N-Myristoyltransferase Inhibition causes Mitochondrial Iron Overload and Parthanatos in TIM17A-dependent Aggressive Lung Carcinoma.

Myristoylation is a type of protein acylation by which the fatty acid myristate is added to the N-terminus of target proteins, a process mediated by N-myristoyltransferases. Myristoylation is emerging as a promising cancer therapeutic target, however the molecular determinants of sensitivity to N-myristoyltransferase inhibition or the mechanism by which it induces cancer cell death are not completely understood. We report that N-myristoyltransferases are a novel therapeutic target in lung carcinoma cells with LKB1 and/or KEAP1 mutations in a KRAS mutant background. Inhibition of myristoylation decreases cell viability in vitro and tumor growth in vivo. Inhibition of myristoylation causes mitochondrial ferrous iron overload, oxidative stress, elevated protein poly (ADP)-ribosylation and death by parthanatos. Furthermore, NMT inhibitors sensitized lung carcinoma cells to platinum-based chemotherapy. Unexpectedly, the mitochondrial transporter Translocase of Inner Mitochondrial Membrane 17 homologue A (TIM17A) is a critical target of myristoylation inhibitors in these cells. TIM17A silencing recapitulated the effects of NMT inhibition at inducing mitochondrial ferrous iron overload and parthanatos. Furthermore, sensitivity of lung carcinoma cells to myristoylation inhibition correlated with their dependency on TIM17A. This study reveals the unexpected connection between protein myristoylation, the mitochondrial import machinery, and iron homeostasis. It also uncovers myristoylation inhibitors as novel inducers of parthanatos in cancer, and the novel axis N-myristoyltransferase-TIM17A as a potential therapeutic target in highly aggressive lung carcinomas.

Analysis of Golgi Morphology Using Immunofluorescence and CellProfiler Software.

The architecture of the Golgi apparatus in mammalian cells changes dynamically in response to internal and external cues and may be permanently altered in disease states. Here, we present a method to quantify changes in Golgi morphology using immunofluorescence and confocal microscopy followed by CellProfiler software analysis. This method will assist researchers in evaluating alterations in the Golgi complex morphology of cultured cells under a variety of different experimental conditions.

Second-language phoneme learning positively relates to voice recognition abilities in the native language: Evidence from behavior and brain potentials.

Previous studies suggest a relationship between second-language learning and voice recognition processes, but the nature of such relation remains poorly understood. The present study investigates whether phoneme learning relates to voice recognition. A group of bilinguals that varied in their discrimination of a second-language phoneme contrast participated in this study. We assessed participants' voice recognition skills in their native language at the behavioral and brain electrophysiological levels during a voice-avatar learning paradigm. Second-language phoneme discrimination positively correlated with behavioral and brain measures of voice recognition. At the electrophysiological level, correlations were present at two time windows and are interpreted within the dual-process model of recognition memory. The results are relevant to understanding the processes involved in language learning as they show a common variability for second-language phoneme and voice recognition processes.

Adaptation of the Golgi Apparatus in Cancer Cell Invasion and Metastasis.

The Golgi apparatus plays a central role in normal cell physiology by promoting cell survival, facilitating proliferation, and enabling cell-cell communication and migration. These roles are partially mediated by well-known Golgi functions, including post-translational modifications, lipid biosynthesis, intracellular trafficking, and protein secretion. In addition, accumulating evidence indicates that the Golgi plays a critical role in sensing and integrating external and internal cues to promote cellular homeostasis. Indeed, the unique structure of the mammalian Golgi can be fine-tuned to adapt different Golgi functions to specific cellular needs. This is particularly relevant in the context of cancer, where unrestrained proliferation and aberrant survival and migration increase the demands in Golgi functions, as well as the need for Golgi-dependent sensing and adaptation to intrinsic and extrinsic stressors. Here, we review and discuss current understanding of how the structure and function of the Golgi apparatus is influenced by oncogenic transformation, and how this adaptation may facilitate cancer cell invasion and metastasis.

Pancreatic Adenocarcinoma Invasiveness and the Tumor Microenvironment: From Biology to Clinical Trials.

Pancreatic adenocarcinoma (PDAC) originates in the glandular compartment of the exocrine pancreas. Histologically, PDAC tumors are characterized by a parenchyma that is embedded in a particularly prominent stromal component or desmoplastic stroma. The unique characteristics of the desmoplastic stroma shape the microenvironment of PDAC and modulate the reciprocal interactions between cancer and stromal cells in ways that have profound effects in the pathophysiology and treatment of this disease. Here, we review some of the most recent findings regarding the regulation of PDAC cell invasion by the unique microenvironment of this tumor, and how new knowledge is being translated into novel therapeutic approaches.

N-myristoyltransferase-1 is necessary for lysosomal degradation and mTORC1 activation in cancer cells.

N-myristoyltransferase-1 (NMT1) catalyzes protein myristoylation, a lipid modification that is elevated in cancer cells. NMT1 sustains proliferation and/or survival of cancer cells through mechanisms that are not completely understood. We used genetic and pharmacological inhibition of NMT1 to further dissect the role of this enzyme in cancer, and found an unexpected essential role for NMT1 at promoting lysosomal metabolic functions. Lysosomes mediate enzymatic degradation of vesicle cargo, and also serve as functional platforms for mTORC1 activation. We show that NMT1 is required for both lysosomal functions in cancer cells. Inhibition of NMT1 impaired lysosomal degradation leading to autophagy flux blockade, and simultaneously caused the dissociation of mTOR from the surface of lysosomes leading to decreased mTORC1 activation. The regulation of lysosomal metabolic functions by NMT1 was largely mediated through the lysosomal adaptor LAMTOR1. Accordingly, genetic targeting of LAMTOR1 recapitulated most of the lysosomal defects of targeting NMT1, including defective lysosomal degradation. Pharmacological inhibition of NMT1 reduced tumor growth, and tumors from treated animals had increased apoptosis and displayed markers of lysosomal dysfunction. Our findings suggest that compounds targeting NMT1 may have therapeutic benefit in cancer by preventing mTORC1 activation and simultaneously blocking lysosomal degradation, leading to cancer cell death.

Identification of invadopodia by TKS5 staining in human cancer lines and patient tumor samples.

Invadopodia, cancer cell protrusive structures with associated proteolytic activity, provide cancer cells with the ability to remodel the extracellular matrix. Invadopodia facilitate invasive migration and their formation correlates with cancer cell invasiveness and metastatic potential. The unambiguous identification of invadopodia is an important step to undergo studies on invadopodia regulatory inputs, functional outputs, as well as the prevalence and significance of invadopodia for cancer cells and human tumors. The adaptor protein TKS5 is a known invadopodia regulatory protein, which is necessary for invadopodia formation and activity. TKS5 is highly enriched at invadopodia and, unlike other commonly used invadopodia markers, it does not accumulate significantly in other types of cellular protrusions. However, the use of TKS5 as a marker of invadopodia has not been generalized, in part due to the availability of suitable antibodies against the human protein. We have evaluated two commercial antibodies raised against human TKS5. Here, we detail protocols for the detection of invadopodia-associated TKS5 in human cells in culture and in paraffin-embedded archived tumor surgical specimens using commercial antibodies. These methods should facilitate the identification and study of human invadopodia. •TKS5 staining identifies invadopodia in human cancer cell lines and archived surgical tumor specimens.

Reduced Structural Connectivity Between Left Auditory Thalamus and the Motion-Sensitive Planum Temporale in Developmental Dyslexia.

Developmental dyslexia is characterized by the inability to acquire typical reading and writing skills. Dyslexia has been frequently linked to cerebral cortex alterations; however, recent evidence also points toward sensory thalamus dysfunctions: dyslexics showed reduced responses in the left auditory thalamus (medial geniculate body, MGB) during speech processing in contrast to neurotypical readers. In addition, in the visual modality, dyslexics have reduced structural connectivity between the left visual thalamus (lateral geniculate nucleus, LGN) and V5/MT, a cerebral cortex region involved in visual movement processing. Higher LGN-V5/MT connectivity in dyslexics was associated with the faster rapid naming of letters and numbers (RANln), a measure that is highly correlated with reading proficiency. Here, we tested two hypotheses that were directly derived from these previous findings. First, we tested the hypothesis that dyslexics have reduced structural connectivity between the left MGB and the auditory-motion-sensitive part of the left planum temporale (mPT). Second, we hypothesized that the amount of left mPT-MGB connectivity correlates with dyslexics RANln scores. Using diffusion tensor imaging-based probabilistic tracking, we show that male adults with developmental dyslexia have reduced structural connectivity between the left MGB and the left mPT, confirming the first hypothesis. Stronger left mPT-MGB connectivity was not associated with faster RANln scores in dyslexics, but was in neurotypical readers. Our findings provide the first evidence that reduced cortico-thalamic connectivity in the auditory modality is a feature of developmental dyslexia and it may also affect reading-related cognitive abilities in neurotypical readers.SIGNIFICANCE STATEMENT Developmental dyslexia is one of the most widespread learning disabilities. Although previous neuroimaging research mainly focused on pathomechanisms of dyslexia at the cerebral cortex level, several lines of evidence suggest an atypical functioning of subcortical sensory structures. By means of diffusion tensor imaging, we here show that dyslexic male adults have reduced white matter connectivity in a cortico-thalamic auditory pathway between the left auditory motion-sensitive planum temporale and the left medial geniculate body. Connectivity strength of this pathway was associated with measures of reading fluency in neurotypical readers. This is novel evidence on the neurocognitive correlates of reading proficiency, highlighting the importance of cortico-subcortical interactions between regions involved in the processing of spectrotemporally complex sound.

TKS5-positive invadopodia-like structures in human tumor surgical specimens.

Invadopodia, cancer cell protrusions with proteolytic activity, are functionally associated with active remodeling of the extracellular matrix. Here, we show that the invadopodia-related protein TKS5 is expressed in human pancreatic adenocarcinoma lines, and demonstrate that pancreatic cancer cells depend on TKS5 for invadopodia formation and function. Immunofluorescence staining of human pancreatic cancer cells reveals that TKS5 is a marker of mature and immature invadopodia. We also analyze the co-staining patterns of TKS5 and the commonly used invadopodia marker Cortactin, and find only partial co-localization of these two proteins at invadopodia, with a large fraction of TKS5-positive invadopodia lacking detectable levels of Cortactin. Whereas compelling evidence exist on the role of invadopodia as mediators of invasive migration in cultured cells and in animal models of cancer, these structures have never been detected inside human tumors. Here, using antibodies against TKS5 and Cortactin, we describe for the first time structures strongly resembling invadopodia in various paraffin-embedded human tumor surgical specimens from pancreas and other organs. Our results strongly suggest that invadopodia are present inside human tumors, and warrants further investigation on their regulation and occurrence in surgical specimens, and on the value of TKS5 antibodies as pathological research and diagnostic tools.

Experimental supporting data on TKS5 and Cortactin expression and localization in human pancreatic cancer cells and tumors.

In this article, using human pancreatic cancer cell lines and tumor specimens, we analyze the expression and localization of the invadopodia-related proteins TKS5 and Cortactin. Specifically, we present data on: a) TKS5 expression and localization by immunofluorescence in human pancreatic tumors, b) Cortactin expression by western blotting in various human pancreatic adenocarcinoma cell lines, c) TKS5 and Cortactin localization at invadopodia in BxPC-3 pancreatic adenocarcinoma cells, and d) TKS5 and Cortactin localization by co-immunofluorescence in human pancreatic cancer specimens. Data presented here is related to and supportive of the research article by Chen et al., "TKS5-positive invadopodia-like structures in human tumor surgical specimens" (Chen et al., 2019), where interpretation of the research data presented here is available.

Task-dependent modulation of the visual sensory thalamus assists visual-speech recognition.

The cerebral cortex modulates early sensory processing via feed-back connections to sensory pathway nuclei. The functions of this top-down modulation for human behavior are poorly understood. Here, we show that top-down modulation of the visual sensory thalamus (the lateral geniculate body, LGN) is involved in visual-speech recognition. In two independent functional magnetic resonance imaging (fMRI) studies, LGN response increased when participants processed fast-varying features of articulatory movements required for visual-speech recognition, as compared to temporally more stable features required for face identification with the same stimulus material. The LGN response during the visual-speech task correlated positively with the visual-speech recognition scores across participants. In addition, the task-dependent modulation was present for speech movements and did not occur for control conditions involving non-speech biological movements. In face-to-face communication, visual speech recognition is used to enhance or even enable understanding what is said. Speech recognition is commonly explained in frameworks focusing on cerebral cortex areas. Our findings suggest that task-dependent modulation at subcortical sensory stages has an important role for communication: Together with similar findings in the auditory modality the findings imply that task-dependent modulation of the sensory thalami is a general mechanism to optimize speech recognition.

The role of Tks adaptor proteins in invadopodia formation, growth and metastasis of melanoma.

Metastatic cancer cells are characterized by their ability to degrade and invade through extracellular matrix. We previously showed that the Tks adaptor proteins, Tks4 and Tks5, are required for invadopodia formation and/or function in Src-transformed fibroblasts and a number of human cancer cell types. In this study, we investigated the role of Tks adaptor proteins in melanoma cell invasion and metastasis. Knockdown of either Tks4 or Tks5 in both mouse and human melanoma cell lines resulted in a decreased ability to form invadopodia and degrade extracellular matrix. In addition, Tks-knockdown melanoma cells had decreased proliferation in a 3-dimensional type l collagen matrix, but not in 2-dimensional culture conditions. We also investigated the role of Tks proteins in melanoma progression in vivo using xenografts and experimental metastasis assays. Consistent with our in vitro results, reduction of Tks proteins markedly reduced subcutaneous melanoma growth as well as metastatic growth in the lung. We explored the clinical relevance of Tks protein expression in human melanoma specimens using a tissue microarray. Compared to non-malignant nevi, both Tks proteins were highly expressed in melanoma tissues. Moreover, metastatic melanoma cases showed higher expression of Tks5 than primary melanoma cases. Taken together, these findings suggest the importance of Tks adaptor proteins in melanoma growth and metastasis in vivo, likely via functional invadopodia formation.

Tris DBA palladium is highly effective against growth and metastasis of pancreatic cancer in an orthotopic model.

Pancreatic carcinoma ranks among the most lethal of human cancers. Besides late detection, other factors contribute to its lethality, including a high degree of chemoresistance, invasion, and distant metastases. Currently, the mainstay of therapy involves resection of local disease in a minority of patients (Whipple procedure) and systemic gemcitabine. While systemic chemotherapy has some benefit, even with optimal treatment, the five year survival after diagnosis is dismal. Thus, treatment of pancreatic carcinoma remains a tremendous unmet need.The organometallic compound tris DBA palladium is a potent inhibitor of N-myristoyltransferase 1 (NMT1), an enzyme that catalyzes the transfer of myristate to protein substrates. This compound is highly effective in vivo against murine models of melanoma with both mutant and wild type b-RAF genotypes. Based upon the signaling similarities between melanoma and pancreatic carcinoma, we evaluated the efficacy of tris DBA palladium in vitro and in vivo against pancreatic carcinoma. We found that tris DBA palladium decreased proliferation and colony formation of pancreatic cancer cells in vitro. In an orthotopic mouse model, tris DBA palladium was highly active in inhibiting growth, ascites production, and distant metastases in vivo. Furthermore, tris DBA palladium impaired chemotaxis and inhibited cilia formation in Pan02 cells in a NMT1-dependent manner. We propose that NMT1 is a novel regulator of cilia formation and tris DBA palladium a novel inhibitor of cilia formation and metastasis in pancreatic cancer. Thus, further evaluation of tris DBA palladium for the treatment of pancreatic cancer is warranted.

Electrophysiological Correlates of Second-Language Syntactic Processes Are Related to Native and Second Language Distance Regardless of Age of Acquisition.

In the present study, we investigate how early and late L2 learners process L2 grammatical traits that are either present or absent in their native language (L1). Thirteen early (AoA = 4 years old) and 13 late (AoA = 18 years old) Spanish learners of Basque performed a grammatical judgment task on auditory Basque sentences while their event-related brain potentials (ERPs) were recorded. The sentences contained violations of a syntactic property specific to participants' L2, i.e., ergative case, or violations of a syntactic property present in both of the participants' languages, i.e., verb agreement. Two forms of verb agreement were tested: subject agreement, found in participants' L1 and L2, and object agreement, present only in participants' L2. Behaviorally, early bilinguals were more accurate in the judgment task than late L2 learners. Early bilinguals showed native-like ERPs for verb agreement, which differed from the late learners' ERP pattern. Nonetheless, approximation to native-likeness was greater for the subject-verb agreement processing, the type of verb-agreement present in participants' L1, compared to object-verb agreement, the type of verb-agreement present only in participants' L2. For the ergative argument alignment, unique to L2, the two non-native groups showed similar ERP patterns which did not correspond to the natives' ERP pattern. We conclude that non-native syntactic processing approximates native processing for early L2 acquisition and high proficiency levels when the syntactic property is common to the L1 and L2. However, syntactic traits that are not present in the L1 do not rely on native-like processing, despite early AoA and high proficiency.

[RIGHT VARICOCELE AS FINDING OF RIGHT RENAL MASS]. / VARICOCELE DERECHO COMO PRESENTACIÓN DE MASA RENAL DERECHA.

Abdominal and scrotal ultrasounds were requested and, in order of the findings watched they were complemented with an abdominal contrast enhanced CT scan (CECT). The CETC demonstrated a large right renal tumor sized 12 cm located in the upper pole of the right kidney, in contact with hepatic parenchyma (Figure 1). Renal vein and artery were not affected. Important peritumoral collateral circulation was noticed. Consequently to the large tumor size and extrinsic compression, there was dilatation of right spermatic vein. Both Collateral circulation and right spermatic dilated vein converged in the inguinal duct explaining the presence of right varicocele. The image was 3D reconstructed.

The invadopodia scaffold protein Tks5 is required for the growth of human breast cancer cells in vitro and in vivo.

The ability of cancer cells to invade underlies metastatic progression. One mechanism by which cancer cells can become invasive is through the formation of structures called invadopodia, which are dynamic, actin-rich membrane protrusions that are sites of focal extracellular matrix degradation. While there is a growing consensus that invadopodia are instrumental in tumor metastasis, less is known about whether they are involved in tumor growth, particularly in vivo. The adaptor protein Tks5 is an obligate component of invadopodia, and is linked molecularly to both actin-remodeling proteins and pericellular proteases. Tks5 appears to localize exclusively to invadopodia in cancer cells, and in vitro studies have demonstrated its critical requirement for the invasive nature of these cells, making it an ideal surrogate to investigate the role of invadopodia in vivo. In this study, we examined how Tks5 contributes to human breast cancer progression. We used immunohistochemistry and RNA sequencing data to evaluate Tks5 expression in clinical samples, and we characterized the role of Tks5 in breast cancer progression using RNA interference and orthotopic implantation in SCID-Beige mice. We found that Tks5 is expressed to high levels in approximately 50% of primary invasive breast cancers. Furthermore, high expression was correlated with poor outcome, particularly in those patients with late relapse of stage I/II disease. Knockdown of Tks5 expression in breast cancer cells resulted in decreased growth, both in 3D in vitro cultures and in vivo. Moreover, our data also suggest that Tks5 is important for the integrity and permeability of the tumor vasculature. Together, this work establishes an important role for Tks5 in tumor growth in vivo, and suggests that invadopodia may play broad roles in tumor progression.

Spanish consensus on premature menopause.

INTRODUCTION: While we recognise that the term premature menopause is more accepted by most non-specialist health care providers and by the general population, 'primary ovarian insufficiency' (POI) is currently considered the most apposite term to explain the loss of ovarian function, because it better explains the variability of the clinical picture, does not specify definitive failure, and highlights the specific ovarian source. Its pathogenesis involves a congenital reduction in the number of primordial follicles, poor follicle recruitment, or accelerated follicular apoptosis. However, its cause is unknown in most cases. AIM: This guide analyses the factors associated with the diagnosis and treatment of POI and provides recommendations on the most appropriate diagnostic and therapeutic measures for women under 40 years of age who experience POI. METHODOLOGY: A panel of experts from various Spanish scientific societies related to POI (Spanish Menopause Society, Spanish Fertility Society, and Spanish Contraception Society) met to reach a consensus on these issues. RESULTS: Hormonal therapy (HT) is considered the treatment of choice to alleviate the symptoms of hypoestrogenism and to prevent long-term consequences. We suggest that HT should be continued until at least age 51, the average age at natural menopause. The best treatment to achieve pregnancy is oocyte/embryo donation. If a patient is to undergo treatment that will reduce her fertility, she should be informed of this issue and the available techniques to preserve ovarian function, mainly vitrification of oocytes.

Genetic disruption of the sh3pxd2a gene reveals an essential role in mouse development and the existence of a novel isoform of tks5.

Tks5 is a scaffold protein and Src substrate involved in cell migration and matrix degradation through its essential role in invadosome formation and function. We have previously described that Tks5 is fundamental for zebrafish neural crest cell migration in vivo. In the present study, we sought to investigate the function of Tks5 in mammalian development by analyzing mice mutant for sh3pxd2a, the gene encoding Tks5. Homozygous disruption of the sh3pxd2a gene by gene-trapping in mouse resulted in neonatal death and the presence of a complete cleft of the secondary palate. Interestingly, embryonic fibroblasts from homozygous gene-trap sh3pxd2a mice lacked only the highest molecular weight band of the characteristic Tks5 triplet observed in protein extracts, leaving the lower molecular weight bands unaffected. This finding, together with the existence of two human Expressed Sequence Tags lacking the first 5 exons of SH3PXD2A, made us hypothesize about the presence of a second alternative transcription start site located in intron V. We performed 5'RACE on mouse fibroblasts and isolated a new transcript of the sh3pxd2a gene encoding a novel Tks5 isoform, that we named Tks5ß. This novel isoform diverges from the long form of Tks5 in that it lacks the PX-domain, which confers affinity for phosphatidylinositol-3,4-bisphosphate. Instead, Tks5ß has a short unique amino terminal sequence encoded by the newly discovered exon 6ß; this exon includes a start codon located 29 bp from the 5'-end of exon 6. Tks5ß mRNA is expressed in MEFs and all mouse adult tissues analyzed. Tks5ß is a substrate for the Src tyrosine kinase and its expression is regulated through the proteasome degradation pathway. Together, these findings indicate the essentiality of the larger Tks5 isoform for correct mammalian development and the transcriptional complexity of the sh3pxd2a gene.

Oscillation encoding of individual differences in speech perception.

Individual differences in second language (L2) phoneme perception (within the normal population) have been related to speech perception abilities, also observed in the native language, in studies assessing the electrophysiological response mismatch negativity (MMN). Here, we investigate the brain oscillatory dynamics in the theta band, the spectral correlate of the MMN, that underpin success in phoneme learning. Using previous data obtained in an MMN paradigm, the dynamics of cortical oscillations while perceiving native and unknown phonemes and nonlinguistic stimuli were studied in two groups of participants classified as good and poor perceivers (GPs and PPs), according to their L2 phoneme discrimination abilities. The results showed that for GPs, as compared to PPs, processing of a native phoneme change produced a significant increase in theta power. Stimulus time-locked analysis event-related spectral perturbation (ERSP) showed differences for the theta band within the MMN time window (between 70 and 240 ms) for the native deviant phoneme. No other significant difference between the two groups was observed for the other phoneme or nonlinguistic stimuli. The dynamic patterns in the theta-band may reflect early automatic change detection for familiar speech sounds in the brain. The behavioral differences between the two groups may reflect individual variations in activating brain circuits at a perceptual level.

The impact of hypoxia in pancreatic cancer invasion and metastasis.

Intratumoral hypoxia is a common feature of solid tumors. Recent advances in cancer biology indicate that hypoxia is not only a consequence of unrestrained tumor growth, but also plays an active role in promoting tumor progression, malignancy, and resistance to therapy. Hypoxia signaling is mediated by the hypoxia-inducible factors (HIFs), which are not only stabilized under hypoxia, but also by activated oncogenes or inactivated tumor suppressors under normoxia. Hypoxia is a prominent feature of the tumor microenvironment of pancreatic tumors, also characterized by the presence of a fibrotic reaction that promotes, and is also modulated by, hypoxia. As the mechanisms by which hypoxia signaling impacts invasion and metastasis in pancreatic cancer are being elucidated, hypoxia is emerging as a key determinant of pancreatic cancer malignancy as well as an important target for therapy. Herein we present an overview of recent advances in the understanding of the impact that hypoxia has in pancreatic cancer invasion and metastasis.