Evolution, Expression Patterns, and Distribution of Novel Ribbon Worm Predatory and Defensive Toxins.

Ribbon worms are active predators that use an eversible proboscis to inject venom into their prey and defend themselves with toxic epidermal secretions. Previous work on nemertean venom has largely focused on just a few species and has not investigated the different predatory and defensive secretions in detail. Consequently, our understanding of the composition and evolution of ribbon worm venoms is still very limited. Here, we present a comparative study of nemertean venom combining RNA-seq differential gene expression analyses of venom-producing tissues, tandem mass spectrometry-based proteomics of toxic secretions, and mass spectrometry imaging of proboscis sections, to shed light onto the composition and evolution of predatory and defensive toxic secretions in Antarctonemertes valida. Our analyses reveal a wide diversity of putative defensive and predatory toxins with tissue-specific gene expression patterns and restricted distributions to the mucus and proboscis proteomes respectively, suggesting that ribbon worms produce distinct toxin cocktails for predation and defense. Our results also highlight the presence of numerous lineage-specific toxins, indicating that venom evolution is highly divergent across nemerteans, producing toxin cocktails that might be finely tuned to subdue different prey. Our data also suggest that the hoplonemertean proboscis is a highly specialized predatory organ that seems to be involved in a variety of biological functions besides predation, including secretion and sensory perception. Overall, our results advance our knowledge into the diversity and evolution of nemertean venoms and highlight the importance of combining different types of data to characterize toxin composition in understudied venomous organisms.

Proteome Analysis Reveals a Significant Host-Specific Response in Rhizobium leguminosarum bv. viciae Endosymbiotic Cells.

The Rhizobium-legume symbiosis is a beneficial interaction in which the bacterium converts atmospheric nitrogen into ammonia and delivers it to the plant in exchange for carbon compounds. This symbiosis implies the adaptation of bacteria to live inside host plant cells. In this work, we apply RP-LC-MS/MS and isobaric tags as relative and absolute quantitation techniques to study the proteomic profile of endosymbiotic cells (bacteroids) induced by Rhizobium leguminosarum bv viciae strain UPM791 in legume nodules. Nitrogenase subunits, tricarboxylic acid cycle enzymes, and stress-response proteins are among the most abundant from over 1000 rhizobial proteins identified in pea (Pisum sativum) bacteroids. Comparative analysis of bacteroids induced in pea and in lentil (Lens culinaris) nodules revealed the existence of a significant host-specific differential response affecting dozens of bacterial proteins, including stress-related proteins, transcriptional regulators, and proteins involved in the carbon and nitrogen metabolisms. A mutant affected in one of these proteins, homologous to a GntR-like transcriptional regulator, showed a symbiotic performance significantly impaired in symbiosis with pea but not with lentil plants. Analysis of the proteomes of bacteroids isolated from both hosts also revealed the presence of different sets of plant-derived nodule-specific cysteine-rich peptides, indicating that the endosymbiotic bacteria find a host-specific cocktail of chemical stressors inside the nodule. By studying variations of the bacterial response to different plant cell environments, we will be able to identify specific limitations imposed by the host that might give us clues for the improvement of rhizobial performance.

A viral RNA motif involved in signaling the initiation of translation on non-AUG codons.

Noncanonical translation, and particularly initiation on non-AUG codons, are frequently used by viral and cellular mRNAs during virus infection and disease. The Sindbis virus (SINV) subgenomic mRNA (sgRNA) constitutes a unique model system to analyze the translation of a capped viral mRNA without the participation of several initiation factors. Moreover, sgRNA can initiate translation even when the AUG initiation codon is replaced by other codons. Using SINV replicons, we examined the efficacy of different codons in place of AUG to direct the synthesis of the SINV capsid protein. The substitution of AUG by CUG was particularly efficient in promoting the incorporation of leucine or methionine in similar percentages at the amino terminus of the capsid protein. Additionally, valine could initiate translation when the AUG is replaced by GUG. The ability of sgRNA to initiate translation on non-AUG codons was dependent on the integrity of a downstream stable hairpin (DSH) structure located in the coding region. The structural requirements of this hairpin to signal the initiation site on the sgRNA were examined in detail. Of interest, a virus bearing CUG in place of AUG in the sgRNA was able to infect cells and synthesize significant amounts of capsid protein. This virus infects the human haploid cell line HAP1 and the double knockout variant that lacks eIF2A and eIF2D. Collectively, these findings indicate that leucine-tRNA or valine-tRNA can participate in the initiation of translation of sgRNA by a mechanism dependent on the DSH. This mechanism does not involve the action of eIF2, eIF2A, or eIF2D.

CD9 inhibition reveals a functional connection of extracellular vesicle secretion with mitophagy in melanoma cells.

Tetraspanins are often used as Extracellular Vesicle (EV) detection markers because of their abundance on these secreted vesicles. However, data on their function on EV biogenesis are controversial and compensatory mechanisms often occur upon gene deletion. To overcome this handicap, we have compared the effects of tetraspanin CD9 gene deletion with those elicited by cytopermeable peptides with blocking properties against tetraspanin CD9. Both CD9 peptide or gene deletion reduced the number of early endosomes. CD9 peptide induced an increase in lysosome numbers, while CD9 deletion augmented the number of MVB and EV secretion, probably because of compensatory CD63 expression upregulation. In vivo, CD9 peptide delayed primary tumour cell growth and reduced metastasis size. These effects on cell proliferation were shown to be concomitant with an impairment in mitochondrial quality control. CD9 KO cells were able to compensate the mitochondrial malfunction by increasing total mitochondrial mass reducing mitophagy. Our data thus provide the first evidence for a functional connection of tetraspanin CD9 with mitophagy in melanoma cells.

Human and Microbial Proteins From Corpora Amylacea of Alzheimer's Disease.

Corpora amylacea (CA) are spherical bodies mainly composed of polyglucans and, to a lesser extent, proteins. They are abundant in brains from patients with neurodegenerative diseases, particularly Alzheimer's disease. Although CA were discovered many years ago, their precise origin and function remain obscure. CA from the insular cortex of two Alzheimer's patients were purified and the protein composition was assessed by proteomic analysis. A number of microbial proteins were identified and fungal DNA was detected by nested PCR.A wide variety of human proteins form part of CA. In addition, we unequivocally demonstrated several fungal and bacterial proteins in purified CA. In addition to a variety of human proteins, CA also contain fungal and bacterial polypeptides.In conclusion, this paper suggests that the function of CA is to scavenge cellular debris provoked by microbial infections.

Extracellular vesicles as a source for non-invasive biomarkers in bladder cancer progression.

Bladder cancer is the second most frequent malignancy of the urinary tract after prostate cancer. Current diagnostic techniques, such as cystoscopy and biopsies are highly invasive and accompanied of undesirable side effects. Moreover, there are no suitable biomarkers for relapse or progression prognosis. We analysed whether the specific composition of microRNAs (miRNAs) and proteins of extracellular vesicles (EVs) that urothelial tumour cells of bladder mucosa release into the urine, could reflect their pathologic condition. For this purpose, urinary EVs were isolated and their protein and miRNA composition evaluated in healthy donors and low or high-grade bladder cancer patients. Using a microarray platform containing probes for 851 human miRNAs we found 26 deregulated miRNAs in high-grade bladder cancer urine EVs, from which 23 were downregulated and 3 upregulated. Real-time PCR analysis pointed to miR-375 as a biomarker for high-grade bladder cancer while miR-146a could identify low-grade patients. Finally, several protein markers were also deregulated in EVs from tumour patients. Our data suggest that the presence of ApoB in the 100,000 pellet is a clear marker for malignancy.

Evidence for fungal infection in cerebrospinal fluid and brain tissue from patients with amyotrophic lateral sclerosis.

Among neurogenerative diseases, amyotrophic lateral sclerosis (ALS) is a fatal illness characterized by a progressive motor neuron dysfunction in the motor cortex, brainstem and spinal cord. ALS is the most common form of motor neuron disease; yet, to date, the exact etiology of ALS remains unknown. In the present work, we have explored the possibility of fungal infection in cerebrospinal fluid (CSF) and in brain tissue from ALS patients. Fungal antigens, as well as DNA from several fungi, were detected in CSF from ALS patients. Additionally, examination of brain sections from the frontal cortex of ALS patients revealed the existence of immunopositive fungal antigens comprising punctate bodies in the cytoplasm of some neurons. Fungal DNA was also detected in brain tissue using PCR analysis, uncovering the presence of several fungal species. Finally, proteomic analyses of brain tissue demonstrated the occurrence of several fungal peptides. Collectively, our observations provide compelling evidence of fungal infection in the ALS patients analyzed, suggesting that this infection may play a part in the etiology of the disease or may constitute a risk factor for these patients.

Fungal infection in patients with Alzheimer's disease.

Alzheimer's disease is a progressive neurodegenerative disorder that leads to dementia mainly among the elderly. This disease is characterized by the presence in the brain of amyloid plaques and neurofibrillary tangles that provoke neuronal cell death, vascular dysfunction, and inflammatory processes. In the present work, we have analyzed the existence of fungal infection in Alzheimer's disease patients. A proteomic analysis provides compelling evidence for the existence of fungal proteins in brain samples from Alzheimer's disease patients. Furthermore, PCR analysis reveals a variety of fungal species in these samples, dependent on the patient and the tissue tested. DNA sequencing demonstrated that several fungal species can be found in brain samples. Together, these results show that fungal macromolecules can be detected in brain from Alzheimer's disease patients. To our knowledge these findings represent the first evidence that fungal infection is detectable in brain samples from Alzheimer's disease patients. The possibility that this may represent a risk factor or may contribute to the etiological cause of Alzheimer's disease is discussed.