Heterozygous variants in USP25 cause genetic generalized epilepsy.

USP25 encodes ubiquitin-specific proteases 25, a key member of deubiquitinating enzyme family and is involved in neural fate determination. Although abnormal expression in Down's syndrome was reported previously, the specific role of USP25 in human diseases has not been defined. In this study, we performed trio-based whole exome sequencing in a cohort of 319 cases (families) with generalized epilepsy of unknown etiology. Five heterozygous USP25 variants including two de novo and three co-segregated variants were determined in eight individuals affected by generalized seizures and/or febrile seizures from five unrelated families. The frequency of USP25 variants showed a significantly high aggregation in this cohort compared to the East Asian population and all populations in the gnomAD database. The mean onset ages of febrile and afebrile seizures were 10 months (infancy) and 11.8 years (juvenile), respectively. The patients achieved seizure freedom except one had occasional nocturnal seizures at the last follow-up. Two patients exhibited intellectual disability. Usp25 was ubiquitously expressed in mouse brain with two peaks on embryonic days (E14‒E16) and postnatal day 21, respectively. Similarly, USP25 expressed in fetus/early childhood stage with a second peak at approximately 12‒20 years old in human brain, consistent with the seizure onset age at infancy and juvenile in the patients. To investigate the functional impact of USP25 deficiency in vivo, we established Usp25 knock-out mice, which showed increased seizure susceptibility compared to wild-type mice in pentylenetetrazol-induced seizure test. To explore the impact of USP25 variants, we employed multiple functional detections. In HEK293T cells, the severe phenotype associated variant (p.Gln889Ter) led to a significant reduction of mRNA and protein expressions but formed a stable truncated dimers with increment of deubiquitinating enzyme activities and abnormal cellular aggregations, indicating a gain-of-function effect. The p.Gln889Ter and p.Leu1045del increased neuronal excitability in mice brain, with a higher firing ability in p.Gln889Ter. These functional impairments align with the severity of the observed phenotypes, suggesting a genotype-phenotype correlation. Hence, a moderate association between USP25 and epilepsy was noted, indicating USP25 is potentially a predisposing gene for epilepsy. Our results from Usp25 null mice and the patient-derived variants indicated that USP25 would play epileptogenic role via loss-of-function or gain-of-function effects. The truncated variant p.Gln889Ter would have profoundly different effect on epilepsy. Together, our results underscore the significance of USP25 heterozygous variants in epilepsy, thereby highlighting the critical role of USP25 in the brain.

NUS1 Variants Cause Lennox-Gastaut Syndrome Related to Unfolded Protein Reaction Activation.

NUS1 encodes the Nogo-B receptor, a critical regulator for unfolded protein reaction (UPR) signaling. Although several loss-of-function variants of NUS1 have been identified in patients with developmental and epileptic encephalopathy (DEE), the role of the NUS1 variant in Lennox-Gastaut syndrome (LGS), a severe child-onset DEE, remains unknown. In this study, we identified two de novo variants of NUS1, a missense variant (c.868 C > T/p.R290C) and a splice site variant (c.792-2 A > G), in two unrelated LGS patients using trio-based whole-exome sequencing performed in a cohort of 165 LGS patients. Both variants were absent in the gnomAD population and showed a significantly higher observed number of variants than expected genome-wide. The R290C variant was predicted to damage NUS1 and decrease its protein stability. The c.792-2 A > G variant caused premature termination of the protein. Knockdown of NUS1 activated the UPR pathway, resulting in apoptosis of HEK293T cells. Supplementing cells with expression of wild-type NUS1, but not the mutant (R290C), rescued UPR activation and apoptosis in NUS1 knockdown cells. Compared to wild-type Drosophila, seizure-like behaviors and excitability in projection neurons were significantly increased in Tango14 (homolog of human NUS1) knockdown and Tango14R290C/+ knock-in Drosophila. Additionally, abnormal development and a small body size were observed in both mutants. Activated UPR signaling was also detected in both mutants. Thus, NUS1 is a causative gene for LGS with dominant inheritance. The pathogenicity of these variants is related to the UPR signaling activation, which may be a common pathogenic mechanism of DEE.

MED12 variants associated with X-linked recessive partial epilepsy without intellectual disability.

OBJECTIVES: The MED12 gene encodes mediator complex subunit 12, which is a component of the mediator complex involved in the transcriptional regulation of nearly all RNA polymerase II-dependent genes. MED12 variants have previously been associated with developmental disorders with or without nonspecific intellectual disability. This study aims to explore the association between MED12 variants and epilepsy. MATERIALS AND METHODS: Trios-based whole-exome sequencing was performed in a cohort of 349 unrelated cases with partial (focal) epilepsy without acquired causes. The genotype-phenotype correlations of MED12 variants were analyzed. RESULTS: Five hemizygous missense MED12 variants, including c.958A>G/p.Ile320Val, c.1757G>A/p.Ser586Asn, c.2138C>T/p.Pro713Leu, c.3379T>C/p.Ser1127Pro, and c.4219A>C/p.Met1407Leu were identified in five unrelated males with partial epilepsy. All patients showed infrequent focal seizures and achieved seizure free without developmental abnormalities or intellectual disability. All the hemizygous variants were inherited from asymptomatic mothers (consistent with the X-linked recessive inheritance pattern) and were absent in the general population. The two variants with damaging hydrogen bonds were associated with early-onset seizures. Further genotype-phenotype analysis revealed that congenital anomaly disorder (Hardikar syndrome) was associated with (de novo) destructive variants in an X-linked dominant inheritance pattern, whereas epilepsy was associated with missense variants in an X-linked recessive inheritance pattern. Phenotypic features of intellectual disability appeared as the intermediate phenotype in terms of both genotype and inheritance. Epilepsy-related variants were located at the MED12-LCEWAV domain and the regions between MED12-LCEWAV and MED12-POL. CONCLUSION: MED12 is a potentially causative gene for X-linked recessive partial epilepsy without developmental or intellectual abnormalities. The genotype-phenotype correlation of MED12 variants explains the phenotypic variations and can help the genetic diagnosis.

Self-Assembly of [3]Catenane and [4]Catenane Based on Neutral Organometallic Scaffolds.

Transition metal-mediated templating and self-assembly have shown great potential to construct mechanically interlocked molecules. Herein, we describe the formation of the bimetallic [3]catenane and [4]catenane based on neutral organometallic scaffolds via the orthogonality of platinum-to-oxygen coordination-driven self-assembly and copper(I) template-directed strategy of a [2]pseudorotaxane. The structures of these bimetallic [3]catenane and [4]catenane were characterized by multinuclear NMR {1H and 31P} spectroscopy, electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS), and PM6 semiempirical molecular orbital theoretical calculations. In addition, single-crystal X-ray analyses of the [3]catenane revealed two asymmetric [2]pseudorotaxane units inside the metallacycle. It was discovered that tubular structures were formed through the stacking of individual [3]catenane molecules driven by the strong π-π interactions.

Molecular mechanism of azoles resistant Candida albicans in a patient with chronic mucocutaneous candidiasis.

BACKGROUND: More and more azole-resistant strains emerged through the development of acquired resistance and an epidemiological shift towards inherently less susceptible species. The mechanisms of azoles resistance of Candida albicans is very complicated. In this study, we aim to investigate the mechanism of azole-resistant C. albicans isolated from the oral cavity of a patient with chronic mucocutaneous candidiasis (CMC). CASE PRESENTATION: CMC diagnosis was given based on clinical manifestations, laboratory test findings and gene sequencing technique. Minimum inhibitory concentration (MIC) of the fungal isolate, obtained from oral cavity termed as CA-R, was obtained by in vitro anti-fungal drugs susceptibility test. To further investigate the resistant mechanisms, we verified the mutations of drug target genes (i.e. ERG11 and ERG3) by Sanger sequencing, and verified the over-expression of ERG11 and drug efflux genes (i.e. CDR1 and CDR2) by RT-PCR. A heterozygous mutation of c.1162A > G resulting in p.K388E was detected in STAT1 of the patient. The expression of CDR1 and CDR2 in CA-R was 4.28-fold and 5.25-fold higher than that of type strain SC5314, respectively. CONCLUSIONS: Up-regulation of CDR1 and CDR2 was mainly responsible for the resistance of CA-R. For CMC or other immunodeficiency patients, drug resistance monitoring is necessary.

Sensitivities of periodic acid-Schiff staining, Grocott's silver staining and calcofluor white staining in the diagnosis of human sporotrichosis.

OBJECTIVE: This study aimed to investigate the sensitivity of periodic acid-Schiff (PAS) staining, Grocott's silver staining (GSS) and calcofluor white (CFW) staining in the diagnosis of sporotrichosis. METHODS: Paraffin embedded tissues (n = 100) which were diagnosed with sporotrichosis by fungal culture were subjected to PAS, GSS, and CFW staining, and the detection rate of sporotrichosis was determined. RESULTS: The sensitivity of PAS, GSS, and CFW staining was 31%, 40% and 74%, respectively, in the diagnosis of sporotrichosis. CONCLUSION: CFW staining has a high sensitivity in the diagnosis of sporotrichosis, and sections are easily observed and can be repeatedly stained after CFW staining. For patients suspected to have sporotrichosis, CFW staining may be employed for early diagnosis before a fungal culture.

[Different subtypes of estrogen receptor α and related signal molecules in the hippocampus are associated with spatial cognitive impairment of diabetic mice].

To study the correlation between the spatial cognitive impairment and different subtypes of estrogen receptor α (ERα) of hippocampus in diabetic mice, we used alloxan (intraperitoneal injection) to induce type 1 diabetes in male Kunming mice and compared the spatial cognitive ability of the model mice with that of control mice through Morris water maze test. Meanwhile, using Western blot, we detected the protein expressions of ER-α36, ER-α66, caveolin-1, PKCα, cAMP-response element binding protein 2 (CREB2), and synaptophysin (Syn) in the hippocampus of the mice. The results showed that on the 3rd and 5th days of training, the ability of spatial learning and memory in the diabetic mice was significantly inferior to that of the control mice (P < 0.05). In the diabetic mice, the protein expressions of caveolin-1 and PKCα were decreased (P < 0.05), but ER-α66 expression was unaffected, while ER-α36 and CREB2 expressions were significantly increased (P < 0.05) compared with those of the control mice. The results suggest that abnormal expression of ER-α36 and related signal molecules may be important factors for diabetes-induced spatial cognitive impairment.