Two-Step Induction of Trabecular Meshwork Cells from Induced Pluripotent Stem Cells for Glaucoma

Glaucoma is a leading cause of irreversible blindness worldwide. Reducing intraocular pressure is currently the only effective treatment. Elevated intraocular pressure is associated with increased resistance of the outflow pathway, mainly the trabecular meshwork (TM). Despite great progress in the field, the development of novel and effective treatment for glaucoma is still challenging. In this study, we reported that human induced pluripotent stem cells (iPSCs) can be cultured as colonies and monolayer cells expressing OCT4, alkaline phosphatase, SSEA4 and SSEA1. After induction to neural crest cells (NCCs) positive to NGFR and HNK1, the iPSCs can differentiate into TM cells. The induced iPSC-TM cells expressed TM cell marker CHI3L1, were responsive to dexamethasone treatment with increased expression of myocilin, ANGPTL7, and formed CLANs, comparable to primary TM cells. To the best of our knowledge, this is the first study that induces iPSCs to TM cells through a middle neural crest stage, which ensures a stable NCC pool and ensures the high output of the same TM cells. This system can be used to develop personalized treatments using patient-derived iPSCs, explore high throughput screening of new drugs focusing on TM response for controlling intraocular pressure, and investigate stem cell-based therapy for TM regeneration

Characterization of novel microsatellite loci in rare minnow (Gobiocypris rarus) and amplification in closely related species in Gobioninae

Rare minnow (Gobiocypris rarus) is an endangered small fish endemic to upper reach of the Yangtze River. From a (GT)n enriched genomic library, 32 microsatellites were isolated and characterized. Nineteen of these loci were polymorphic in a test population with alleles ranging from 2-7, and observed and expected heterozygosities from zero to 0.8438, and 0.2679 to 0.8264, respectively. In the cross-species amplifications, 13 out of 19 polymorphic loci were found to be also polymorphic in at least one of the 7 closely related species of the subfamily Gobioninae. These polymorphic microsatellite loci should provide sufficient level of genetic diversity to evaluate the fine-scale population structure in rare minnow and its closely related species for the conservation purpose.Rare minnow (Gobiocypris rarus) is an endangered small fish endemic to upper reach of the Yangtze River. From a (GT)n enriched genomic library, 32 microsatellites were isolated and characterized. Nineteen of these loci were polymorphic in a test population with alleles ranging from 2-7, and observed and expected heterozygosities from zero to 0.8438, and 0.2679 to 0.8264, respectively. In the cross-species amplifications, 13 out of 19 polymorphic loci were found to be also polymorphic in at least one of the 7 closely related species of the subfamily Gobioninae. These polymorphic microsatellite loci should provide sufficient level of genetic diversity to evaluate the fine-scale population structure in rare minnow and its closely related species for the conservation purpose

Identification of the Xenopus DNA2 protein as a major nuclease for the 5′→3′ strand-specific processing of DNA ends

The first step of homology-dependent DNA double-strand break (DSB) repair is the 5′ strand-specific processing of DNA ends to generate 3′ single-strand tails. Despite extensive effort, the nuclease(s) that is directly responsible for the resection of 5′ strands in eukaryotic cells remains elusive. Using nucleoplasmic extracts (NPE) derived from the eggs of Xenopus laevis as the model system, we have found that DNA processing consists of at least two steps: an ATP-dependent unwinding of ends and an ATP-independent 5′→3′ degradation of single-strand tails. The unwinding step is catalyzed by DNA helicases, the major one of which is the Xenopus Werner syndrome protein (xWRN), a member of the RecQ helicase family. In this study, we report the purification and identification of the Xenopus DNA2 (xDNA2) as one of the nucleases responsible for the 5′→3′ degradation of single-strand tails. Immunodepletion of xDNA2 resulted in a significant reduction in end processing and homology-dependent DSB repair. These results provide strong evidence that xDNA2 is a major nuclease for the resection of DNA ends for homology-dependent DSB repair in eukaryotes

More is less: Effect of ICF-based early progressive mobilization on severe aneurysmal subarachnoid hemorrhage in the NICU

IntroductionAneurysmal subarachnoid hemorrhage (aSAH) is a type of stroke that occurs due to a ruptured intracranial aneurysm. Although advanced therapies have been applied to treat aSAH, patients still suffer from functional impairment leading to prolonged stays in the NICU. The effect of early progressive mobilization as an intervention implemented in the ICU setting for critically ill patients remains unclear.MethodsThis retrospective study evaluated ICF-based early progressive mobilization's validity, safety, and feasibility in severe aSAH patients. Sixty-eight patients with aSAH with Hunt-Hess grades III-IV were included. They were divided into two groups—progressive mobilization and passive movement. Patients in the progressive mobilization group received progressive ICF-based mobilization intervention, and those in the passive movement group received passive joint movement training. The incidence of pneumonia, duration of mechanical ventilation, length of NICU stay, and incidence of deep vein thrombosis were evaluated for validity. In contrast, the incidence of cerebral vasospasm, abnormally high ICP, and other safety events were assessed for safety. We also described the feasibility of the early mobilization initiation time and the rate of participation at each level for patients in the progressive mobilization group.ResultsThe results showed that the incidence of pneumonia, duration of mechanical ventilation, and length of NICU stay were significantly lower among patients in the progressive mobilization group than in the passive movement group (P = 0.031, P = 0.004, P = 0.012), but the incidence of deep vein thrombosis did not significantly differ between the two groups. Regarding safety, patients in the progressive mobilization group had a lower incidence of cerebral vasospasm than those in the passive movement group. Considering the effect of an external ventricular drain on cerebral vasospasm (P = 0.015), we further analyzed those patients in the progressive mobilization group who had a lower incidence of cerebral vasospasm in patients who did not have an external ventricular drain (P = 0.011). Although we found 2 events of abnormally increased intracranial pressure in the progressive mobilization group, there was no abnormal decrease in cerebral perfusion pressure in the 2 events. In addition, among other safety events, there was no difference in the occurrence of adverse events between the two groups (P = 0.073), but the number of potential adverse events was higher in the progressive mobilization group (P = 0.001). Regarding feasibility, patients in the progressive mobilization group were commonly initiated 72 h after admission to the NICU, and 47.06% were in the third level of the mobilization protocol.DiscussionWe conclude that the ICF-based early progressive mobilization protocol is an effective and feasible intervention tool. For validity, more mobilization interventions might lead to less pneumonia, duration of mechanical ventilation and length of stay for patients with severe aSAH in the NICU, Moreover, it is necessary to pay attention over potential adverse events (especially line problems), although we did not find serious safety events

Acute combined effects of concurrent physical activities on autonomic nervous activation during cognitive tasks

Backgrounds: The validity of heart rate variability (HRV) has been substantiated in mental workload assessments. However, cognitive tasks often coincide with physical exertion in practical mental work, but their synergic effects on HRV remains insufficiently established. The study aims were to investigate the combined effects of cognitive and physical load on autonomic nerve functions.Methods: Thirty-five healthy male subjects (aged 23.5 ± 3.3 years) were eligible and enrolled in the study. The subjects engaged in n-back cognitive tasks (1-back, 2-back, and 3-back) under three distinct physical conditions, involving isotonic contraction of the left upper limb with loads of 0 kg, 3 kg, and 5 kg. Electrocardiogram signals and cognitive task performance were recorded throughout the tasks, and post-task assessment of subjective experiences were conducted using the NASA-TLX scale.Results: The execution of n-back tasks resulted in enhanced perceptions of task-load feelings and increased reaction times among subjects, accompanied by a decline in the accuracy rate (p < 0.05). These effects were synchronously intensified by the imposition of physical load. Comparative analysis with a no-physical-load scenario revealed significant alterations in the HRV of the subjects during the cognitive task under moderate and high physical conditions. The main features were a decreased power of the high frequency component (p < 0.05) and an increased low frequency component (p < 0.05), signifying an elevation in sympathetic activity. This physiological response manifested similarly at both moderate and high physical levels. In addition, a discernible linear correlation was observed between HRV and task-load feelings, as well as task performance under the influence of physical load (p < 0.05).Conclusion: HRV can serve as a viable indicator for assessing mental workload in the context of physical activities, making it suitable for real-world mental work scenarios

Replication intermediates that escape Dna2 activity are processed by Holliday junction resolvase Yen1

Cells have evolved mechanisms to protect, restart and repair perturbed replication forks, allowing full genome duplication, even under replication stress. Interrogating the interplay between nuclease-helicase Dna2 and Holliday junction (HJ) resolvase Yen1, we find the Dna2 helicase activity acts parallel to homologous recombination (HR) in promoting DNA replication and chromosome detachment at mitosis after replication fork stalling. Yen1, but not the HJ resolvases Slx1-Slx4 and Mus81-Mms4, safeguards chromosome segregation by removing replication intermediates that escape Dna2. Post-replicative DNA damage checkpoint activation in Dna2 helicase-defective cells causes terminal G2/M arrest by precluding Yen1-dependent repair, whose activation requires progression into anaphase. These findings explain the exquisite replication stress sensitivity of Dna2 helicase-defective cells, and identify a non-canonical role for Yen1 in the processing of replication intermediates that is distinct from HJ resolution. The involvement of Dna2 helicase activity in completing replication may have implications for DNA2-associated pathologies, including cancer and Seckel syndrome