Klinefelter syndrome (KS) is one of the most frequent genetic abnormalities and the leading genetic cause of nonobstructive azoospermia. The breeding and study of KS mouse models are essential to ...advancing our knowledge of the underlying pathological mechanism. Karyotyping and fluorescence in situ hybridization are reliable methods for identifying chromosomal contents. However, technical issues associated with these methods can decrease the efficiency of breeding KS mouse models and limit studies that require rapid identification of target mice. To overcome these limitations, we developed three polymerase chain reaction-based assays to measure specific genetic information, including presence or absence of the sex determining region of chromosome Y (Sry), copy number of amelogenin, X-linked (Amelx), and inactive X specific transcripts (Xist) levels. Through a combined analysis of the assay results, we can infer the karyotype of target mice. We confirmed the utility of our assays with the successful generation of KS mouse models. Our assays are rapid, inexpensive, high capacity, easy to perform, and only require small sample amounts. Therefore, they facilitate the breeding and study of KS mouse models and help advance our knowledge of the pathological mechanism underlying KS.
Genetically encoded tags for single-molecule imaging in electron microscopy (EM) are long-awaited. Here, we report an approach for directly synthesizing EM-visible gold nanoparticles (AuNPs) on ...cysteine-rich tags for single-molecule visualization in cells. We first uncovered an auto-nucleation suppression mechanism that allows specific synthesis of AuNPs on isolated tags. Next, we exploited this mechanism to develop approaches for single-molecule detection of proteins in prokaryotic cells and achieved an unprecedented labeling efficiency. We then expanded it to more complicated eukaryotic cells and successfully detected the proteins targeted to various organelles, including the membranes of endoplasmic reticulum (ER) and nuclear envelope, ER lumen, nuclear pores, spindle pole bodies and mitochondrial matrices. We further implemented cysteine-rich tag-antibody fusion proteins as new immuno-EM probes. Thus, our approaches should allow biologists to address a wide range of biological questions at the single-molecule level in cellular ultrastructural contexts.
•The mutation in OsAGPL2 resulted in aberrant storage substances in the w24 endosperm.•Expression of AGPase, activities of Pho1 and SuSy were enhanced in the w24 endosperm.•Expression of OsAGPL2 and ...OsAGPS2b was highly coordinated in rice endosperm.•Direct interactions of OsAGPL1:OsAGPS1 as well as OsAGPS2b:OsAGPL2 were verified.•Self-binding was evident for the OsAGPL2 subunits but not the OsAGPS2b subunits.
ADP-glucose pyrophosphorylase (AGPase) controls a rate-limiting step in the starch biosynthetic pathway in higher plants. Here we isolated a shrunken rice mutant w24. Map-based cloning identified OsAGPL2, a large subunit of the cytosolic AGPase in rice endosperm, as the gene responsible for the w24 mutation. In addition to severe inhibition of starch synthesis and significant accumulation of sugar, the w24 endosperm showed obvious defects in compound granule formation and storage protein synthesis. The defect in OsAGPL2 enhanced the expression levels of the AGPase family. Meanwhile, the elevated activities of starch phosphorylase 1 and sucrose synthase in the w24 endosperm might possibly partly account for the residual starch content in the mutant seeds. Moreover, the expression of OsAGPL2 and its counterpart, OsAGPS2b, was highly coordinated in rice endosperm. Yeast two-hybrid and BiFC assays verified direct interactions between OsAGPL2 and OsAGPS2b as well as OsAGPL1 and OsAGPS1, supporting the model for spatiotemporal complex formation of AGPase isoforms in rice endosperm. Besides, our data provided no evidence for the self-binding of OsAGPS2b, implying that OsAGPS2b might not interact to form higher molecular mass aggregates in the absence of OsAGPL2. Therefore, the molecular mechanism of rice AGPase assembly might differ from that of Arabidopsis.
Saccharomyces cerevisiae
is a widely used microorganism and a greatly popular cell factory for the production of various chemicals. In order to improve the yield of target chemicals, it is often ...necessary to increase the copy numbers of key genes or engineer the related metabolic pathways, which traditionally required time-consuming repetitive rounds of gene editing. With the development of gene-editing technologies such as meganucleases, TALENs, and the CRISPR/Cas system, multiplex genome editing has entered a period of rapid development to speed up cell factory optimization. Multi-copy insertion and removing bottlenecks in biosynthetic pathways can be achieved through gene integration and knockout, for which multiplexing can be accomplished by targeting repetitive sequences and multiple sites, respectively. Importantly, the development of the CRISPR/Cas system has greatly increased the speed and efficiency of multiplex editing. In this review, the various multiplex genome editing technologies in
S. cerevisiae
were summarized, and the principles, advantages, and the disadvantages were analyzed and discussed. Finally, the practical applications and future prospects of multiplex genome editing were discussed.
Key points
• The development of multiplex genome editing in S. cerevisiae was summarized.
• The pros and cons of various multiplex genome editing technologies are discussed.
• Further prospects on the improvement of multiplex genome editing are proposed.
•Three potential factors were investigated to control ice crystal growth during aerogel formation.•Low temperature polarizing microscopy and scanning electron microscopy were used to view the ice ...crystal and pore structures.•Size distribution figures of ice crystals and aerogel pores were drawn and could be used to support the discussions.
Konjac glucomannan (KGM)-based aerogels were prepared using a combination of sol-gel and freeze-drying methods. Preparation conditions were chosen to control ice crystal growth and aerogel structure formation. The ice crystals formed during pre-freezing were observed by low temperature polarizing microscopy, and images of aerogel pores were obtained by scanning electron microscopy. The size of ice crystals were calculated and size distribution maps were drawn, and similarly for aerogel pores. Results showed that ice crystal growth and aerogel pore sizes may be controlled by varying pre-freezing temperatures, KGM concentration and glyceryl monostearate concentration. The impact of pre-freezing temperatures on ice crystal growth was explained as combining ice crystal growth rate with nucleation rate, while the impacts of KGM and glyceryl monostearate concentration on ice crystal growth were interpreted based on their influences on sol network structure.
The effects of virgin coconut oil (VCO) contents (10%, 30%, 50%, and 70%, based on the mass of gum Arabic) on the properties of the emulsions and the films of corn starch/sodium alginate/gum Arabic ...composite were investigated. Results showed that increased VCO content led to larger droplet size and weakened emulsion stability, as evidenced by the microscopic images. Regarding the film properties, the higher the VCO content, the lower the water vapor permeability (WVP), oxygen permeability, and water adsorption. The incorporation of VCO decreased the tensile strength but effectively increased the elongation at break of the films. Moreover, high linearity against VCO content was observed for WVP, water adsorption, and water contact angle. This study contributes to the understanding of modulating the properties of the emulsion and the resultant film by altering the oil content.
Display omitted
•VCO addition affected the properties of film-forming emulsions and the films.•Increased VCO content caused increases in the droplet size and physical instability.•VCO incorporation improved the water and oxygen barrier properties of the films.•VCO addition can positively influence the EAB of the film.
Konjac glucomannan (KGM)/ethyl cellulose (EC) blend films were prepared at different blend ratios (KGM/EC = 10/0, 9/1, 8/2, 7/3, 6/4, and 0/10, w/w) in the presence of dibutyl sebacate at 25% (w/w) ...of total solids content.
Hydrogen bond interactions between KGM and EC occurred. An ethyl cellulose phase consisting of oval-shaped particles was uniformly embedded in the KGM continuous phase. The maximum values of tensile strength and elongation at break of blend films were respectively 48 MPa and 12.7% when the ratio of KGM and EC was 7/3 (w/w). Compared to KGM film, the moisture resistance, thermal stability, tensile strength, and elongation at break of blend films were increased. In addition, water vapor transmission was decreased, but the oxygen permeability was increased by blending. Applications of the blend film of KGM and EC for food packaging are suggested.
Display omitted
•Films based on konjac glucomannan and ethyl cellulose were prepared and characterized.•Blend films were composed of the KGM continuous phase with oval-shaped EC particles embedded in films.•Increased contents of konjac glucomannan affected film forming properties.
What is the physiological function of Yes-associated protein-1 (Yap1), a susceptibility gene for polycystic ovary syndrome (PCOS), in ovarian granulosa cells (GCs)?
Physiologically, steroid sex ...hormones stimulate follicle growth by activating YAP1; however, the preovulatory inhibition of YAP1 activity in GCs is a prerequisite of LH actions.
PCOS is a common gynecologic and endocrine disease with multiple short and long-term consequences. Many PCOS patients suffer anovulation caused by hyperandrogenism, but its etiology remains unclear.
To study the effect of acute hyperandrogenism on ovulation, we injected pregnant mare serum gonadotrophin (PMSG)-primed (44 h) pubertal mice with dihydrotestosterone (DHT), the major biologically active form of androgen, in a superovulation assay. We investigated if YAP1 is regulated by testosterone and if it is potentially involved in follicle development and ovulation. Cultured primary GCs were subjected to Yap1 depletion by RNA interference and Yap1 overexpression by adenoviral infections.
Female mice at postnatal day (PD)-21~23 were analyzed to avoid the complexity of ovarian functions associated with estrous cycles and endogenous surges of gonadotropins. Immature mice were injected intraperitoneally with five IU PMSG to stimulate preovulatory follicle development followed 44 h later with five IU hCG to stimulate ovulation. For DHT treatments, female mice at PD23 were injected intraperitoneally with five IU PMSG followed 44 h later with five IU hCG alone (as control) or five IU hCG plus 100 μg DHT, which was dissolved in 0.1 ml DMSO. Methods of gene expression detection used include immunohistochemistry, immunofluorescence, Western blotting and quantitative PCR. More than three biological and technical replicates were included in each experiments.
we provide novel evidence in a mouse model that YAP1 is required for proliferation of ovarian GCs, but is down-regulated by LH through the extracellular-regulated kinase-1/2 (ERK1/2) cascade. Acute hyperandrogenism blocks LH actions and causes oligo-ovulation by activating YAP1.
N/A.
Results shown were obtained only in mouse, and need to be further confirmed in human samples.
These findings not only elucidated the role of YAP1 in maintaining normal ovarian functions, but also link the YAP1 deregulation to the pathogenesis of PCOS.
This study is funded by the National Key Research and Development Program of China (2016YFC1000600 and 2017YFSF1001500) and National Natural Science Foundation of China (31528016, 31371449 and 31671558). The authors have no competing interests.
Some patients with diabetic foot ulcers (DFUs) may suffer from depression, but the latest information regarding the incidence of depression in patients with DFUs is limited. This review aimed to ...provide up-to-date information concerning the incidence of depression in patients with DFUs. We searched the literature in PubMed and Web of Science databases, limited to English publications. 11 eligible studies with a total of 2117 participants were included in this review. A random-effects model was applied due to high heterogeneity. The incidence of depression in patients with DFUs ranged from 26% (95% confidence interval CI = 19% to 33%) to 85% (95% CI = 78% to 92%), and was 47% (95% CI = 36% to 58%) after systematically summarizing. Subgroup analyses suggested that the incidence of depression were 49% (95%CI = 35% to 63%) in Europe, 37% (95% CI = 23% to 51%) in Asia, 62% (95% CI = 48% to 76%) in North America. Additionally, the incidence of depression were 40% (95% CI = 29% to 50%) in prospective studies, 55% (95% CI = 28% to 82%) in retrospective studies, 40% (95% CI = 29% to 50%) in cross-sectional studies. Furthermore, the incidence of depression were 43% (95% CI = 25% to 60%), 49% (95% CI = 35% to 63%), 68% (95% CI = 35% to 102%), 32% (95% CI = 26% to 38%), and 28% (95% CI = 18% to 38%) in patients with DFUs assessed by the Hospital Anxiety and Depression Scale, EuroQol 5-Dimension Questionnaire, Geriatric Depression Scale, Diagnostic and Statistical Manual of Mental Disorders, 4th edition, and the Center for Epidemiologic Studies Depression Scale, respectively. The estimates were robust in the sensitivity analysis. According to the meta-regression analyses, diabetes mellitus duration (t = 0.93, P = .422), publication years (t = −0.72, P = .488), and age of subjects (t = 0.01, P = .989) were not the sources of high heterogeneity. Our meta-analysis showed nearly half of patients with DFUs had depression problems.
•Genistein exerts neuroprotective effects on dopaminergic neurons in LPS-induced PD rat model.•Genistein inhibits LPS-induced activation of MAPKs and NF-κB pathways and reduces neuroinflammation in ...substantia nigra.•GPER and IGF-1R signaling pathways might be involved in the anti-inflammatory and neuroprotective effects of genistein.
Neuroinflammation plays an important role in the pathogenesis of Parkinson's disease (PD). Genistein is an estrogen-like phytoestrogen that can exert biological effects via the crosstalk of estrogen receptor and insulin-like growth factor 1 receptor (IGF-1R). The present study aimed to evaluate the involvement of G protein-coupled estrogen receptor (GPER) and IGF-1R in the anti-inflammatory effects of genistein against lipopolysaccharide (LPS)-induced nigrostriatal injury in ovariectomized rats. Our results showed that genistein treatment could ameliorate the apomorphine-induced rotational behavior in LPS-induced inflammatory PD rat model. Genistein attenuated LPS-induced decrease of the contents of dopamine (DA) and its metabolites in striatum as well as the loss of tyrosine hydroxylase-immunoreactive (TH-IR) neurons in the substantia nigra (SN) of the lesioned side, which could be blocked by GPER antagonist G15 or IGF-1R antagonist JB1. Meanwhile, G15 or JB1 could attenuate the anti-inflammatory effects of genistein in LPS-induced microglial activation and production of tumor necrosis factor-α (TNF-α), interleukin 1β (IL-1β), inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Moreover, genistein could inhibit the LPS-induced phosphorylation of p38, JNK, ERK and IκB in the lesioned side of SN and these effects could also be blocked by G15 or JB1. Taken together, our data provide the first evidence that genistein can inhibit the increase of microglia and protect dopaminergic neurons at least in part via GPER and IGF-1R signaling pathways in ovariectomized PD rat model.
PDF
