Improving single-cell cloning workflow for gene editing in human pluripotent stem cells

The availability of human pluripotent stem cells (hPSCs) and progress in genome engineering technology have altered the way we approach scientific research and drug development screens. Unfortunately, the procedures for genome editing of hPSCs often subject cells to harsh conditions that compromise viability: a major problem that is compounded by the innate challenge of single-cell culture. Here we describe a generally applicable workflow that supports single-cell cloning and expansion of hPSCs after genome editing and single-cell sorting. Stem-Flex and RevitaCell supplement, in combination with Geltrex or Vitronectin (VN), promote reliable single-cell growth in a feeder-free and defined environment. Characterization of final genome-edited clones reveals that pluripotency and normal karyotype are retained following this single-cell culture protocol. This time-efficient and simplified culture method paves the way for high-throughput hPSC culture and will be valuable for both basic research and clinical applications. Keywords: Human pluripotent stem cells, Embryonic stem cells, Single-cell cloning, Induced pluripotent stem cells, hPSCs, hESCs, Genome engineering, CRISPR-Cas

Correlation of Copper Interaction, Copper-Driven Aggregation, and Copper-Driven H2O2 Formation with Aβ40 Conformation

The neurotoxicity of Aβ is associated with the formation of free radical by interacting with redox active metals such as Cu2+. However, the relationship between ion-interaction, ion-driven free radical formation, and Aβ conformation remains to be further elucidated. In the present study, we investigated the correlation of Cu2+ interaction and Cu2+-driven free radical formation with Aβ40 conformation. The Cu2+-binding affinity for Aβ40 in random coiled form is 3-fold higher than that in stable helical form. Unexpectedly but interestingly, we demonstrate in the first time that the stable helical form of Aβ40 can induce the formation of H2O2 by interacting with Cu2+. On the other hand, the H2O2 generation is repressed at Aβ/Cu2+ molar ratio ≥1 when Aβ40 adopts random coiled structure. Taken together, our result demonstrates that Aβ40 adopted a helical structure that may play a key factor for the formation of free radical with Cu2+ ions

Mesoderm induction in Ambystoma mexicanum, a urodele amphibian

Understanding how the germ layers are formed is one of the key questions of developmental biology. Abundant studies in the anuran amphibian Xenopus laevis have described that maternal and vegetally localised mRNAs for VegT and Vg1 contribute greatly to the formation of mesoderm and endoderm in the developing embryo. Within Xenopus mesendoderm gene-regulatory network (GRN), Wnt/β-catenin as well as Nodal and Mix family members have been shown to play important roles. The involvement of several members of the Nodal and Mix gene families with redundant functions makes the mesendoderm GRN surprisingly complex and difficult to study in Xenopus laevis. By contrast, mouse and humans have only single copies of Nodal and Mix. Since urodeles have an embryology that is basal to amphibians and that has most likely also been conserved during the evolution of amniotes, including mammals, we have investigated the Mix and Nodal genes in the urodele Axolotl in the hope that their gene families contained fewer members. We cloned one Mix and two Nodal orthologs from the axolotl and showed by Southern blot analysis that there are likely no further copies in the axolotl genome. Morpholino and rescue experiments furthermore showed that AxNodal-1, Mix and Brachyury play essential roles in mesoderm specification in axolotl embryos, suggesting that the urodele Axolotl has a more simplified mesendoderm GRN. In this context, we demonstrate that Mix acts to induce Brachyury expression during mesoderm induction. Mixl1 shRNA knowdown in mouse embryonic stem cells (mESCs) shows that Mixl1 is involved in the production of mesoderm in mESCs too. Analysis of the localisation of the VegT and Vg1 mRNAs in oocytes revealed that they are neither vegetally localised in the Axolotl, nor in the basal fish species lungfish and sturgeon. Furthermore, gain and loss of function assays examining the roles of maternal VegT and β-catenin demonstrated that VegT is not required for mesoderm induction, whereas β-catenin is necessary and sufficient for mesoderm induction by activating AxNodal-1 expression in the axolotl. As these results reveal additional similarities to the GRN in mammals they further support our hypothesis that the regulatory network in the axolotl is more closely related to that in amniotes rather than anuran amphibians

The Study and Implementation of Network-Based Auditing System with Session Tracking and Monitoring

With consideration of the increasing importance of auditing system and the present auditing systems’ incapability of performing packet reassembling analysis, this research attempts to develop a “network-based auditing system with session tracking and monitoring” to assist network administrators to analyze and rearrange the packets into separate session groups. This developed system is able to reveal every single step of the unauthorized activities. As a result, the administrators can investigate each network session and its transferred data more efficiently, and reduced greatly the time for auditing data analysis. In addition, the event reconstruction simulates the actual event occurred at that time; this feature provides network administrators with more detailed and realistic insight concerning vulnerabilities in network security that need to be fixed. Also, this system keeps track of all network events, and collects related information in a set of auditing files (log files). Moreover, the collected records and reassembled files can serve as evidences in tracing cyber-crimes and as references for recovery process

Neurofibromatosis-1 regulation of neural stem cell proliferation and multilineage differentiation operates through distinct RAS effector pathways

Neurofibromatosis type 1 (NF1) is a common neurodevelopmental disorder caused by impaired function of the neurofibromin RAS regulator. Using a combination of Nf1 genetically engineered mice and pharmacological/genetic inhibition approaches, we report that neurofibromin differentially controls neural stem cell (NSC) proliferation and multilineage differentiation through the selective use of the PI3K/AKT and RAF/MEK pathways. While PI3K/AKT governs neurofibromin-regulated NSC proliferation, multilineage differentiation is MEK-dependent. Moreover, whereas MEK-regulated multilineage differentiation requires Smad3-induced Jagged-1 expression and Notch activation, MEK/Smad3-regulated Hes1 induction is only responsible for astrocyte and neuronal differentiation. Collectively, these findings establish distinct roles for the RAS effector pathways in regulating brain NSC function

Coprescription of Chinese Herbal Medicine and Western Medications among Prostate Cancer Patients: A Population-Based Study in Taiwan

Use of herbal medicine is popular among cancer patients. This study aimed to explore the coprescription of CHM and WM among prostate cancer patients in Taiwan. This cross-sectional retrospective study used a population-based database containing one million beneficiaries of National Health Insurance. Claims and prescriptions were analyzed. In 2007, 218 (22.4%) prostate cancer patients were CHM users. Among CHM users, 200 (91.7%) patients with 5618 (79.5%) CHM prescriptions were on coprescription of CHM and WM. A total of 484 types of CHM and 930 types of WM were used. The most commonly used CHMs on coprescription were Shu Jing Huo Xue Tang, Ma Zi Ren Wan, and Xue Fu Zhu Yu Tang. The most commonly used WMs on coprescription were magnesium oxide, amlodipine, and aspirin. The average number of prescriptions per user per year was 261.2 versus 151.7 in all (P < 0.001), 123.6 versus 76.9 in WM (P = 0.033), and 34.8 versus 5.1 in CHM (P < 0.001) for patients with and without coprescription, respectively. In conclusion, use of CHM among prostate cancer patients was popular in Taiwan. Most CHMs were used with WM concurrently. The potential drug-herb interactions should be investigated, especially for patients with more prescriptions

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