Determinants of minor satellite RNA function in chromosome segregation in mouse embryonic stem cells.

The centromere is a fundamental higher-order structure in chromosomes ensuring their faithful segregation upon cell division. Centromeric transcripts have been described in several species and suggested to participate in centromere function. However, low sequence conservation of centromeric repeats appears inconsistent with a role in recruiting highly conserved centromeric proteins. Here, we hypothesized that centromeric transcripts may function through a secondary structure rather than sequence conservation. Using mouse embryonic stem cells (ESCs), we show that an imbalance in the levels of forward or reverse minor satellite (MinSat) transcripts leads to severe chromosome segregation defects. We further show that MinSat RNA adopts a stem-loop secondary structure, which is conserved in human α-satellite transcripts. We identify an RNA binding region in CENPC and demonstrate that MinSat transcripts function through the structured region of the RNA. Importantly, mutants that disrupt MinSat secondary structure do not cause segregation defects. We propose that the conserved role of centromeric transcripts relies on their secondary RNA structure.

Personalized Prediction of Postconcussive Working Memory Decline: A Feasibility Study.

Concussion, also known as mild traumatic brain injury (mTBI), commonly causes transient neurocognitive symptoms, but in some cases, it causes cognitive impairment, including working memory (WM) deficit, which can be long-lasting and impede a patient's return to work. The predictors of long-term cognitive outcomes following mTBI remain unclear, because abnormality is often absent in structural imaging findings. Previous studies have demonstrated that WM functional activity estimated from functional magnetic resonance imaging (fMRI) has a high sensitivity to postconcussion WM deficits and may be used to not only evaluate but guide treatment strategies, especially targeting brain areas involved in postconcussion cognitive decline. The purpose of the study was to determine whether machine learning-based models using fMRI biomarkers and demographic or neuropsychological measures at the baseline could effectively predict the 1-year cognitive outcomes of concussion. We conducted a prospective, observational study of patients with mTBI who were compared with demographically matched healthy controls enrolled between September 2015 and August 2020. Baseline assessments were collected within the first week of injury, and follow-ups were conducted at 6 weeks, 3 months, 6 months, and 1 year. Potential demographic, neuropsychological, and fMRI features were selected according to their significance of correlation with the estimated changes in WM ability. The support vector machine classifier was trained using these potential features and estimated changes in WM between the predefined time periods. Patients demonstrated significant cognitive recovery at the third month, followed by worsened performance after 6 months, which persisted until 1 year after a concussion. Approximately half of the patients experienced prolonged cognitive impairment at the 1-year follow up. Satisfactory predictions were achieved for patients whose WM function did not recover at 3 months (accuracy = 87.5%), 6 months (accuracy = 83.3%), and 1 year (accuracy = 83.3%) and performed worse at the 1-year follow-up compared to the baseline assessment (accuracy = 83.3%). This study demonstrated the feasibility of personalized prediction for long-term postconcussive WM outcomes based on baseline fMRI and demographic features, opening a new avenue for early rehabilitation intervention in selected individuals with possible poor long-term cognitive outcomes.

Inactivation of the tight junction gene CLDN11 by aberrant hypermethylation modulates tubulins polymerization and promotes cell migration in nasopharyngeal carcinoma.

BACKGROUND: Aberrant hypermethylation of cellular genes is a common phenomenon to inactivate genes and promote tumorigenesis in nasopharyngeal carcinoma (NPC). METHODS: Methyl binding domain (MBD)-ChIP sequencing of NPC cells, microarray data of NPC biopsies and gene ontology analysis were conducted to identify a potential tumor suppressor gene CLDN11 that was both hypermethylated and downregulated in NPC. Bisulfite sequencing, qRT-PCR, immunohistochemistry staining of the NPC clinical samples and addition of methylation inhibitor, 5'azacytidine, in NPC cells were performed to verify the correlation between DNA hypermethylation and expression of CLDN11. Promoter reporter and EMSA assays were used to dissect the DNA region responsible for transcription activator binding and to confirm whether DNA methylation could affect activator's binding, respectively. CLDN11 was transiently overexpressed in NPC cells followed by cell proliferation, migration, invasion assays to characterize its biological roles. Co-immunoprecipitation experiments and proteomic approach were carried out to identify novel interacting protein(s) and the binding domain of CLDN11. Anti-tumor activity of the CLDN11 was elucidated by in vitro functional assay. RESULTS: A tight junction gene, CLDN11, was identified as differentially hypermethylated gene in NPC. High methylation percentage of CLDN11 promoter in paired NPC clinical samples was correlated with low mRNA expression level. Immunohistochemistry staining of NPC paired samples tissue array demonstrated that CLDN11 protein expression was relatively low in NPC tumors. Transcription activator GATA1 bound to CLDN11 promoter region - 62 to - 53 and its DNA binding activity was inhibited by DNA methylation. Re-expression of CLDN11 reduced cell migration and invasion abilities in NPC cells. By co-immunoprecipitation and liquid chromatography-tandem mass spectrometry LC-MS/MS, tubulin alpha-1b (TUBA1B) and beta-3 (TUBB3), were identified as the novel CLDN11-interacting proteins. CLDN11 interacted with these two tubulins through its intracellular loop and C-terminus. Furthermore, these domains were required for CLDN11-mediated cell migration inhibition. Treatment with a tubulin polymerization inhibitor, nocodazole, blocked NPC cell migration. CONCLUSIONS: CLDN11 is a hypermethylated and downregulated gene in NPC. Through interacting with microtubules TUBA1B and TUBB3, CLDN11 blocks the polymerization of tubulins and cell migration activity. Thus, CLDN11 functions as a potential tumor suppressor gene and silencing of CLDN11 by DNA hypermethylation promotes NPC progression.

Measurement of gp130 cytokines oncostatin M and IL-6 in gingival crevicular fluid of patients with chronic periodontitis.

Several proinflammatory cytokines can induce periodontal tissue destruction and are thought to be useful indicators or diagnostic markers for periodontitis. Here, we aimed to investigate whether oncostatin M (OSM) was present in gingival crevicular fluid (GCF) and to clarify the correlation of GCF OSM and interleukin-6 (IL-6) levels with the severity of periodontitis. Sixty-two sites in 14 patients were divided into 4 groups based on probing depth (PD) and bleeding on probing (BOP). GCF was collected using paper strips from clinically health sites (PD < or = 3 mm, CAL: 1-3 mm, without BOP, n = 31), mildly diseased sites (PD < or = 3 mm, CAL: 3-5 mm, with BOP, n = 11), moderately diseased sites (PD = 4-6 mm, CAL: 5-8 mm, with BOP, n = 11), and severely diseased sites (PD > 6 mm, CAL: 8-12 mm, with BOP, n = 9). IL-6 and OSM in GCF were quantified by enzyme-linked immunosorbent assay and are expressed as concentrations (pg/ml) and total amounts (pg/site). Correlations of OSM and IL-6 levels with the severity of periodontitis in all groups were determined using Spearman rank correlation (r(s)). Our results showed that OSM and IL-6 were detected in most GCF samples. The total amounts of OSM and IL-6 were significantly positive correlated with severity of diseased sites (OSM: r(s) = 0.526, p < 0.01; IL-6: r(s) = 0.729, p < 0.01). No correlations of OSM or IL-6 concentration in GCF were found with disease severity. OSM and IL-6 levels in GCF were positively correlated to each other when expressed as either concentrations or total amounts (concentrations: r = 0.485, p < 0.01; total amounts r = 0.490, p < 0.01). In conclusion, our findings suggest that IL-6 and OSM may play a role in modulating the inflammatory cascade of chronic periodontitis.

Effect of Polygala tenuifolia root on behavioral disorders by lesioning nucleus basalis magnocellularis in rat.

UNLABELLED: We investigated whether an aqueous extract of Polygala tenuifolia Willd (PTW) could improve the rats' memory and behavioral disorders produced by lesioning nucleus basalis magnocellularis (NBM) in rats. The animals were divided into four groups for surgery, and following that they were orally administered PTW extract for 7 and 21 days. Each group consisted of eight male Sprague-Dawley rats and were treated as follows: CONTROL: no surgery (n = 8), PBS: 1M (mol/L) phosphate buffered saline (n = 8), IBO: 0.12 M (n = 8), QUIS: 0.12 M (n = 8). Two 0.5 microL injections were made in the vicinity of the bilateral side of the nucleus basalis magnocellularis (NBM). All rats were tested in the neurological tests and the step-through passive avoidance memory test during pre-surgery, surgery and post-surgery drug treatment. The results suggest that PTW extract has some repairing effects on the memory and behavioral disorders produced by lesioning of the NBM in rats.