Heat shock protein Hspa13 regulates endoplasmic reticulum and cytosolic proteostasis through modulation of protein translocation.

Most eukaryotic secretory proteins are cotranslationally translocated through Sec61 into the endoplasmic reticulum (ER). Because these proteins have evolved to fold in the ER, their mistargeting is associated with toxicity. Genetic experiments have implicated the ER heat shock protein 70 (Hsp70) Hspa13/STCH as involved in processing of nascent secretory proteins. Herein, we evaluate the role of Hspa13 in protein import and the maintenance of cellular proteostasis in human cells, primarily using the human embryonic kidney 293T cell line. We find that Hspa13 interacts primarily with the Sec61 translocon and its associated factors. Hspa13 overexpression inhibits translocation of the secreted protein transthyretin, leading to accumulation and aggregation of immature transthyretin in the cytosol. ATPase-inactive mutants of Hspa13 further inhibit translocation and maturation of secretory proteins. While Hspa13 overexpression inhibits cell growth and ER quality control, we demonstrate that HSPA13 knockout destabilizes proteostasis and increases sensitivity to ER disruption. Thus, we propose that Hspa13 regulates import through the translocon to maintain both ER and cytosolic protein homeostasis. The raw mass spectrometry data associated with this article have been deposited in the PRIDE archive and can be accessed at PXD033498.

Monitoring Protein Import into the Endoplasmic Reticulum in Living Cells with Proximity Labeling.

The proper trafficking of eukaryotic proteins is essential to cellular function. Genetic, environmental, and other stresses can induce protein mistargeting and, in turn, threaten cellular protein homeostasis. Current methods for measuring protein mistargeting are difficult to translate to living cells, and thus the role of cellular signaling networks in stress-dependent protein mistargeting processes, such as ER pre-emptive quality control (ER pQC), is difficult to parse. Herein, we use genetically encoded peroxidases to characterize protein import into the endoplasmic reticulum (ER). We show that the ERHRP/cytAPEX pair provides good selectivity and sensitivity for both multiplexed protein labeling and for identifying protein mistargeting, using the known ER pQC substrate transthyretin (TTR). Although ERHRP labeling induces formation of detergent-resistant TTR aggregates, this is minimized by using low ERHRP expression, without loss of labeling efficiency. cytAPEX labeling recovers TTR that is mistargeted as a consequence of Sec61 inhibition or ER stress-induced ER pQC. Furthermore, we discover that stress-free activation of the ER stress-associated transcription factor ATF6 recapitulates the TTR import deficiency of ER pQC. Hence, proximity labeling is an effective strategy for characterizing factors that influence ER protein import in living cells.

Efficacy comparison of intraperitoneal anaesthesia and post-operative analgesia regimens in laboratory rats.

INTRODUCTION: Effective and safe anesthesia for rodents has long been a leading concern among biomedical researchers. Intraperitoneal injection constitutes an alternative to inhalant anesthesia. PURPOSE: The aim of this study was to identify a safe, reliable, and effective anesthesia and postoperative analgesia protocol for laboratory rats exposed to painful procedures. MATERIAL AND METHODS: Twenty-seven female Wistar rats in an ongoing study that required surgery were randomized into groups for three different intraperitoneal anesthesia protocols and three different analgesia regimens. The anesthesia groups were (1) medetomidine + ketamine (MK), (2) ketamine + xylacine (KX), and (3) fentanyl + medetomidine (FM). Three analgesia groups were equally distributed among the anesthesia groups: (1) local mepivacaine + oral ibuprofen (MI), (2) oral tramadol + oral ibuprofen (TI), and (3) local tramadol + oral tramadol + + oral ibuprofen (TTI). A core was assigned to measure anesthesia (0-3) and analgesia (0-2) effectiveness; the lower the score, the more effective the treatment. RESULTS: The mean MK score was 0.44 versus 2.00 for FM and 2.33 for KX. Mean score for analgesia on the first postoperative day was TTI (4.66) TI (9.13), and MI (10.14). Mean score 48 hours after surgery was TTI (3.4), TI (6.71), and MI (9.5). These differences were statistically significant. CONCLUSION: MK was shown to be a reliable, safe, and effective method of anesthesia. The TTI analgesia regimen is strongly recommended in light of these results.

Stem cell-like transcriptional reprogramming mediates metastatic resistance to mTOR inhibition.

Inhibitors of the mechanistic target of rapamycin (mTOR) are currently used to treat advanced metastatic breast cancer. However, whether an aggressive phenotype is sustained through adaptation or resistance to mTOR inhibition remains unknown. Here, complementary studies in human tumors, cancer models and cell lines reveal transcriptional reprogramming that supports metastasis in response to mTOR inhibition. This cancer feature is driven by EVI1 and SOX9. EVI1 functionally cooperates with and positively regulates SOX9, and promotes the transcriptional upregulation of key mTOR pathway components (REHB and RAPTOR) and of lung metastasis mediators (FSCN1 and SPARC). The expression of EVI1 and SOX9 is associated with stem cell-like and metastasis signatures, and their depletion impairs the metastatic potential of breast cancer cells. These results establish the mechanistic link between resistance to mTOR inhibition and cancer metastatic potential, thus enhancing our understanding of mTOR targeting failure.

The acetyltransferase Tip60 contributes to mammary tumorigenesis by modulating DNA repair.

The acetyltransferase Tip60/Kat5 acetylates both histone and non-histone proteins, and is involved in a variety of biological processes. By acetylating p53, Tip60 controls p53-dependent transcriptional activity and so is implicated as a tumor suppressor. However, many breast cancers with low Tip60 also show p53 mutation, implying that Tip60 has a tumor suppressor function independent of its acetylation of p53. Here, we show in a p53-null mouse model of sporadic invasive breast adenocarcinoma that heterozygosity for Tip60 deletion promotes mammary tumorigenesis. Low Tip60 reduces DNA repair in normal and tumor mammary epithelial cells, both under resting conditions and following genotoxic stress. We demonstrate that Tip60 controls homologous recombination (HR)-directed DNA repair, and that Tip60 levels correlate inversely with a gene expression signature associated with defective HR-directed DNA repair. In human breast cancer data sets, Tip60 mRNA is downregulated, with low Tip60 levels correlating with p53 mutations in basal-like breast cancers. Our findings indicate that Tip60 is a novel breast tumor suppressor gene whose loss results in genomic instability leading to cancer formation.

Reproductive characteristics of a captive colony of big-eared climbing rats (Ototylomys phyllotis).

The authors analyzed the breeding characteristics of a colony of Ototylomys phyllotis (big-eared climbing rat) from Campeche, México, that was bred in captivity for 6 y. The big-eared climbing rat is a reservoir of Leishmania (Leishmania) mexicana, a causal agent of localized cutaneous leishmaniasis on the Yucatán Peninsula. The colony had been established to facilitate studies analyzing the effectiveness of O. phyllotis as an experimental model for L. (L.) mexicana. The authors describe the housing and husbandry of the colony, the procedures used for mating the animals and the behavior of the animals during mating. They report that the animals showed social behavior and could be bred successfully. Most breeding pairs successfully produced litters; some pairs produced more than one litter. The authors also report data for other parameters, such as the interval between pairing and birth or between births of consecutive litters, litter size, survival to weaning, the timing of sexual maturity and the effects of breeder age on breeding success.

Solución del caso 24. Isquemia aguda de miembros inferiores secundaria a intoxicación crónica por ingesta de ergotamínicos / No disponible

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Changes in ochratoxin A and type B trichothecenes contained in wheat flour during dough fermentation and bread-baking.

Ochratoxin A (OTA) and type B trichothecenes are mycotoxins that occur frequently in cereals and thus can be found in cereal by-products such as bread. The aim of this work was to study the variation of the levels of OTA, deoxynivalenol (DON), 3-acetyldeoxynivalenol (3-ADON) and nivalenol (NIV) during the bread-making process. This was done by using wheat flour spiked with different levels of toxins. Mycotoxin levels were controlled after fermentation of the dough with yeasts (Saccharomyces cerevisiae) and after further baking at different temperature-time combinations. Analysis of variance (ANOVA) of the results showed a significant reduction in OTA level (p < 0.05) during fermentation of the dough. The reduction ranged between 29.8% and 33.5%, depending on the initial concentration of toxin in the flour. During this period, the level of the other mycotoxins studied was not modified. By contrast, in the baking phase there were significant changes in the levels of the four mycotoxins, although the reduction was similar under all the baking conditions. Considering all the temperature-time conditions tested, it can be concluded that during the baking period the average reduction of OTA, NIV, 3-ADON, and DON was 32.9%, 76.9%, 65.6%, and 47.9%, respectively.

[Hereditary and acquired angioedema: clinical characteristics in 8 patients and review of the literature]. / Angioedema hereditario y adquirido: características clínicas de ocho pacientes y revisión de la literatura.

C1 inhibitor disorders are a group of rare conditions in which the C1 inhibitor is deficient or defective. We present the clinical characteristics of 8 patients and a review of the literature. These are characterized by recurrent episodes of angioedema, which most often affect the skin or mucosal tissues of the upper respiratory and gastrointestinal tract. Laryngeal involvement may cause fatal asphyxiation. These disorders may be divided into two broad categories: hereditary angioedema (HAE) and acquired C1 inhibitor disorders. Indications for screening for HAE include: recurrent angioedema, episodic abdominal pain, laryngeal, a family background of angioedema, and a low C4 level. Acquired C1 inhibitor disorders are similar, but lack a family background. Treatment is divided into short and long-term prophylaxis with androgens, antifibrinolytics and C1 inhibitor replacement. First line therapy of acute attacks is C1 inhibitor.

Predictive assessment of ochratoxin A accumulation in grape juice based-medium by Aspergillus carbonarius using neural networks.

AIMS: To study the ability of multi-layer perceptron artificial neural networks (MLP-ANN) and radial-basis function networks (RBFNs) to predict ochratoxin A (OTA) concentration over time in grape-based cultures of Aspergillus carbonarius under different conditions of temperature, water activity (a(w)) and sub-inhibitory doses of the fungicide carbendazim. METHODS AND RESULTS: A strain of A. carbonarius was cultured in a red grape juice-based medium. The input variables to the network were temperature (20-28 degrees C), a(w) (0.94-0.98), carbendazim level (0-450 ng ml(-1)) and time (3-15 days after the lag phase). The output of the ANNs was OTA level determined by liquid chromatography. Three algorithms were comparatively tested for MLP. The lowest error was obtained by MLP without validation. Performance decreased when hold-out validation was accomplished but the risk of over-fitting is also lower. The best MLP architecture was determined. RBFNs provided similar performances but a substantially higher number of hidden nodes were needed. CONCLUSIONS: ANNs are useful to predict OTA level in grape juice cultures of A. carbonarius over a range of a(w), temperature and carbendazim doses. SIGNIFICANCE AND IMPACT OF THE STUDY: This is a pioneering study on the application of ANNs to forecast OTA accumulation in food based substrates. These models can be similarly applied to other mycotoxins and fungal species.

Degradation of cyclin A is regulated by acetylation.

Cyclin A accumulates at the onset of S phase, remains high during G(2) and early mitosis and is degraded at prometaphase. Here, we report that the acetyltransferase P/CAF directly interacts with cyclin A that as a consequence becomes acetylated at lysines 54, 68, 95 and 112. Maximal acetylation occurs simultaneously to ubiquitylation at mitosis, indicating importance of acetylation on cyclin A stability. This was further confirmed by the observation that the pseudoacetylated cyclin A mutant can be ubiquitylated whereas the nonacetylatable mutant cannot. The nonacetylatable mutant is more stable than cyclin A WT (cycA WT) and arrests cell cycle at mitosis. Moreover, in cells treated with histone deacetylase inhibitors cyclin A acetylation increases and its stability decreases, thus supporting the function of acetylation on cyclin A degradation. Although the nonacetylatable mutant cannot be ubiquitylated, it interacts with the proteins needed for its degradation (cdks, Cks, Cdc20, Cdh1 and APC/C). In fact, its association with cdks is increased and its complexes with these kinases display higher activity than control cycA WT-cdk complexes. All these results indicate that cyclin A acetylation at specific lysines is crucial for cyclin A stability and also has a function in the regulation of cycA-cdk activity.

Genome sequence and comparative genome analysis of Lactobacillus casei: insights into their niche-associated evolution.

Lactobacillus casei is remarkably adaptable to diverse habitats and widely used in the food industry. To reveal the genomic features that contribute to its broad ecological adaptability and examine the evolution of the species, the genome sequence of L. casei ATCC 334 is analyzed and compared with other sequenced lactobacilli. This analysis reveals that ATCC 334 contains a high number of coding sequences involved in carbohydrate utilization and transcriptional regulation, reflecting its requirement for dealing with diverse environmental conditions. A comparison of the genome sequences of ATCC 334 to L. casei BL23 reveals 12 and 19 genomic islands, respectively. For a broader assessment of the genetic variability within L. casei, gene content of 21 L. casei strains isolated from various habitats (cheeses, n = 7; plant materials, n = 8; and human sources, n = 6) was examined by comparative genome hybridization with an ATCC 334-based microarray. This analysis resulted in identification of 25 hypervariable regions. One of these regions contains an overrepresentation of genes involved in carbohydrate utilization and transcriptional regulation and was thus proposed as a lifestyle adaptation island. Differences in L. casei genome inventory reveal both gene gain and gene decay. Gene gain, via acquisition of genomic islands, likely confers a fitness benefit in specific habitats. Gene decay, that is, loss of unnecessary ancestral traits, is observed in the cheese isolates and likely results in enhanced fitness in the dairy niche. This study gives the first picture of the stable versus variable regions in L. casei and provides valuable insights into evolution, lifestyle adaptation, and metabolic diversity of L. casei.