A new xanthene‐based two‐photon fluorescent probe for the imaging of 1,4‐dithiothreitol (DTT) in living cells

1,4‐Dithiothreitol (DTT) has wide applications in cell biology and biochemistry. Development of effective methods for monitoring DTT in biological systems is important for the safe handling and study of toxicity to humans. Herein, we describe a two‐photon fluorescence probe (Rh‐DTT) to detect DTT in living systems for the first time. Rh‐DTT showed high selectivity and sensitivity to DTT. Rh‐DTT can be successfully used for the two‐photon imaging of DTT in living cells, and also can detect DTT in living tissues and mice.     

The effect of 7,8,4´‐trihydroxyflavone on tyrosinase activity and conformation: Spectroscopy and docking studies

Tyrosinase is a ubiquitous enzyme that plays an essential role in the production of melanin. Effective inhibitors of tyrosinase have extensive applications in the medical, cosmetic and food industries. In this study, a combination of enzyme kinetics, ultraviolet (UV)‐visible absorption, fluorescence spectroscopic techniques and a computational simulation method was used to characterize the inhibitory mechanism of 7,8,4´‐trihydroxyflavone on tyrosinase. 7,8,4´‐Trihydroxyflavone was found to strongly inhibit the oxidation of l ‐DOPA by tyrosinase with an IC₅₀ value of 10.31 ± 0.41 μM. The inhibitory mechanism was determined to be reversible and non‐competitive with a K_i of 9.50 ± 0.40 μM. The UV absorption spectra showed that 7,8,4´‐trihydroxyflavone could chelate with copper ions and form a complex with tyrosinase. The intrinsic fluorescence of tyrosinase was quenched by 7,8,4´‐trihydroxyflavone through a static quenching mechanism. 7,8,4´‐Trihydroxyflavone was found to occupy a single binding site with a binding constant of 7.50 ± 1.20 × 10⁴ M^?1 at 298 K. The conformation of tyrosinase changed, and the microenvironment became more hydrophilic after 7,8,4´‐trihydroxyflavone binding. Thermodynamics parameters indicated that the binding was a spontaneous process and involved hydrogen bonds and van der Waals forces. The binding distance was evaluated to be 4.54 ± 0.05 nm. Docking simulation analysis further authenticated that 7,8,4´‐trihydroxyflavone could form hydrogen bonds with the residues His244 and Met280 within the tyrosinase active site. Our results will contribute to further understanding of the inhibitory mechanisms of 7,8,4´‐trihydroxyflavone against tyrosinase and will facilitate future screening for tyrosinase inhibitors.     

Identification of a specific molecular marker for the rice blast-resistant gene <Emphasis Type="Italic">Pigm</Emphasis> and molecular breeding of thermo-sensitive genic male sterile leaf-color marker lines

Rice blast is a damaging disease caused by Magnaportheoryzae. Marker-assisted selection of blast resistance genes could help develop cultivars with blast resistance. Pigm is a broad-spectrum blast-resistant gene. However, few rice resources contain Pigm. In this study, the Pigm gene donor Gumei4 (GM4) was investigated. By analyzing different regions of Pigm sequences, we found that marker G8900 was a specific molecular marker of Pigm gene in GM4. Correlation analysis between molecular marker detection and identification of rice blast disease nursery revealed that G8900 could be used in marker-assisted selection (MAS) of Pigm. Furthermore, we introduced Pigm gene into the KT27S line (a blast-susceptible yellow-green-leaf-color mutant) in G8900-assisted breeding and identified three new yellow-green-leaf-color marker lines that are resistant to blast. The agronomic and economic traits of the three new lines are similar to those of their parental lines. The identification and application of Pigm-specific molecular marker in breeding of yellow-green-leaf-color marker line could play an important role in the production of disease-resistant hybrid rice.     

Identifying sequence features that drive ribosomal association for lncRNA

Background

With the increasing number of annotated long noncoding RNAs (lncRNAs) from the genome, researchers are continually updating their understanding of lncRNAs. Recently, thousands of lncRNAs have been reported to be associated with ribosomes in mammals. However, their biological functions or mechanisms are still unclear.

Results

In this study, we tried to investigate the sequence features involved in the ribosomal association of lncRNA. We have extracted ninety-nine sequence features corresponding to different biological mechanisms (i.e., RNA splicing, putative ORF, k-mer frequency, RNA modification, RNA secondary structure, and repeat element). An \(\mathcal {L}1\)-regularized logistic regression model was applied to screen these features. Finally, we obtained fifteen and nine important features for the ribosomal association of human and mouse lncRNAs, respectively.

Conclusion

To our knowledge, this is the first study to characterize ribosome-associated lncRNAs and ribosome-free lncRNAs from the perspective of sequence features. These sequence features that were identified in this study may shed light on the biological mechanism of the ribosomal association and provide important clues for functional analysis of lncRNAs.

     

Vaginal microbiome variances in sample groups categorized by clinical criteria of bacterial vaginosis

Background

One of the most common and recurrent vaginal infections is bacterial vaginosis (BV). The diagnosis is based on changes to the “normal” vaginal microbiome; however, the normal microbiome appears to differ according to reproductive status and ethnicity, and even among individuals within these groups. The Amsel criteria and Nugent score test are widely used for diagnosing BV; however, these tests are based on different criteria, and so may indicate distinct changes in the vaginal microbial community. Nevertheless, few studies have compared the results of these test against metagenomics analysis.

Methods

Vaginal flora samples from 77 participants were classified according to the Amsel criteria and Nugent score test. The microbiota composition was analyzed using 16S ribosome RNA gene amplicon sequencing. Bioinformatics analysis and multivariate statistical analysis were used to evaluate the microbial diversity and function.

Results

Only 3 % of the participants diagnosed BV negative using the Amsel criteria (A−) were BV-positive according to the Nugent score test (N+), while over half of the BV-positive patients using the Amsel criteria (A+) were BV-negative according to the Nugent score test (N−). Thirteen genera showed significant differences in distribution among BV status defined by BV tests (e.g., A − N−, A + N− and A + N+). Variations in the four most abundant taxa, Lactobacillus, Gardnerella, Prevotella, and Escherichia, were responsible for most of this dissimilarity. Furthermore, vaginal microbial diversity differed significantly among the three groups classified by the Nugent score test (N−, N+, and intermediate flora), but not between the Amsel criteria groups. Numerous predictive microbial functions, such as bacterial chemotaxis and bacterial invasion of epithelial cells, differed significantly among multiple BV test, but not between the A− and A+ groups.

Conclusions

Metagenomics analysis can greatly expand our current understanding of vaginal microbial diversity in health and disease. Metagenomics profiling may also provide more reliable diagnostic criteria for BV testing.

     

The InR/Akt/TORC1 Growth-Promoting Signaling Negatively Regulates JAK/STAT Activity and Migratory Cell Fate during Morphogenesis

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Negative pressure wound therapy promotes muscle‐derived stem cell osteogenic differentiation through MAPK pathway

Negative pressure wound therapy (NPWT) has been revealed to be effective in the treatment of open fractures, although the underlying mechanism is not clear. This article aimed to investigate the effects of NPWT on muscle‐derived stem cell (MDSC) osteoblastic differentiation and the related potential mechanism. The cell proliferation rate was substantially increased in NPWT‐treated MDSCs in comparison with a static group for 3 days. There was no observable effect on the apoptosis of MDSC treated with NPWT compared with the control group for 3 days. The expression levels of HIF‐1α, BMP‐2, COL‐I, OST and OPN were increased on days 3, 7 and 14, but the expression level of Runx2 was increased on days 3 and 7 in the NPWT group. Pre‐treatment, the specific inhibitors were added into the MDSCs treated with NPWT and the control group. ALP activity and mineralization were reduced by inhibiting the ERK1/2, p38 and JNK pathways. The expression levels of Runx2, COL‐I, OST and OPN genes and proteins were also decreased using the specific MAPK pathway inhibitors on days 3, 7 and 14. There were no significant effects on the expression of BMP‐2 except on day 3. However, the expressions of the HIF‐1α gene and protein slightly increased when the JNK pathway was inhibited. Therefore, NPWT promotes the proliferation and osteogenic differentiation of MDSCs through the MAPK pathway.     

Induced pluripotent stem cell‐derived conditional medium promotes Leydig cell anti‐apoptosis and proliferation via autophagy and Wnt/β‐catenin pathway

Leydig cell transplantation is a better alternative in the treatment of androgen‐deficient males. The main purpose of this study was to investigate the effects of induced pluripotent stem cell‐derived conditioned medium (iPS‐CM) on the anti‐apoptosis, proliferation and function of immature Leydig cells (ILCs), and illuminate the underlying mechanisms. ILCs were exposed to 200 μmol/L hydrogen peroxide (H₂O₂) for 24 hours with or without iPS‐CM treatments. Cell apoptosis was detected by flow cytometric analysis. Cell proliferation was assessed using cell cycle assays and EdU staining. The steroidogenic enzyme expressions were quantified with Western blotting. The results showed that iPS‐CM significantly reduced H₂O₂‐induced ILC apoptosis through down‐regulation of autophagic and apoptotic proteins LC3‐I/II, Beclin‐1, P62, P53 and BAX as well as up‐regulation of BCL‐2, which could be inhibited by LY294002 (25 μmol/L). iPS‐CM could also promote ILC proliferation through up‐regulation of β‐catenin and its target proteins cyclin D1, c‐Myc and survivin, but was inhibited by XAV939 (10 μmol/L). The level of bFGF in iPS‐CM was higher than that of DMEM‐LG. Exogenous bFGF (20 ng/mL) or Wnt signalling agonist lithium chloride (LiCl) (20 mmol/L) added into DMEM‐LG could achieve the similar effects of iPS‐CM. Meanwhile, iPS‐CM could improve the medium testosterone levels and up‐regulation of LHCGR, SCARB1, STAR, CYP11A1, HSD3B1, CYP17A1, HSD17B3 and SF‐1 in H₂O₂‐induced ILCs. In conclusion, iPS‐CM could reduce H₂O₂‐induced ILC apoptosis through the activation of autophagy, promote proliferation through up‐regulation of Wnt/β‐catenin pathway and enhance testosterone production through increasing steroidogenic enzyme expressions, which might be used in regenerative medicine for future.     

Complete genome sequencing of exopolysaccharide-producing Lactobacillus plantarum K25 provides genetic evidence for the probiotic functionality and cold endurance capacity of the strain

Lactobacillus plantarum (L. plantarum) K25 is a probiotic strain isolated from Tibetan kefir. Previous studies showed that this exopolysaccharide (EPS)-producing strain was antimicrobial active and cold tolerant. These functional traits were evidenced by complete genome sequencing of strain K25 with a circular 3,175,846-bp chromosome and six circular plasmids, encoding 3365 CDSs, 16 rRNA genes and 70 tRNA genes. Genomic analysis of L. plantarum K25 illustrates that this strain contains the previous reported mechanisms of probiotic functionality and cold tolerance, involving plantaricins, lysozyme, bile salt hydrolase, chaperone proteins, osmoprotectant, oxidoreductase, EPSs and terpenes. Interestingly, strain K25 harbors more genes that function in defense mechanisms, and lipid transport and metabolism, in comparison with other L. plantarum strains reported. The present study demonstrates the comprehensive analysis of genes related to probiotic functionalities of an EPS-producing L. plantarum strain based on whole genome sequencing.     

UCH-L1 Inhibition Suppresses tau Aggresome Formation during Proteasomal Impairment

In conditions of proteasomal impairment, the damaged or misfolded proteins, collectively known as aggresome, can accumulate in the perinuclear space and be subsequently eliminated by autophagy. Abnormal aggregation of microtubule-associated protein tau in the cytoplasm is a common neuropathological feature of tauopathies. The deficiency in ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), a proteasomal deubiquitinating enzyme, is closely related to tau aggregation; however, the associated mechanisms remain unclear. Here, we showed that UCH-L1 inhibition interrupts proteasomal impairment-induced tau aggresome formation. By reducing the production of lysine (K63)-linked ubiquitin chains, UCH-L1 inhibition decreases HDAC6 deacetylase activity and attenuates the interaction of HDAC6 and tau protein, finally leading to tau aggresome formation impairment. All these results indicated that UCH-L1 plays a key role in the process of tau aggresome formation by regulating HDAC6 deacetylase activity and implied that UCH-L1 may act as a signaling molecule to coordinate the effects of the ubiquitin-proteasome system and the autophagy-lysosome pathway, which mediate protein aggregates degradation in the cytoplasm.