Co-treatment of betulin and gefitinib is effective against EGFR wild-type/KRAS-mutant non-small cell lung cancer by inducing ferroptosis.

Clinical trials suggest that non-small-cell lung cancer (NSCLC) patients with KRAS mutations and wild-type EGFR have reduced benefits from gefitinib treatment. Ferroptosis is a new form of cell death that plays an important role in mediating the sensitivity of EGFR-TIKs. Here, we explored the antitumor ability of gefitinib in combination with betulin to overcome drug resistance through ferroptosis in wild-type EGFR/KRAS-mutant NSCLC cells. A549 and H460 cells were treated with gefitinib and betulin, and cell viability, apoptosis, and migration ability were assessed using the CCK-8 assay, flow cytometry, and wound-healing assay, respectively. Several cell death inhibitors were used to study the form of cell death. Ferroptosis-related events were detected by performing reactive oxygen species (ROS) and iron level detection, malondialdehyde (MDA) assay, and glutathione (GSH) assay. EMT-associated proteins and ferroptosis-related proteins were detected by using western blotting. A xenograft model was constructed in vivo to investigate the role of the combination treatment of betulin and gefitinib in NSCLC tumor growth. Gefitinib in combination with betulin exhibited antagonistic effects on cellular viability and induced cell apoptosis. It also induced ROS accumulation, lipid peroxidation, and GSH depletion and induced ferroptosis-related gene expression. Moreover, ferroptosis inhibitors, but not inhibitors of other forms of cell death, abrogated the effect of gefitinib in combination with betulin. Moreover, it also inhibited the tumor growth of NSCLC in vivo. Our findings suggest that gefitinib in combination with betulin is a novel therapeutic approach to overcome gefitinib resistance in EGFR wild-type/KRAS-mutant NSCLC cells by inducing ferroptosis.

Impaired meningeal lymphatic drainage in patients with idiopathic Parkinson's disease.

Animal studies implicate meningeal lymphatic dysfunction in the pathogenesis of neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease (PD). However, there is no direct evidence in humans to support this role1-5. In this study, we used dynamic contrast-enhanced magnetic resonance imaging to assess meningeal lymphatic flow in cognitively normal controls and patients with idiopathic PD (iPD) or atypical Parkinsonian (AP) disorders. We found that patients with iPD exhibited significantly reduced flow through the meningeal lymphatic vessels (mLVs) along the superior sagittal sinus and sigmoid sinus, as well as a notable delay in deep cervical lymph node perfusion, compared to patients with AP. There was no significant difference in the size (cross-sectional area) of mLVs in patients with iPD or AP versus controls. In mice injected with α-synuclein (α-syn) preformed fibrils, we showed that the emergence of α-syn pathology was followed by delayed meningeal lymphatic drainage, loss of tight junctions among meningeal lymphatic endothelial cells and increased inflammation of the meninges. Finally, blocking flow through the mLVs in mice treated with α-syn preformed fibrils increased α-syn pathology and exacerbated motor and memory deficits. These results suggest that meningeal lymphatic drainage dysfunction aggravates α-syn pathology and contributes to the progression of PD.

Autonomic ganglionic injection of α-synuclein fibrils as a model of pure autonomic failure α-synucleinopathy.

α-Synucleinopathies are characterized by autonomic dysfunction and motor impairments. In the pure autonomic failure (PAF), α-synuclein (α-Syn) pathology is confined within the autonomic nervous system with no motor features, but mouse models recapitulating PAF without motor dysfunction are lacking. Here, we show that in TgM83+/- mice, inoculation of α-Syn preformed fibrils (PFFs) into the stellate and celiac ganglia induces spreading of α-Syn pathology only through the autonomic pathway to both the central nervous system (CNS) and the autonomic innervation of peripheral organs bidirectionally. In parallel, the mice develop autonomic dysfunction, featured by orthostatic hypotension, constipation, hypohidrosis and hyposmia, without motor dysfunction. Thus, we have generated a mouse model of pure autonomic dysfunction caused by α-Syn pathology. This model may help define the mechanistic link between transmission of pathological α-Syn and the cardinal features of autonomic dysfunction in α-synucleinopathy.

Long-read sequencing identified intronic repeat expansions in SAMD12 from Chinese pedigrees affected with familial cortical myoclonic tremor with epilepsy.

BACKGROUND: The locus for familial cortical myoclonic tremor with epilepsy (FCMTE) has long been mapped to 8q24 in linkage studies, but the causative mutations remain unclear. Recently, expansions of intronic TTTCA and TTTTA repeat motifs within SAMD12 were found to be involved in the pathogenesis of FCMTE in Japanese pedigrees. We aim to identify the causative mutations of FCMTE in Chinese pedigrees. METHODS: We performed genetic linkage analysis by microsatellite markers in a five-generation Chinese pedigree with 55 members. We also used array-comparative genomic hybridisation (CGH) and next-generation sequencing (NGS) technologies (whole-exome sequencing, capture region deep sequencing and whole-genome sequencing) to identify the causative mutations in the disease locus. Recently, we used low-coverage (~10×) long-read genome sequencing (LRS) on the PacBio Sequel and Oxford Nanopore platforms to identify the causative mutations, and used repeat-primed PCR for validation of the repeat expansions. RESULTS: Linkage analysis mapped the disease locus to 8q23.3-24.23. Array-CGH and NGS failed to identify causative mutations in this locus. LRS identified the intronic TTTCA and TTTTA repeat expansions in SAMD12 as the causative mutations, thus corroborating the recently published results in Japanese pedigrees. CONCLUSIONS: We identified the pentanucleotide repeat expansion in SAMD12 as the causative mutation in Chinese FCMTE pedigrees. Our study also suggested that LRS is an effective tool for molecular diagnosis of genetic disorders, especially for neurological diseases that cannot be positively diagnosed by conventional clinical microarray and NGS technologies.

[Genetic analysis and prenatal diagnosis for non-syndromic hearing impairment].

OBJECTIVE: To detect genetic mutations underlying non-syndromic hearing impairment (NSHI) and establish a method for prenatal diagnosis. METHODS: Sixty six NSHI patients were included in this study. DNA was extracted from peripheral blood. Genetic mutations were detected by gene chip analysis and direct sequencing of GJB2 gene. For 7 pregnant women at high risk, prenatal genetic diagnosis was provided. RESULTS: Fourteen cases (21.21%) were found to have GJB2 mutations by both methods (homozygous 235delC mutation in 3 cases, homozygous 176del16 mutation in 2 cases, 235delC and 299delAT compound heterozygous mutation in 2 cases, 299delAT and 176del16 compound heterozygous mutation in 1 case, c.339T > G and 313del12bp compound heterozygous mutation 1 case, and 235delC heterozygous mutation in 5 cases). 13 (19.70%) had SLC26A4 mutations (IVS7-2 A >G homozygous mutation in 2 cases, IVS7-2 A > G homozygous mutation in 2 cases, IVS7-2 A > G and 2168A > G compound heterozygous mutation in 3 cases, 2168A>G heterozygous mutation in 3 cases, and IVS7-2 heterozygous mutation in 3 cases); and 3 had mtDNA12S rRNA mutation (1555A > G mutation in 2 cases, 1494C > T mutation in 1 case). Prenatal diagnosis suggested that 3 fetuses have carried a heterozygous mutation. Two fetuses were detected as normal and confirmed to have normal hearing after birth. Two fetuses were found to have carried compound mutations of GJB2. CONCLUSION: Gene chip combined with GJB2 gene analysis is an accurate and effective method for the diagnosis of NSHI. The results can facilitate accurate prenatal diagnosis.

[Efficiency of multiplex ligation-dependent probe amplification combined with short tandem repeat linkage analysis for the prenatal diagnosis for Duchenne muscular dystrophy].

OBJECTIVE: To investigate the efficiency of multiplex ligation-dependent probe amplification (MLPA) combined with short tandem repeat (STR) linkage analysis for the prenatal diagnosis for Duchenne muscular dystrophy (DMD). METHODS: Gender of the fetus was first determined by the presence of Y chromosome sex-determining gene (SRY). Subsequently, combined MLPA and STR linkage analysis were applied for the probands, pregnant women and fetuses in 45 affected families. RESULTS: Among the 45 families, 31 SRY-positive fetuses were identified, among whom six were diagnosed with DMD. For 14 SRY-negative fetuses, four were diagnosed as carriers. The remainders were normal. CONCLUSION: MLPA can detect mutations in the exons of dystrophin gene, whilst STR linkage analysis can determine whether the fetus has inherited the maternal X chromosome bearing the mutant gene. As the result, the method can detect affected fetuses in which no exonic mutations are detected with MLPA. By combining the two methods, the diagnostic rate for DMD can be greatly improved.

[Association of methionine synthase reductase gene polymorphism with unexplained recurrent spontaneous abortion].

OBJECTIVE: To explore the relationship between the polymorphism of methionine synthase reductase (MTRR) A66G and the susceptibility to unexplained repeated spontaneous abortion (URSA). METHODS: Total of 200 Henan Han couples with URSA (URSA group) and 76 Henan Han healthy couples without URSA (control group) were enrolled in this study. Their MTRR A66G genotypes were determined by PCR restriction fragment length polymorphism (PCR-RFLP). RESULTS: (1) The allele frequencies of MTRR A66G: the frequencies of allele A and allele G in URSA group were 76.5% (153/200) in husband and 72.8% (146/200) in wife, 23.5% (47/200) in husband and 27.2% (54/200) in wife, respectively. The frequencies of allele A and allele G in control group were 78.9% (60/76) in husband and 78.3% (59/76) in wife, 21.1% (16/76) in husband and 21.7% (16/76) in wife, respectively. The frequencies of allele A and allele G were not significantly different between female and male subjects within the same experimental group (P > 0.05), and also there were not significantly different between the same gender subjects at URAS and control groups (P > 0.05). (2) The genotype frequencies of MTRR A66G: the frequencies of genotype AA, AG and GG in URSA group were 57.0% (114/200) in husband and 52.0% (104/200) in wife, 39.0% (78/200) in husband and 41.5% (83/200) in wife, 4.0% (8/200) in husband and 6.5% (13/200) in wife, prepectively. The frequencies of genotype AA, AG and GG in control group were 59.2% (45/76) in husband and 59.2% (50/76) in wife, 39.5% (30/76) in husband and 38.2% (29/76) in wife; 1.3% (1/76) in husband and 2.6% (2/76) in wife, prepectively. The frequencies of genotype AA, AG and GG were not significantly different between female and male subjects within the same group (P > 0.05), and also there were not significantly different between the same gender subjects at URSA and control groups (P > 0.05).(3)Combined genotype of couples: the combined genotype frequencies of GG + GG, GG + AG, GG + AA, AG + AG, AG + AA and AA + AA in URSA group were 1.0% (2/200), 2.5% (5/200), 6.0% (12/200), 20.0% (40/200), 38.0% (76/200), and 32.5% (65/200), prepectively; the combined genotype frequencies in control group were 0, 1.3% (1/76), 2.6% (2/76), 17.1% (13/76), 42.1% (32/76), 36.8% (28/76), prepectively. The combined genotype analysis between the two groups were also not significantly different (P > 0.05). CONCLUSION: The polymorphism of MTRR A66G gene was not associated with the susceptibility to URSA (P > 0.05), and so it was not the inherited genetic risk factor of URSA.