Dual-functional, receptor-targeted fluorogenic probe for in vivo imaging of extracellular protease expressions

Herein, we report a new type of in vivo fluorogenic probe that enables simultaneous and active targeting of overexpressed receptors, α(V)β(3) integrins, and extracellular proteases, matrix metalloproteinases (MMPs), in the tumor regions. This c(RGDyK)-conjugated MMP fluorogenic probe efficiently targets the tumor regions with high retention time while maintaining receptor binding affinity and substrate activity. The probe minimizes nonspecific accumulation, thus demonstrating improved tumor-to-background signal ratio (T/N) in both α(V)β(3) integrin- and MMP-overexpressing U87MG tumor-bearing mouse model. This strategy can be easily tuned for a wide array of applications targeting various receptors and extracellular proteases in vivo.     

Trypanosoma (Herpetosoma) grosi: first isolation from Chinese striped field mouse (Apodemus agrarius)

A "lewisi-like" Trypanosoma parasite was isolated from the blood of Chinese striped field mice (Apodemus agrarius) trapped in the fields in the Gannan Tibet area, Gansu province, China. The parasite was successfully cultivated in vitro in HL-1 medium supplemented 20% fetal bovine serum (FBS). Full formed spheromastigote, metacyclic trypomastigote and trypomastigote structures were all visible in films made from the culture. A nucleotide fragment of 2159-bp length was amplified from genomic DNA of the parasite using specific primers for the 18S rRNA gene of trypanosomes. The alignment indicated that this parasite had higher identities with T. (Herpetosoma) grosi (more than 99.6%) than other Herpetosoma species (less than 98.5%), which suggest that the parasite should be classified as T. (Herpetosoma) grosi. This is the first time in China that an isolation of T. (Herpetosoma) grosi is reported although several strains of T. (Herpetosoma) lewisi have been isolated from rodents of family Muridae in various provinces. Thus, it was designated as T. (Herpetosoma) grosi Cha1 and deposited in the center of parasite strain collection and preservation in our laboratory for future study. In addition, this culture method will be used to isolate, maintain and study the long-term development of this parasite in vitro.     

SSR-based linkage map of flax (<Emphasis Type="Italic">Linum usitatissimum</Emphasis> L.) and mapping of QTLs underlying fatty acid composition traits

Flax (Linum usitatissimum L.) seeds contain nearly 50% oil which is high in linolenic acid (an omega-3 fatty acid). In this study, a genetic linkage map was constructed based on 114 expressed sequence tag-derived simple sequence repeat (SSR) markers in addition to five single nucleotide polymorphism markers, five genes (fad2A, fad2B, fad3A, fad3B and dgat1) and one phenotypic trait (seed coat color), using a doubled haploid (DH) population of 78 individuals generated from a cross between SP2047 (a yellow-seeded Solin™ line with 2–4% linolenic acid) and UGG5-5 (a brown-seeded flax line with 63–66% linolenic acid). This map consists of 24 linkage groups with 113 markers spanning ~833.8 cM. Quantitative trait locus (QTL) analysis detected two major QTLs each for linoleic acid (LIO, QLio.crc-LG7, QLio.crc-LG16), linolenic acid (LIN, QLin.crc-LG7, QLin.crc-LG16) and iodine value (IOD, QIod.crc-LG7, QIod.crc-LG16), and one major QTL for palmitic acid (PAL, QPal.crc-LG9). The mutant allele of fad3A, mapped to the chromosomal segment inherited from the parent SP2047, underlies the QTL on linkage group 7 and was positively associated with high LIO content but negatively associated with LIN and IOD. This fad3A locus accounted for approximately 34, 25 and 29% of the phenotypic variation observed in this DH population for these three traits, respectively. The QTL localized on linkage group 16 explained approximately 20, 25 and 13% of the phenotypic variation for these same traits, respectively. For palmitic acid, QPal.crc-LG9 accounted for ~42% of the phenotypic variation. This first SSR-based linkage map in flax will serve as a resource for mapping additional markers, genes and traits, in map-based cloning and in marker-assisted selection.     

Multicolor combinatorial probe coding for real-time PCR

The target volume of multiplex real-time PCR assays is limited by the number of fluorescent dyes available and the number of fluorescence acquisition channels present in the PCR instrument. We hereby explored a probe labeling strategy that significantly increased the target volume of real-time PCR detection in one reaction. The labeling paradigm, termed "Multicolor Combinatorial Probe Coding" (MCPC), uses a limited number (n) of differently colored fluorophores in various combinations to label each probe, enabling one of 2(n)-1 genetic targets to be detected in one reaction. The proof-of-principle of MCPC was validated by identification of one of each possible 15 human papillomavirus types, which is the maximum target number theoretically detectable by MCPC with a 4-color channel instrument, in one reaction. MCPC was then improved from a one-primer-pair setting to a multiple-primer-pair format through Homo-Tag Assisted Non-Dimer (HAND) system to allow multiple primer pairs to be included in one reaction. This improvement was demonstrated via identification of one of the possible 10 foodborne pathogen candidates with 10 pairs of primers included in one reaction, which had limit of detection equivalent to the uniplex PCR. MCPC was further explored in detecting combined genotypes of five β-globin gene mutations where multiple targets were co-amplified. MCPC strategy could expand the scope of real-time PCR assays in applications which are unachievable by current labeling strategy.     

Long noncoding RNA lncMREF promotes myogenic differentiation and muscle regeneration by interacting with the Smarca5/p300 complex

Long noncoding RNAs (lncRNAs) play important roles in the spatial and temporal regulation of muscle development and regeneration. Nevertheless, the determination of their biological functions and mechanisms underlying muscle regeneration remains challenging. Here, we identified a lncRNA named lncMREF (lncRNA muscle regeneration enhancement factor) as a conserved positive regulator of muscle regeneration among mice, pigs and humans. Functional studies demonstrated that lncMREF, which is mainly expressed in differentiated muscle satellite cells, promotes myogenic differentiation and muscle regeneration. Mechanistically, lncMREF interacts with Smarca5 to promote chromatin accessibility when muscle satellite cells are activated and start to differentiate, thereby facilitating genomic binding of p300/CBP/H3K27ac to upregulate the expression of myogenic regulators, such as MyoD and cell differentiation. Our results unravel a novel temporal-specific epigenetic regulation during muscle regeneration and reveal that lncMREF/Smarca5-mediated epigenetic programming is responsible for muscle cell differentiation, which provides new insights into the regulatory mechanism of muscle regeneration.     

Establishment of a five‐enzalutamide‐resistance‐related‐gene‐based classifier for recurrence‐free survival predicting of prostate cancer

To identify prostate cancer (PCa) patients with a high risk of recurrence is critical before delivering adjuvant treatment. We developed a classifier based on the Enzalutamide treatment resistance‐related genes to assist the currently available staging system in predicting the recurrence‐free survival (RFS) prognosis of PCa patients. We overlapped the DEGs from two datasets to obtain a more convincing Enzalutamide‐resistance‐related‐gene (ERRG) cluster. The five‐ERRG‐based classifier obtained good predictive values in both the training and validation cohorts. The classifier precisely predicted RFS of patients in four cohorts, independent of patient age, pathological tumour stage, Gleason score and PSA levels. The classifier and the clinicopathological factors were combined to construct a nomogram, which had an increased predictive accuracy than that of each variable alone. Besides, we also compared the differences between high‐ and low‐risk subgroups and found their differences were enriched in cancer progression‐related pathways. The five‐ERRG‐based classifier is a practical and reliable predictor, which adds value to the existing staging system for predicting the RFS prognosis of PCa after radical prostatectomy, enabling physicians to make more informed treatment decisions concerning adjuvant therapy.