Somatic and intergenerational G4C2 hexanucleotide repeat instability in a human C9orf72 knock-in mouse model.

Expansion of a G4C2 repeat in the C9orf72 gene is associated with familial Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD). To investigate the underlying mechanisms of repeat instability, which occurs both somatically and intergenerationally, we created a novel mouse model of familial ALS/FTD that harbors 96 copies of G4C2 repeats at a humanized C9orf72 locus. In mouse embryonic stem cells, we observed two modes of repeat expansion. First, we noted minor increases in repeat length per expansion event, which was dependent on a mismatch repair pathway protein Msh2. Second, we found major increases in repeat length per event when a DNA double- or single-strand break (DSB/SSB) was artificially introduced proximal to the repeats, and which was dependent on the homology-directed repair (HDR) pathway. In mice, the first mode primarily drove somatic repeat expansion. Major changes in repeat length, including expansion, were observed when SSB was introduced in one-cell embryos, or intergenerationally without DSB/SSB introduction if G4C2 repeats exceeded 400 copies, although spontaneous HDR-mediated expansion has yet to be identified. These findings provide a novel strategy to model repeat expansion in a non-human genome and offer insights into the mechanism behind C9orf72 G4C2 repeat instability.

[Origin and relationship of jing-well points and Shixuan (EX-UE 11)].

Both the twelve jing-well points and Shixuan (EX-UE 11) are the commonly used first-aid points. These two kinds of acupoints are located closely and similar in function, hence, they are often confused in application. In order to explore the origin of their location and theory as well as their relationship, the relevant data were retrieved. It has been found that the relationship between jing-well points and Shixuan (EX-UE 11) is traced at the earliest time to Huangdi Neijing (Yellow Emperor's Internal Classics). It is believed that the jing-well points refer to the starting points or the ending points of the twelve regular meridians and Shixuan (EX-UE 11) are located at the crossing sites of yin-yang related meridians of the regular meridians. These two kinds of acupoints are interconnected, share the same source and are also different from each other. Qiduan (EX-LE 12) is also named as foot-Shixuan. Shixuan (EX-UE 11) and Qiduan (EX-LE 12) can be regarded as the same category, just like jing-well points. In clinical practice, the jing-well points are generally selected in treatment of internal diseases, local diseases and those on the running course of meridians. They can be used separately in treatment. Shixuan (EX-UE 11) is the first option, or combined with Qiduan (EX-LE 12) in the emergent treatment of tense syndrome and syncope. The jing-well points and Shixuan (EX-UE 11) are different even though sharing the same origin. They are mutually benefited and supplemented with each other in clinical practice.

[Acupuncture strategy for Bu-yi in Inner Canon of Yellow Emperor].

This article focuses on the Bu-yi (the disease has not been cured after treatment) recorded in the Inner Canon of Yellow Emperor and discusses four aspects, including acupuncture feasibility, acupuncture treatment transformation, acupuncture point selection and acupuncture treatment principles. We hope to explain the virtual acupuncture in the Inner Canon of Yellow Emperor from a deeper level and provide new ideas for clinical acupuncture treatment.

[Exploration into the simplex reinforcing-reducing manipulation of acupuncture].

Four controversial types of simplex reinforcing-reducing manipulation of acupuncture and their possible meanings were summarized to explore several key elements of reinforcing-reducing manipulation of acupuncture, in addition, the simplex reinforcing-reducing manipulation of acupuncture was classified by single factor. It is concluded that the definition of simplex reinforcing-reducing manipulation of acupuncture should try not to include other non-manipulative elements. According to single factor, it can be divided into: needle-oriented reinforcing-reducing manipulation, twisting reinforcing-reducing manipulation, lifting and interpolating reinforcing-reducing manipulation, fast and slow reinforcing-reducing manipulation, breathing reinforcing-reducing manipulation, opening and closing reinforcing-reducing manipulation. In addition, after considering the effect and principle of number reinforcing-reducing manipulation, it can be considered.

Hyaluronan microenvironment enhances cartilage regeneration of human adipose-derived stem cells in a chondral defect model.

Hyaluronan (HA) is an important extracellular matrix component in the early stage of chondrogenesis. This study aimed to investigate the application of an HA microenvironment for human adipose-derived stem cells (hADSCs)-based articular cartilage regeneration. HA-enriched fibrin (HA/Fibrin) hydrogels were synthesized and characterized for use as HA microenvironments. The cell viability and chondrogenic gene expression of hADSCs cultured in HA/Fibrin (HA/Fibrin/hADSC) and Fibrin (Fibrin/hADSC) hydrogels were tested in vitro. A chondral defect created in osteochondral core explants ex vivo was used to test chondral defect regeneration by HA/Fibrin/hADSC or Fibrin/hADSC hydrogels. The results showed that HA/Fibrin hydrogels exhibited an increased swelling ratio and matrix stiffness and a smoother surface with more interconnected pores than in Fibrin hydrogels. The viability of hADSCs in HA/Fibrin/hADSC hydrogels was not altered, but they exhibited higher chondrogenic gene expression than those in Fibrin/hADSC hydrogels. For chondral defect regeneration, the HA/Fibrin/hADSC hydrogels exhibited the most cartilaginous tissue neo-formation, chondral integration and sGAG content in the surrounding tissue. This study demonstrated that an HA microenvironment enhances hADSC-mediated cartilage regeneration in chondral defects and thus may be used for ADSC-based articular cartilage tissue engineering.

Light-Up of Rhodamine Hydrazide to Generate Emissive Initiator for Polymerization and to Afford Photochromic Polypeptide Metal Complex.

Ring-opening polymerization (ROP) of cyclic peptide monomer of γ-propargyl-l-glutamate N-carboxyanhydride (PLG⁻NCA) was originally initiated by non-emissive, ring-close rhodamine 6G hydrazide (R-C). However, instantaneously after adding PLG⁻NCA to R-C, the spirolactam ring of R-C was opened by PLG⁻NCA, rendering emissive, ring-open R-O to initiate ROP of PLG⁻NCA. The emissive R-O moiety therefore produced fluorescent R⁻PLG with aggregation-induced emission (AIE) properties. Moreover, R⁻PLG was found to exhibit photochromic properties with good fatigue resistance and long lifetime when forming metal complexes with Sn(II) and Fe(III). In the dark, irradiated metal complexes slowly (~50 min) restored to the initial state. This research provides foundation for the development of new photochromic materials with long lifetime.

Ionic complex of a rhodamine dye with aggregation-induced emission properties.

An AIE-active rhodamine based luminogen was prepared via a complexation reaction between non-emissive rhodamine hydrazide (RdH) and bulky camphorsulfonic acid (CSA). Besides acting to open the spirolactam ring of RdH, CSA also imposes a rotational restriction on the resultant ionic complex, RdH(CSA)x. Without CSA, the analogous complex RdH(HCl)3 is a luminogen with aggregation-caused quenching (ACQ) properties. The ionic bonds of RdH(CSA)3 are sensitive to several external stimuli and therefore it is a luminescent sensor for metal ions, organic amines and the blood protein bovine serum albumin (BSA). Besides being a sensor for BSA, the ionic RdH(CSA)3 is also a denaturant capable of uncoiling the peptide chain of BSA.

Induction of HIV Neutralizing Antibody Lineages in Mice with Diverse Precursor Repertoires.

The design of immunogens that elicit broadly reactive neutralizing antibodies (bnAbs) has been a major obstacle to HIV-1 vaccine development. One approach to assess potential immunogens is to use mice expressing precursors of human bnAbs as vaccination models. The bnAbs of the VRC01-class derive from the IGHV1-2 immunoglobulin heavy chain and neutralize a wide spectrum of HIV-1 strains via targeting the CD4 binding site of the envelope glycoprotein gp120. We now describe a mouse vaccination model that allows a germline human IGHV1-2(∗)02 segment to undergo normal V(D)J recombination and, thereby, leads to the generation of peripheral B cells that express a highly diverse repertoire of VRC01-related receptors. When sequentially immunized with modified gp120 glycoproteins designed to engage VRC01 germline and intermediate antibodies, IGHV1-2(∗)02-rearranging mice, which also express a VRC01-antibody precursor light chain, can support the affinity maturation of VRC01 precursor antibodies into HIV-neutralizing antibody lineages.

A Rapid Embryonic Stem Cell-Based Mouse Model for B-cell Lymphomas Driven by Epstein-Barr Virus Protein LMP1.

The Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) contributes to oncogenic human B-cell transformation. Mouse B cells conditionally expressing LMP1 are not predisposed to B-cell malignancies, as LMP1-expressing B cells are eliminated by T cells. However, mice with conditional B-cell LMP1 expression and genetic elimination of α/ß and γ/δ T cells ("CLT" mice) die early in association with B-cell lymphoproliferation and lymphomagenesis. Generation of CLT mice involves in-breeding multiple independently segregating alleles. Thus, although introduction of additional activating or knockout mutations into the CLT model is desirable for further B-cell expansion and immunosurveillance studies, doing such experiments by germline breeding is time-consuming, expensive, and sometimes unfeasible. To generate a more tractable model, we generated clonal CLT embryonic stem (ES) cells from CLT embryos and injected them into RAG2-deficient blastocysts to generate chimeric mice, which, like germline CLT mice, harbor splenic CLT B cells and lack T cells. CLT chimeric mice generated by this RAG2-deficient blastocyst complementation ("RDBC") approach die rapidly in association with B-cell lymphoproliferation and lymphoma. Because CLT lymphomas routinely express the activation-induced cytidine deaminase (AID) antibody diversifier, we tested potential AID roles by eliminating the AID gene in CLT ES cells and testing them via RDBC. We found that CLT and AID-deficient CLT ES chimeras had indistinguishable phenotypes, showing that AID is not essential for LMP1-induced lymphomagenesis. Beyond expanding accessibility and utility of CLT mice as a cancer immunotherapy model, our studies provide a new approach for facilitating generation of genetically complex mouse cancer models.

Lin28a regulates germ cell pool size and fertility.

Overexpression of LIN28A is associated with human germ cell tumors and promotes primordial germ cell (PGC) development from embryonic stem cells in vitro and in chimeric mice. Knockdown of Lin28a inhibits PGC development in vitro, but how constitutional Lin28a deficiency affects the mammalian reproductive system in vivo remains unknown. Here, we generated Lin28a knockout (KO) mice and found that Lin28a deficiency compromises the size of the germ cell pool in both males and females by affecting PGC proliferation during embryogenesis. Interestingly however, in Lin28a KO males, the germ cell pool partially recovers during postnatal expansion, while fertility remains impaired in both males and females mated to wild-type mice. Embryonic overexpression of let-7, a microRNA negatively regulated by Lin28a, reduces the germ cell pool, corroborating the role of the Lin28a/let-7 axis in regulating the germ lineage.