RANBP17 Overexpression Restores Nucleocytoplasmic Transport and Ameliorates Neurodevelopment in Induced DYT1 Dystonia Motor Neurons.

DYT1 dystonia is a debilitating neurological movement disorder, and it represents the most frequent and severe form of hereditary primary dystonia. There is currently no cure for this disease due to its unclear pathogenesis. In our previous study utilizing patient-specific motor neurons (MNs), we identified distinct cellular deficits associated with the disease, including a deformed nucleus, disrupted neurodevelopment, and compromised nucleocytoplasmic transport (NCT) functions. However, the precise molecular mechanisms underlying these cellular impairments have remained elusive. In this study, we revealed the genome-wide changes in gene expression in DYT1 MNs through transcriptomic analysis. We found that those dysregulated genes are intricately involved in neurodevelopment and various biological processes. Interestingly, we identified that the expression level of RANBP17, a RAN-binding protein crucial for NCT regulation, exhibited a significant reduction in DYT1 MNs. By manipulating RANBP17 expression, we further demonstrated that RANBP17 plays an important role in facilitating the nuclear transport of both protein and transcript cargos in induced human neurons. Excitingly, the overexpression of RANBP17 emerged as a substantial mitigating factor, effectively restoring impaired NCT activity and rescuing neurodevelopmental deficits observed in DYT1 MNs. These findings shed light on the intricate molecular underpinnings of impaired NCT in DYT1 neurons and provide novel insights into the pathophysiology of DYT1 dystonia, potentially leading to the development of innovative treatment strategies.

Pretreatment level of serum sialic acid predicts both qualitative and quantitative bone metastases of prostate cancer.

Background: Recently, serum sialic acid (SA) has emerged as a distinct prognostic marker for prostate cancer (PCa) and bone metastases, warranting differential treatment and prognosis for low-volume (LVD) and high-volume disease (HVD). In clinical settings, evaluating bone metastases can prove advantageous. Objectives: We aimed to establish the correlation between SA and both bone metastasis and HVD in newly diagnosed PCa patients. Methods: We conducted a retrospective analysis of 1202 patients who received a new diagnosis of PCa between November 2014 and February 2021. We compared pretreatment SA levels across multiple groups and investigated the associations between SA levels and the clinical parameters of patients. Additionally, we compared the differences between HVD and LVD. We utilized several statistical methods, including the non-parametric Mann-Whitney U test, Spearman correlation, receiver operating characteristic (ROC) curve analysis, and logistic regression. Results: The results indicate that SA may serve as a predictor of bone metastasis in patients with HVD. ROC curve analysis revealed a cut-off value of 56.15 mg/dL with an area under the curve of 0.767 (95% CI: 0.703-0.832, P < 0.001) for bone metastasis versus without bone metastasis and a cut-off value of 65.80 mg/dL with an area under the curve of 0.766 (95% CI: 0.644-0.888, P = 0.003) for HVD versus LVD. Notably, PCa patients with bone metastases exhibited significantly higher SA levels than those without bone metastases, and HVD patients had higher SA levels than LVD patients. In comparison to the non-metastatic and LVD cohorts, the cohort with HVD exhibited higher levels of alkaline phosphatase (AKP) (median, 122.00 U/L), fibrinogen (FIB) (median, 3.63 g/L), and prostate-specific antigen (PSA) (median, 215.70 ng/mL), as well as higher Gleason scores (> 7). Multivariate logistic regression analysis demonstrated that an SA level of > 56.15 mg/dL was independently associated with the presence of bone metastases in PCa patients (OR = 2.966, P = 0.018), while an SA level of > 65.80 mg/dL was independently associated with HVD (OR = 1.194, P = 0.048). Conclusion: The pretreatment serum SA level is positively correlated with the presence of bone metastases.

Comparison of Fine-Needle Biopsy (FNB) versus Fine-Needle Aspiration (FNA) Combined with Flow Cytometry in the Diagnosis of Deep-Seated Lymphoma.

Evidence comparing ultrasound endoscopy-guided fine-needle biopsy (EUS-FNB) with EUS-guided fine-needle aspiration (EUS-FNA) in deep-seated lymphoma tissue sampling is insufficient. This study aims to evaluate the diagnostic efficacy of immunohistochemistry (IHC) or flow cytometry (FCM) on specimens obtained from EUS-FNB and EUS-FNA in the diagnosis and staging of deep-seated lymphomas. This real-world, dual-center study prospectively evaluated all eligible specimens from patients who underwent EUS-FNB/FNA over an 8-year period. 53 patients were enrolled, with 23 patients in the EUS-FNB group and 30 patients in the EUS-FNA group. FNB yielded specimens with longer core tissues (0.80 mm [0.55, 1.00] vs. 0.45 mm [0.30, 0.50], p = 0.009) and higher scores of specimen adequacy [4 (3.75, 4.00) vs. 3 (1.00, 4.00), p = 0.025]. Overall analysis revealed that the diagnostic accuracy of IHC based on specimens acquired from EUS-FNB was significantly higher than that of EUS-FNA (91.30% vs. 60.00%, p = 0.013). After controlling confounding factors including lesion size and endoscopists, EUS-FNB with IHC maintained a higher-level diagnostic accuracy compared to EUS-FNA (OR = 1.292 [1.037-1.609], p = 0.023). When FCM was additionally used to analyze the specimen acquired from EUS-FNA, the diagnostic yield was significantly improved (ROC AUC: 0.733 vs. 0.550, p = 0.015), and the AUC of FNB alone or combined with FCM was 0.739 and 0.761. Conclusions: FNB needles generate higher histopathological diagnostic accuracy and specimen quality than FNA for the deep-seated lymphoma. Though the application of FCM significantly improves the diagnostic efficacy of EUS-FNA, FNB was still the preferred diagnostic modality with a shorter procedure time, comparable diagnostic accuracy, and better cost-effectiveness.

Generation of two induced pluripotent stem cell lines with heterozygous and homozygous amyotrophic lateral sclerosis-causing mutation P525L (c.1574C > T) in FUS gene.

Mutations in the FUS (fused in sarcoma) gene are implicated in the neurodegenerative disease amyotrophic lateral sclerosis (ALS). However, the pathophysiology underlying these mutations remains elusive. In this study, we created two induced pluripotent stem cell (iPSC) lines through genetic modification of a healthy hiPSC line (WTC11, UCSFi001-A). These iPSC lines carry the heterozygous and homozygous P525L (c.1574C > T) mutation in the FUS gene. We confirmed that both cell lines possess typical stem cell morphology, normal karyotype, and pluripotency. Our iPSC lines offer a valuable resource for investigating the pathological mechanisms underlying the FUS mutation P525L in ALS.

Generation of two induced pluripotent stem cell lines with heterozygous and homozygous amyotrophic lateral sclerosis-causing mutation R521G (c.1561C > G) in FUS gene.

Mutations in the RNA-binding protein FUS (fused in sarcoma) are linked to amyotrophic lateral sclerosis (ALS), but the pathogenesis is not fully understood. For modeling ALS, here we generated two induced pluripotent stem cell (iPSC) lines carrying the heterozygous and homozygous R521G (c.1561C > G) mutation in the FUS gene via genetic modification of a healthy hiPSC line (WTC11, UCSFi001-A). Both lines show normal stem cell morphology and karyotype, express pluripotent markers, and can differentiate into three germ layers, providing a valuable resource in determining the pathological mechanisms underlying the FUS mutation of R521G in ALS.

Protocol to image and quantify nucleocytoplasmic transport in cultured cells using fluorescent in situ hybridization and a dual reporter system.

Nucleocytoplasmic transport (NCT) plays critical roles in maintaining cellular homeostasis. Here, we present a protocol to measure NCT for both transcript and protein cargos in cultured cells. We first describe the fluorescent in situ hybridization (FISH) assay to measure the nuclear mRNA export. We then detail a dual reporter system to measure the protein NCT. This protocol also includes image analysis and data output using CellProfiler™. The combined approach can be used to unbiasedly analyze NCT activities in cultured cells. For complete details on the use and execution of this protocol, please refer to Ding et al. (2020, 2021).

Precolumn Derivatization High-Performance Liquid Chromatography for Determination of Perfluorocarboxylic Acids in Catalytic Degradation Solutions.

Perfluoroalkyl carboxylic acids (PFCAs), a series of ubiquitous contaminants in the global environment, attracted much attention due to their potential for high bioaccumulation and toxicity to various organisms. There are a lot of measurement requests in currently increasing degradation studies of PFCAs, which usually rely on expensive liquid chromatography-mass spectrometry (LC-MS). The degradation solutions containing high-concentration PFCAs can easily cause the pipeline pollution of the LC/MS instrument, which is usually used for trace analysis of environmental samples. In this study, a simple and reliable precolumn derivatization LC method coupled with an ultraviolet detector (UV) was developed for the determination of the main PFCAs (C4-9) of environmental concern. These PFCAs in degradation solutions were crosslinked to UV-responsive 3, 4-diphenylamine (DCA) by a carbodiimidization method, followed by a simple solid-phase extraction (SPE) cleanup, and quantitatively measured using a conventional LC-UV instrument. Compared to previously reported precolumn derivatization methods, this new derivatization approach has the advantages such as mild reaction conditions, easy operation, enhanced stability of derivatives, and low cost. The instrumental limits of detection (ILDs) for the targeted PFCAs in organic and aqueous mediums were 0.2-0.5 and 0.6-1.5 mg/L, respectively. The method has been successfully applied to the determination of PFCAs in catalytic degradation solutions and recommended for use in other assays involving relatively high-concentration PFCAs.

Generation of gene-corrected isogenic control cell lines from a DYT1 dystonia patient iPSC line carrying a heterozygous GAG mutation in TOR1A gene.

Childhood-onset torsin dystonia (DYT1) is a rare hereditary movement disorder and usually caused by a heterozygous GAG deletion (c.907-909) in the TOR1A gene (ΔE, p.Glu303del). The neuronal functions of torsin proteins and the pathogenesis of ΔE mutation are not clear. Previously, we have generated a hiPSC line from DYT1 patient fibroblast cells. In this study, we genetically corrected GAG deletion and obtained two isogenic control lines. These hiPSC lines contain the wild-type TOR1A sequence, showed the normal stem cell morphology and karyotype, expressed pluripotency markers, and differentiated into three germ layers, providing a valuable resource in DYT1 research.

Generation of highly pure motor neurons from human induced pluripotent stem cells.

Generation of human motor neurons (MNs) overcomes the inaccessibility to patient brain tissues and greatly facilitates the research in MN-related diseases. Here, we describe a protocol for generation of neural progenitor cells (NPCs) from human induced pluripotent stem cells (hiPSCs), followed by preparation of functional MNs. The optimized induction condition with the expression of three transcription factors in a single lentiviral vector significantly improved the yield and purity, making it possible to biochemically identify dysregulated factors in diseased neurons. For complete details on the use and execution of this protocol, please refer to Ding (2021), Ding et al. (2021), and Sepehrimanesh and Ding (2020).

Generation of two induced pluripotent stem cell lines with heterozygous and homozygous GAG deletion in TOR1A gene from a healthy hiPSC line.

A typical DYT1 dystonia is caused by a heterozygous GAG deletion (c.907-909) in the TOR1A gene (ΔE, p.Glu303del) and the pathogenesis is not clear. In this study, human induced pluripotent stem cell (hiPSC) lines carrying the heterozygous or homozygous GAG deletion in TOR1A gene were generated by genetic modification of a healthy hiPSC line (WTC11, UCSFi001-A). These hiPSC lines showed the normal stem cell morphology and karyotype, expressed the same pluripotency markers as their parental line, and had the capacity to differentiate into three germ layers, providing a valuable resource in determining the pathogenesis of human DYT1 dystonia.

Identification and quantification of perfluorooctane sulfonamide isomers by liquid chromatography-tandem mass spectrometry.

Characterization of the isomers perfluorooctane sulfonamide (PFOSA), a key intermediate of the perfluorooctane sulfonate (PFOS) precursors, is a prerequisite to understand the contribution of precursors to PFOS in the environmental and biological matrices. However, the lack of commercial standards makes quantification of the PFOSA isomers in complex matrices a big challenge. For the first time, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was established to identify and quantify PFOSA isomers. Improvement on chromatographic separation with selected reaction monitoring allowed full resolution of six monomethyl branched isomers of PFOSA. The isomers were identified and quantified using a series of characteristic fragment ions and the specific product ions of molecular anion m/z 498: m/z 78 (n-), m/z 169 (iso-), m/z 419 (1m-), m/z 164 (2m-), m/z 259 (3m-), m/z 269 (4m-) and m/z 219 (5m-PFOSA). With the aid of 19F nuclear magnetic resonance to define one technical product as standard, the developed method was used to quantify PFOSA isomers in other technical products and spiked fish blood samples. The results indicated that the developed method displayed strong application prospect for measuring trace level of PFOSA isomers in complex samples. The method detection limits for all isomers were in the range of 0.1-1 pg/g ww for blood samples.

A new less invasive surgical technique in the management of acute Achilles tendon rupture through limited-open procedure combined with a single-anchor and "circuit" suture technique.

BACKGROUND: Traditional incision repair and minimally invasive repair for acute Achilles tendon repair have limitations. This study aimed to present our series of 23 patients with acute Achilles tendon rupture that was repaired using two small incisions to assist the anchor repair of the tear and a new "circuit" suture technique. METHODS: This was a retrospective study of 23 patients with acute Achilles tendon rupture treated with the new technique at Changhai Hospital between January 2015 and December 2016 and followed up for 14-33 months. Clinical outcome was assessed using the AOFAS, Leppilahti, and Arner-Lindholm scores. Complications, range of motion (ROM), and time to return to work and light sport activity were assessed. RESULTS: The AOFAS score was 85-96 at 3 months and 92-100 at 12 months. The 3-month ROM was 27°-37°, and the 12-month ROM was 36°-48°. The Leppilahti score was 85-95 at 3 months and 90-100 at 12 months. The recovery time of the patients was 10-18 weeks. The postoperative recovery time to exercise was 16-24 weeks. There was only one case of deep venous thrombosis. According to the Arner-Lindholm assessment criteria, patient outcomes were rated as excellent in 20 (87.0%) cases, good in three (13.0%) cases, and poor in 0 cases. The excellent-to-good rate was 100%. CONCLUSION: The limited-open procedure combined with a single-anchor and "circuit" suture technique could be used to repair torn Achilles sites, with a low occurrence of complications. This new and minimally invasive technique could be an alternative in the management of acute Achilles tendon rupture.

miR-422a inhibits osteosarcoma proliferation by targeting BCL2L2 and KRAS.

Osteosarcoma is the most common primary malignant bone tumor in children and adolescents. However, the underlying mechanism of osteosarcoma carcinogenesis and progression remains unknown. In the present study, we evaluated the expression profile of miRNAs in osteosarcoma tissues and the adjacent normal tissues. We found that the expression of miR-422a was down-regulated in osteosarcoma tissues and cell lines. In addition, we observed significantly elevated levels of repressive H3K9me3 and H3K27me3 and decreased active H3K4me3 on the promote region of miR-422a in osteosarcoma cells and clinical samples. Furthermore, up-regulation of miR-422a exhibited both in vitro and in vivo anti-tumor effects by inhibiting osteosarcoma cell growth and inducing apoptosis and cell cycle arrest. We also found that miR-422a targeted BCL2L2 and KRAS and negatively regulated their protein expression. Furthermore, restoration of miR-422a and knockdown of BCL2L2 and KRAS promoted apoptosis and induce cell cycle arrest in osteosarcoma cells. Taken together, the present study demonstrates that miR-422a may serve as a tumor suppressor in osteosarcoma via inhibiting BCL2L2 and KRAS translation both in vitro and in vivo Therefore, miR-422a could be developed as a novel therapeutic target in osteosarcoma.

Increased circular RNA UBAP2 acts as a sponge of miR-143 to promote osteosarcoma progression.

Deregulated expression of circular RNA (circRNA) has been determined to be important in carcinogenesis and progression; however, in the most common type of primary malignant bone tumor osteosarcoma, the roles of circRNA in cancer development still remain to be elucidated. Here, we found that circRNA UBAP2 (circUBAP2) expression is significantly increased in human osteosarcoma tissues as compared to those in matched controls. Increased circUBAP2 expression was significantly correlated with human osteosarcoma progression and prognosis. Furthermore, increased circUBAP2 could promote osteosarcoma growth and inhibit apoptosis both in vitro and in vivo. Mechanistically, circUBAP2 was found to inhibit the expression of microRNA-143 (miR-143), thus enhancing the expression and function of anti-apoptotic Bcl-2, which is a direct target of miR-143. Together, our results suggest the roles of circUBAP2 in osteosarcoma development and implicate its potential in prognosis prediction and cancer therapy.

Topological Analysis and Gaussian Decision Tree: Effective Representation and Classification of Biosignals of Small Sample Size.

GOAL: Bucking the trend of big data, in microdevice engineering, small sample size is common, especially when the device is still at the proof-of-concept stage. The small sample size, small interclass variation, and large intraclass variation, have brought biosignal analysis new challenges. Novel representation and classification approaches need to be developed to effectively recognize targets of interests with the absence of a large training set. METHODS: Moving away from the traditional signal analysis in the spatiotemporal domain, we exploit the biosignal representation in the topological domain that would reveal the intrinsic structure of point clouds generated from the biosignal. Additionally, we propose a Gaussian-based decision tree (GDT), which can efficiently classify the biosignals even when the sample size is extremely small. RESULTS: This study is motivated by the application of mastitis detection using low-voltage alternating current electrokinetics (ACEK) where five categories of bisignals need to be recognized with only two samples in each class. Experimental results demonstrate the robustness of the topological features as well as the advantage of GDT over some conventional classifiers in handling small dataset. CONCLUSION: Our method reduces the voltage of ACEK to a safe level and still yields high-fidelity results with a short assay time. SIGNIFICANCE: This paper makes two distinctive contributions to the field of biosignal analysis, including performing signal processing in the topological domain and handling extremely small dataset. Currently, there have been no related works that can efficiently tackle the dilemma between avoiding electrochemical reaction and accelerating assay process using ACEK.

Rapid and sensitive detection of bisphenol a from serum matrix.

Bisphenol A (BPA) is an endocrine disrupting compound that may have adverse developmental, reproductive, neurological, and immune system effects. Low-level exposure to BPA is ubiquitous in human populations due to its widespread use in consumer products. Therefore, highly sensitive methods are needed to quantify BPA in various matrices including water, serum, and food products. In this study, we developed a simple, rapid, highly sensitive and specific sensor based on an aptamer probe and AC electrokinetics capacitive sensing method that successfully detected BPA at femto molar (fM) levels, which is an improvement over prior work by a factor of 10. We were able to detect BPA spiked in human serum as well as in maternal and cord blood within 30s. The sensor is responsive to BPA down to femto molar levels, but not to structurally similar compounds including bisphenol F (BPF) or bisphenol S (BPS) even at much higher concentration. Further development of this platform may prove useful in monitoring exposure to BPA and other small molecules in various matrices.

Combinatorial Histone Readout by the Dual Plant Homeodomain (PHD) Fingers of Rco1 Mediates Rpd3S Chromatin Recruitment and the Maintenance of Transcriptional Fidelity.

The plant homeodomain (PHD) finger is found in many chromatin-associated proteins and functions to recruit effector proteins to chromatin through its ability to bind both methylated and unmethylated histone residues. Here, we show that the dual PHD fingers of Rco1, a member of the Rpd3S histone deacetylase complex recruited to transcribing genes, operate in a combinatorial manner in targeting the Rpd3S complex to histone H3 in chromatin. Although mutations in either the first or second PHD finger allow for Rpd3S complex formation, the assembled complexes from these mutants cannot recognize nucleosomes or function to maintain chromatin structure and prevent cryptic transcriptional initiation from within transcribed regions. Taken together, our findings establish a critical role of combinatorial readout in maintaining chromatin organization and in enforcing the transcriptional fidelity of genes.

Homodimeric PHD Domain-containing Rco1 Subunit Constitutes a Critical Interaction Hub within the Rpd3S Histone Deacetylase Complex.

Recognition of histone post-translational modifications is pivotal for directing chromatin-modifying enzymes to specific genomic regions and regulating their activities. Emerging evidence suggests that other structural features of nucleosomes also contribute to precise targeting of downstream chromatin complexes, such as linker DNA, the histone globular domain, and nucleosome spacing. However, how chromatin complexes coordinate individual interactions to achieve high affinity and specificity remains unclear. The Rpd3S histone deacetylase utilizes the chromodomain-containing Eaf3 subunit and the PHD domain-containing Rco1 subunit to recognize nucleosomes that are methylated at lysine 36 of histone H3 (H3K36me). We showed previously that the binding of Eaf3 to H3K36me can be allosterically activated by Rco1. To investigate how this chromatin recognition module is regulated in the context of the Rpd3S complex, we first determined the subunit interaction network of Rpd3S. Interestingly, we found that Rpd3S contains two copies of the essential subunit Rco1, and both copies of Rco1 are required for full functionality of Rpd3S. Our functional dissection of Rco1 revealed that besides its known chromatin-recognition interfaces, other regions of Rco1 are also critical for Rpd3S to recognize its nucleosomal substrates and functionin vivo. This unexpected result uncovered an important and understudied aspect of chromatin recognition. It suggests that precisely reading modified chromatin may not only need the combined actions of reader domains but also require an internal signaling circuit that coordinates the individual actions in a productive way.

Nucleosome contact triggers conformational changes of Rpd3S driving high-affinity H3K36me nucleosome engagement.

The Rpd3S histone deacetylase complex utilizes two subunits, Eaf3 and Rco1, to recognize nucleosomes methylated at H3K36 (H3K36me) with high affinity and strong specificity. However, the chromobarrel domain of Eaf3 (CHD) that is responsible for H3K36me recognition only binds weakly and with little specificity to histone peptides. Here, using deuterium exchange mass spectrometry (DXMS), we detected conformational changes of Rpd3S upon its contact with chromatin. Interestingly, we found that the Sin3-interacting domain of Rco1 (SID) allosterically stimulates preferential binding of Eaf3 to H3K36-methylated peptides. This activation is tightly regulated by an autoinhibitory mechanism to ensure optimal multivalent engagement of Rpd3S with nucleosomes. Lastly, we identified mutations at the interface between SID and Eaf3 that do not disrupt complex integrity but severely compromise Rpd3S functions in vitro and in vivo, suggesting that the nucleosome-induced conformational changes are essential for chromatin recognition.

Alternating current electrokinetics enhanced in situ capacitive immunoassay.

A rapid in situ capacitive immunoassay is presented herein. Conventional immunoassay typically relies on diffusion for transport of analytes in many cases causing long detection time and lack of sensitivity. By integrating alternating current electrokinetics (ACEK) and impedance sensing, this work provides a rapid in situ capacitive affinity biosensing. ACEK induces both fluid flow and particle motion, conveying target molecules toward electrodes immobilized with probes, resulting in rapid enrichment of target molecules and a capacitance change at the ''electrode-fluid'' interface. The benefit of ACEK enhanced immunoassay was demonstrated using the antigen and antibody from Johne's disease (JD) as an example. To clarify the importance of DEP and ACET effects for binding reaction, two different electrode pattern designs for capacitive immunoassay are studied. The asymmetric array and symmetric electrodes exhibit very similar response at lower electric field due to DEP effects, while asymmetric array has remarkable higher response at high-electric field because the convection becomes more important at high field. The disease positive and negative serum samples are distinguished in few minutes.