger sequencing was performed to validate the variants of candidate genes in the patient and his parents. In vitro expression analysis was further conducted to examine the potential biological function of the gene variant. RESULTS: A heterozygous nonsense variant c.292C > T (p.R98X) of CRX gene was identified to be present in the affected male. The c.292C > T variant of CRX was absent in all of the searched databases, including the 10,000 Chinese exome database. The nonsense variant was supposed to result in a truncated CRX protein with a destroyed homedomain (HD), which is essential for CRX translation. Interestingly, the following assay showed that the potential truncated protein was not detected, indicating that the variant may cause a loss-of-function mutation of CRX gene. CONCLUSION: We identified a novel heterozygous null mutation in the CRX gene which was the first evidence of a nonsense mutation in the HD domain of CRX. Our finding suggested that the haploinsufficiency mutation of CRX gene contributed to the atypical and mild manifestations of the autosomal dominant RP in the Chinese family.