Accumulation of poly(A) RNA in nuclear granules enriched in Sam68 in motor neurons from the SMNA7 mouse model of SMA

Spinal muscular atrophy (SMA) is a severe motor neuron (MN) disease caused by the deletion or mutation of the survival motor neuron 1 (SMN1) gene, which results in reduced levels of the SMN protein and the selective degeneration of lower MNs. The best-known function of SMN is the biogenesis of spliceosomal snRNPs, the major components of the pre-mRNA splicing machinery. Therefore, SMN deficiency in SMA leads to widespread splicing abnormalities. We used the SMN?7 mouse model of SMA to investigate the cellular reorganization of polyadenylated mRNAs associated with the splicing dysfunction in MNs. We demonstrate that SMN deficiency induced the abnormal nuclear accumulation in euchromatin domains of poly(A) RNA granules (PARGs) enriched in the splicing regulator Sam68. However, these granules lacked other RNA-binding proteins, such as TDP43, PABPN1, hnRNPA12B, REF and Y14, which are essential for mRNA processing and nuclear export. These effects were accompanied by changes in the alternative splicing of the Sam68-dependent Bcl-x and Nrnx1 genes, as well as changes in the relative accumulation of the intron-containing Chat, Chodl, Myh9 and Myh14 mRNAs, which are all important for MN functions. PARG-containing MNs were observed at presymptomatic SMA stage, increasing their number during the symptomatic stage. Moreover, the massive accumulations of poly(A) RNA granules in MNs was accompanied by the cytoplasmic depletion of polyadenylated mRNAs for their translation. We suggest that the SMN-dependent abnormal accumulation of polyadenylated mRNAs and Sam68 in PARGs reflects a severe dysfunction of both mRNA processing and translation, which could contribute to SMA pathogenesis.This work was supported by grants from: “Dirección General de Investigación” of Spain (BFU2014-54754-P and SAF2015-70801-R, cofinanced by FEDER) and “Instituto de Investigación Marqués de Valdecilla-IDIVAL (NVAL17/22). Dr. Tapia is the recipient of a grant from SMA Europe and FundAME (Spain)

Mechanism of Splicing Regulation of Spinal Muscular Atrophy Genes

Spinal muscular atrophy (SMA) is one of the major genetic disorders associated with infant mortality. More than 90% cases of SMA result from deletions or mutations of Survival Motor Neuron 1 (SMN1) gene. SMN2, a nearly identical copy of SMN1, does not compensate for the loss of SMN1due to predominant skipping of exon 7. However, correction of SMN2 exon 7 splicing has proven to confer therapeutic benefits in SMA patients. The only approved drug for SMA is an antisense oligonucleotide (Spinraza™/Nusinersen), which corrects SMN2 exon 7 splicing by blocking intronic splicing silencer N1 (ISS-N1) located immediately downstream of exon 7. ISS-N1 is a complex regulatory element encompassing overlapping negative motifs and sequestering a cryptic splice site. More than 40 protein factors have been implicated in the regulation of SMN exon 7 splicing. There is evidence to support that multiple exons of SMN are alternatively spliced during oxidative stress, which is associated with a growing number of pathological conditions. Here, we provide the most up to date account of the mechanism of splicing regulation of the SMN genes

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

In vitro fermentation of forage prickly pear cactus with yeast inoculum of Kluyveromyces lactis from apple waste [Fermentación in vitro de nopal forrajero con un ináculo de levadura Kluyveromyces lactis obtenida a partir de manzana de desecho]

The aim of this study was to evaluate the effect of an inoculum of yeast Kluyveromyces lactis, from apple waste on the variety Golden Delicious in solid state fermentation (SSF) of forage prickly pear cactus Opuntia spp. During the fermentation period, 3 samples were taken per treatment at different times (0, 6, 12, 24 and 48 h), which was determined by temperature, pH, ammonia nitrogen, lactic acid, yeast count, soluble sugars and crude protein. The data were evaluated with the procedure Proc Mixed for a random design of 4 treatments with 2 2 factorial arrangements. The temperature was increased (P<0.01) of the h0 to h12 in all treatments. The ammonia nitrogen increased (P<0.01) differences between inoculums by time interaction. The lactic acid showed difference (P<0.01) between treatments with calcium interaction between inoculum inoculum by time. Yeast counts showed a treatment effect (P<0.01) by inoculum interaction time, the highest concentration of yeasts was observed from the increase of h0 8.3 10 6 0.22 to 1.8 107 0.22 cel/mL h48 t1 to the witness, and 3.2 10 7 0.22 to 4.9 10 7 0.22 h0 to h48 cel/mL treatment with inoculum. Soluble sugars in effect was observed (P<0.01) by time. Crude protein (CP) treatment effect was found (P <0.01) in the use of inoculum and inoculum interaction time. It can be conclude, that the use of yeast inoculum in the prickly pear cactus SSF subject to significantly increase crude protein value of 9.35 to 19.36% PC in 12 hours of fermentation, being an excellent alternative for use in animal feeding

In vitro fermentation of forage prickly pear cactus with yeast inoculum of Kluyveromyces lactis from apple waste [Fermentación in vitro de nopal forrajero con un inóculo de levadura Kluyveromyces lactis obtenida a partir de manzana de desecho]

The aim of this study was to evaluate the effect of an inoculum of yeast Kluyveromyces lactis, from apple waste on the variety Golden Delicious in solid state fermentation (SSF) of forage prickly pear cactus Opuntia spp. During the fermentation period, 3 samples were taken per treatment at different times (0, 6, 12, 24 and 48 h), which was determined by temperature, pH, ammonia nitrogen, lactic acid, yeast count, soluble sugars and crude protein. The data were evaluated with the procedure Proc Mixed for a random design of 4 treatments with 2×2 factorial arrangements. The temperature was increased (P<0.01) of the h0 to h12 in all treatments. The ammonia nitrogen increased (P<0.01) differences between inoculums by time interaction. The lactic acid showed difference (P<0.01) between treatments with calcium interaction between inoculum inoculum by time. Yeast counts showed a treatment effect (P<0.01) by inoculum interaction time, the highest concentration of yeasts was observed from the increase of h0 8.3×10 6±0.22 to 1.8×107±0.22 cel/mL h48 t1 to the witness, and 3.2×10 7±0.22 to ×4.9×10 7±0.22 h0 to h48 cel/mL treatment with inoculum. Soluble sugars in effect was observed (P<0.01) by time. Crude protein (CP) treatment effect was found (P <0.01) in the use of inoculum and inoculum interaction time. It can be conclude, that the use of yeast inoculum in the prickly pear cactus SSF subject to significantly increase crude protein value of 9.35 to 19.36% PC in 12 hours of fermentation, being an excellent alternative for use in animal feeding

Why autophagy is good for retinal ganglion cells?

13 p.-3 fig.Autophagy is a catabolic pathway that promotes the degradation and recycling of cellular components. Proteins, lipids, and even whole organelles are engulfed in autophagosomes and delivered to the lysosome for elimination. In response to stress, autophagy mediates the degradation of cell components, which are recycled to generate the nutrients and building blocks required to sustain cellular homeostasis. Moreover, it has an important role in cellular quality control, particularly in neurons, in which the total burden of altered proteins and damaged organelles cannot be reduced by redistribution to daughter cells through cell division. Autophagy occurs in all cells and tissues, and it is regulated by the Atg genes. The importance of this pathway has been recently recognized by the Nobel Prize in Physiology and Medicine award to Professor Yoshinori Ohsumi who was the discoverer of the first Atg genes in yeast in the 1990s. Research has only begun to examine the role of autophagy in the visual system. Both the retina and the eye are exposed to a variety of environmental insults and stressors, including genetic mutations and age-associated alterations that impair their function. Here, we review studies that have sought to explain autophagy’s importance for retinal ganglion cells, and their implications for diseases like glaucoma and optic neuropathies.Peer reviewe

Minimally invasive tumor bed implant (MITBI) and peri-operative high-dose-rate brachytherapy (PHDRBT) for accelerated minimal breast irradiation (AMBI) or anticipated boost (A-PHDRBT-boost) in breast-conserving surgery for ductal carcinoma in situ

Purpose: To evaluate our institutional experience of minimally invasive tumor bed implantation (MITBI) during breast-conserving surgery (BCS) for ductal carcinoma in situ (DCIS) to deliver peri-operative high-dose-rate brachytherapy (PHDRBT) as accelerated minimal breast irradiation (AMBI) or anticipated boost (A-PHDRBT-boost). Material and methods: Patients older than 40, with clinical and radiological unifocal DCIS < 3 cm were considered potential candidates for accelerated partial breast irradiation (APBI) and were implanted during BCS using MITBItechnique. Patients who in final pathology reports showed free margins and no other microscopic tumor foci, received AMBI with PHDRBT (3.4 Gy BID in 5 days). Patients with adverse features received A-PHDRBT-boost with post-operative external beam radiotherapy (EBRT). Results: Forty-one patients were implanted, and 36 were treated and analyzed. According to final pathology, 24 (67%) patients were suitable for AMBI and 12 (33%) were qualified for A-PHDRBT-boost. Reoperation rate for those with clear margins was 16.6% (6/36); this rate increased to 33% (4/12) for G3 histology, and 66% (4/6) were rescued using AMBI. Early complications were documented in 5 patients (14%). With a median follow-up of 97 (range, 42-138) months, 5-year rates of local, elsewhere, locoregional, and distant control were all 97.2%. 5-year ipsilateral breast tumor recurrence rates (IBTR) were 5.6% (2/36), 8.3% (2/24) for AMBI, and 0% (0/12) for A-PHDRBT-boost patients. Both instances of IBTR were confirmed G3 tumors in pre-operative biopsies; no IBTR was documented in G1-2 tumors. Cosmetic outcomes were excellent/good in 96% of AMBI vs. 67% in A-PHDRBT-boost (p = 0.034). Conclusions: The MITBI-PHDRBT program allows selection of patients with excellent prognoses (G1-2 DCIS with negative margins and no multifocality), for whom AMBI could be a good alternative with low recurrence rate, decrease of unnecessary radiation, treatment logistics improvement, and over-treatment reduction. Patients whose pre-operative biopsy showed G3 tumor, presents with inferior local control and more risk of reoperation due to positive margins