Crystal structure of the coxsackievirus A16 RNA-dependent RNA polymerase elongation complex reveals novel features in motif A dynamics

<正>Dear Editor,Coxsackievirus A16(CV A16)and enterovirus 71(EV71)are currently the two primary causative agents of handfoot-and-mouth disease(HFMD)(Solomon et al.,2010;Mao et al.,2014),threatening health of children worldwide.They both belong to the Enterovirus genus of the     

The <Emphasis Type="Italic">OPA1</Emphasis> Gene Mutations Are Frequent in Han Chinese Patients with Suspected Optic Neuropathy

While many patients with hereditary optic neuropathies are caused by mitochondrial DNA (mtDNA) mutations of Leber’s hereditary optic neuropathy (LHON), a significant proportion of them does not have mtDNA mutation and is caused by mutations in genes of the nuclear genome. In this study, we investigated whether the OPA1 gene, which is a pathogenic gene for autosomal dominant optic atrophy (ADOA), is frequently mutated in these patients. We sequenced all 29 exons of the OPA1 gene in 105 Han Chinese patients with suspected LHON. mtDNA copy number was quantified in blood samples from patients with and without OPA1 mutation and compared to healthy controls. In silico program-affiliated prediction, evolutionary conservation analysis, and in vitro cellular assays were performed to show the potential pathogenicity of the mutations. We identified nine OPA1 mutations in eight patients; six of them are located in exons and three are located in splicing sites. Mutation c.1172T?>?G has not been reported before. When we combined our data with 193 reported Han Chinese patients with optic neuropathy and compared to the available data of 4327 East Asians by the Exome Aggregation Consortium (ExAC), we found a significant enrichment of potentially pathogenic OPA1 mutations in Chinese patients. Cellular assays for OPA1 mutants c.869G?>?A and c.2708_2711del showed abnormalities in OPA1 isoforms, mitochondrial morphology, and cellular reactive oxygen species (ROS) level. Our results indicated that screening OPA1 mutation is needed for clinical diagnosis of patients with suspected optic neuropathy.     

Repurposing of Proton Pump Inhibitors as first identified small molecule inhibitors of endo-β-N-acetylglucosaminidase (ENGase) for the treatment of NGLY1 deficiency,a rare genetic disease

N-Glycanase deficiency, or NGLY1 deficiency, is an extremely rare human genetic disease. N-Glycanase, encoded by the gene NGLY1, is an important enzyme involved in protein deglycosylation of misfolded proteins. Deglycosylation of misfolded proteins precedes the endoplasmic reticulum (ER)-associated degradation (ERAD) process. NGLY1 patients produce little or no N-glycanase (Ngly1), and the symptoms include global developmental delay, frequent seizures, complex hyperkinetic movement disorder, difficulty in swallowing/aspiration, liver dysfunction, and a lack of tears. Unfortunately, there has not been any therapeutic option available for this rare disease so far. Recently, a proposed molecular mechanism for NGLY1 deficiency suggested that endo-β-N-acetylglucosaminidase (ENGase) inhibitors may be promising therapeutics for NGLY1 patients. Herein, we performed structure-based virtual screening utilizing FDA-approved drug database on this ENGase target to enable repurposing of existing drugs. Several Proton Pump Inhibitors (PPIs), a series of substituted 1H-benzo [d] imidazole, and 1H-imidazo [4,5-b] pyridines, among other scaffolds, have been identified as potent ENGase inhibitors. An electrophoretic mobility shift assay was employed to assess the inhibition of ENGase activity by these PPIs. Our efforts led to the discovery of Rabeprazole Sodium as the most promising hit with an IC₅₀ of 4.47 ± 0.44 μM. This is the first report that describes the discovery of small molecule ENGase inhibitors, which can potentially be used for the treatment of human NGLY1 deficiency.     

Synthesis and antibacterial activity of 5-methylphenanthridium derivatives as FtsZ inhibitors

5-Methylphenanthridium derivatives were designed, synthesized and evaluated for their in vitro antibacterial activity and cell division inhibitory activity against various Gram-positive and -negative bacteria. Among them, compounds 5A2, 5B1, 5B2, 5B3, 5C1 and 5C2 displayed the best on-target antibacterial activity with an MIC value of 4 µg/mL against B. subtilis ATCC9372 and S. pyogenes PS, showing over 2-fold better activity than sanguinarine. The SARs showed that the 5-methylphenanthridium derivatives with the alkyl side chains at the 2-postion, especially the straight alkyl side chains exerted better on-target antibacterial activity.     

Low frequency of BRAF and KRAS mutations in Chinese patients with low-grade serous carcinoma of the ovary

Background

Mounting evidence has shown that KRAS and BRAF are somatic mutations associated with low grade serous carcinoma (LGSC) of the ovary. However, the frequency of KRAS or BRAF mutation was variable in literatures, with a frequency of 16–54% for KRAS mutation and 2–33% for BRAF mutation. Meanwhile, the prognostic significance of KRAS or BRAF mutation remains controversial.

Methods

Codons 12 and 13 of exon 2 of KRAS gene and exon 15 of BRAF gene were analyzed using direct Sanger sequencing in 32 cases of LGSC of the ovary. The associations between KRAS or BRAF mutation and clinicopathological characteristics, overall survival (OS) and disease-free survival (DFS) were statistically analyzed.

Results

KRAS mutation was observed in nine cases (9/32, 28%) and BRAF mutation in two cases (2/32, 6%). KRAS and BRAF mutations were mutually exclusive. Neither KRAS nor BRAF mutation was statistically associated with OS or DFS in our cohort, although there was a favorable prognostic trend in patients with KRAS G12D mutation than those with KRAS G12 V mutation or wild-type KRAS for OS.

Conclusions

The present study indicated a low frequency of BRAF or KRAS mutation in Chinese patients with LGSC of the ovary, and neither KRAS nor BRAF mutation is a prognostic factor.

     

A novel protein derived from lamprey supraneural body tissue with efficient cytocidal actions against tumor cells

Background

In previous research, we found that cell secretion from the adult lamprey supraneural body tissues possesses cytocidal activity against tumor cells, but the protein with cytocidal activity was unidentified.

Methods

A novel lamprey immune protein (LIP) as defense molecule was first purified and identified in jawless vertebrates (cyclostomes) using hydroxyapatite column and Q Sepharose Fast Flow column. After LIP stimulation, morphological changes of tumor cells were analysed and measured whether in vivo or in vitro.

Results

LIP induces remarkable morphological changes in tumor cells, including cell blebbing, cytoskeletal alterations, mitochondrial fragmentation and endoplasmic reticulum vacuolation, and most of the cytoplasmic and organelle proteins are released following treatment with LIP. LIP evokes an elevation of intracellular calcium and inflammatory molecule levels. Our analysis of the cytotoxic mechanism suggests that LIP can upregulate the expression of caspase 1, RIPK1, RIP3 to trigger pyroptosis and necroptosis. To examine the effect of LIP in vivo, tumor xenograft experiments were performed, and the results indicated that LIP inhibits tumor growth without damage to mice. In addition, the cytotoxic action of LIP depended on the phosphatidylserine (PS) content of the cell membrane.

Conclusions

These observations suggest that LIP plays a crucial role in tumor cell survival and growth. The findings will also help to elucidate the mechanisms of host defense in lamprey.