GbEXPATR,a species‐specific expansin,enhances cotton fibre elongation through cell wall restructuring

Cotton provides us the most important natural fibre. High fibre quality is the major goal of cotton breeding, and introducing genes conferring longer, finer and stronger fibre from Gossypium barbadense to Gossypium hirsutum is an important breeding strategy. We previously analysed the G. barbadense fibre development mechanism by gene expression profiling and found two homoeologous fibre‐specific α‐expansins from G. barbadense, GbEXPA2 and GbEXPATR. GbEXPA2 (from the D_T genome) is a classical α‐expansin, while its homoeolog, GbEXPATR (A_T genome), encodes a truncated protein lacking the normal C‐terminal polysaccharide‐binding domain of other α‐expansins and is specifically expressed in G. barbadense. Silencing EXPA in G. hirsutum induced shorter fibres with thicker cell walls. GbEXPA2 overexpression in G. hirsutum had no effect on mature fibre length, but produced fibres with a slightly thicker wall and increased crystalline cellulose content. Interestingly, GbEXPATR overexpression resulted in longer, finer and stronger fibres coupled with significantly thinner cell walls. The longer and thinner fibre was associated with lower expression of a number of secondary wall‐associated genes, especially chitinase‐like genes, and walls with lower cellulose levels but higher noncellulosic polysaccharides which advocated that a delay in the transition to secondary wall synthesis might be responsible for better fibre. In conclusion, we propose that α‐expansins play a critical role in fibre development by loosening the cell wall; furthermore, a truncated form, GbEXPATR, has a more dramatic effect through reorganizing secondary wall synthesis and metabolism and should be a candidate gene for developing G. hirsutum cultivars with superior fibre quality.     

The absence of dysferlin induces the expression of functional connexin-based hemichannels in human myotubes

Background

Mutations in the gene encoding for dysferlin cause recessive autosomal muscular dystrophies called dysferlinopathies. These mutations induce several alterations in skeletal muscles, including, inflammation, increased membrane permeability and cell death. Despite the fact that the etiology of dysferlinopathies is known, the mechanism that explains the aforementioned alterations is still elusive. Therefore, we have now evaluated the potential involvement of connexin based hemichannels in the pathophysiology of dysferlinopathies.

Results

Human deltoid muscle biopsies of 5 Chilean dysferlinopathy patients exhibited the presence of muscular connexins (Cx40.1, Cx43 and Cx45). The presence of these connexins was also observed in human myotubes derived from immortalized myoblasts derived from other patients with mutated forms of dysferlin. In addition to the aforementioned connexins, these myotubes expressed functional connexin based hemichannels, evaluated by ethidium uptake assays, as opposed to myotubes obtained from a normal human muscle cell line, RCMH. This response was reproduced in a knock-down model of dysferlin, by treating RCMH cell line with small hairpin RNA specific for dysferlin (RCMH-sh Dysferlin). Also, the presence of P2X₇ receptor and the transient receptor potential channel, TRPV2, another Ca²⁺ permeable channels, was detected in the myotubes expressing mutated dysferlin, and an elevated resting intracellular Ca²⁺ level was found in the latter myotubes, which was in turn reduced to control levels in the presence of the molecule D4, a selective Cx HCs inhibitor.

Conclusions

The data suggests that dysferlin deficiency, caused by mutation or downregulation of dysferlin, promotes the expression of Cx HCs. Then, the de novo expression Cx HC causes a dysregulation of intracellular free Ca²⁺ levels, which could underlie muscular damage associated to dysferlin mutations. This mechanism could constitute a potential therapeutical target in dysferlinopathies.

     

Identifying and annotating human bifunctional RNAs reveals their versatile functions

Bifunctional RNAs that possess both protein-coding and noncoding functional properties were less explored and poorly understood. Here we systematically explored the characteristics and functions of such human bifunctional RNAs by integrating tandem mass spectrometry and RNA-seq data. We first constructed a pipeline to identify and annotate bifunctional RNAs, leading to the characterization of 132 high-confidence bifunctional RNAs. Our analyses indicate that bifunctional RNAs may be involved in human embryonic development and can be functional in diverse tissues. Moreover, bifunctional RNAs could interact with multiple miRNAs and RNA-binding proteins to exert their corresponding roles. Bifunctional RNAs may also function as competing endogenous RNAs to regulate the expression of many genes by competing for common targeting miRNAs. Finally, somatic mutations of diverse carcinomas may generate harmful effect on corresponding bifunctional RNAs. Collectively, our study not only provides the pipeline for identifying and annotating bifunctional RNAs but also reveals their important gene-regulatory functions.     

Potential use of high levels of vegetal proteins in diets for market-sized gilthead sea bream (Sparus aurata)

The effect of partial or total dietary substitution of fishmeal (FM) by vegetal protein sources on growth and feed efficiency was carried out in on-growing gilthead sea bream (mean initial weight 131 g). The Control diet (FM 100) contained FM as the primary protein source, while in Diets FM 25 and FM 0 the FM protein was replaced at 75% and 100%, respectively, by a vegetable protein mixture consisting of wheat gluten, soybean meal, rapeseed meal and crystalline amino acids. Diets FM 25 and FM 0 also contained krill meal at 47 g/kg in order to improve palatability. At the end of the trial (after 158 d), fish survival was above 90%. Final weight and the specific growth rate were statistically lower in fish fed the Control diet (361 g and 0.64%/d), compared with 390–396 g and 0.69–0.70%/d after feeding vegetal diets. No significant differences were found regarding feed intake and feed conversion ratio. The digestibility of protein and amino acids (determined with chromium oxide as indicator) was similar in all diets. The blood parameters were not significantly affected by treatments. The activity of trypsin and pepsin was significantly reduced after feeding Diet FM 0. In the distal intestine, the villi length in fish fed Diet FM 25 was significantly longer and the intestine of the fish fed the FM 100 diet showed a smaller number of goblet cells. In conclusion, a total FM substitution by a vegetal mix supplemented with synthetic amino acids in on-growing sea bream is feasible.     

Dysfunctional mitochondrial fission impairs cell reprogramming

We have recently shown that mitochondrial fission is induced early in reprogramming in a Drp1-dependent manner; however, the identity of the factors controlling Drp1 recruitment to mitochondria was unexplored. To investigate this, we used a panel of RNAi targeting factors involved in the regulation of mitochondrial dynamics and we observed that MiD51, Gdap1 and, to a lesser extent, Mff were found to play key roles in this process. Cells derived from Gdap1-null mice were used to further explore the role of this factor in cell reprogramming. Microarray data revealed a prominent down-regulation of cell cycle pathways in Gdap1-null cells early in reprogramming and cell cycle profiling uncovered a G2/M growth arrest in Gdap1-null cells undergoing reprogramming. High-Content analysis showed that this growth arrest was DNA damage-independent. We propose that lack of efficient mitochondrial fission impairs cell reprogramming by interfering with cell cycle progression in a DNA damage-independent manner.     

Focal adhesion kinase: predictor of tumour response and risk factor for recurrence after neoadjuvant chemoradiation in rectal cancer

Rectal cancer represents about 30% of colorectal cancers, being around 50% locally advanced at presentation. Chemoradiation (CRT) followed by total mesorectal excision is the standard of care for these locally advanced stages. However, it is not free of adverse effects and toxicity and the complete pathologic response rate is between 10% and 30%. This makes it extremely important to define factors that can predict response to this therapy. Focal adhesion kinase (FAK) expression has been correlated with worse prognosis in several tumours and its possible involvement in cancer radio‐ and chemosensitivity has been suggested; however, its role in rectal cancer has not been analysed yet. To analyse the association of FAK expression with tumour response to CRT in locally advanced rectal cancer. This study includes 73 patients with locally advanced rectal cancer receiving standard neoadjuvant CRT followed by total mesorectal excision. Focal adhesion kinase protein levels were immunohistochemically analysed in the pre‐treatment biopsies of these patients and correlated with tumour response to CRT and patients survival. Low FAK expression was significantly correlated with local and distant recurrence (P = 0.013). Low FAK expression was found to be a predictive marker of tumour response to neoadjuvant therapy (P = 0.007) and patients whose tumours did not express FAK showed a strong association with lower disease‐free survival (P = 0.01). Focal adhesion kinase expression predicts neoadjuvant CRT response in rectal cancer patients and it is a clinically relevant risk factor for local and distant recurrence.