Inka2 expression in smooth muscle cells and its involvement in cell migration.

The cell motility of smooth muscle cells (SMCs) is essential for vascular and internal organ development and tissue regeneration in response to damage. Cell migration requires dynamic changes in the actin-cytoskeleton via the p-21 activated kinase (Pak)-Cofilin signaling cascade, which is the central axis of the actin filaments. We previously identified that the Inka2 gene was preferentially expressed in the central nervous system (CNS) and revealed that Inka2 directly binds Pak4 to suppress its kinase activity, thereby regulating actin de-polymerization in dendritic spine formation of the forebrain neurons. However, its physiological significance outside the CNS remains unclear. Here we determined the Inka2 expression profile in various organs using in situ hybridization analysis and lacZ staining on Inka2flox/+ mice. Robust Inka2 expression was consistently detected in the SMCs of many peripheral organs, including the arteries, esophagus, stomach, intestine, and bladder. The scratch assay was used on primary cultured SMCs and revealed that Inka2-/- SMC exhibits accelerated cell migration ability without a change in the cell proliferation rate. Inka2-/- SMCs displayed Cofilin activation/phosphorylation, a downstream molecule of Pak4 signal cascade. These results suggest that Inka2 regulates SMC motility through modulating actin reorganization as the endogenous inhibitor of Pak4.

Control of cell migration by the novel protein phosphatase-2A interacting protein inka2.

Cell migration is essential for many physiological and pathological processes, including embryonic development, wound healing, immune response and cancer metastasis. Inka2 transcripts are observed in migrating cells during embryonic development, suggesting the involvement of inka2 in cell migration. However, its precise role remains unclear. Here, we found that inka2 controlled focal adhesion dynamics and cell migration, likely by regulating protein phosphatase-2A (PP2A) function. A scratch assay revealed that inka2 shRNA-transfected NIH3T3 cells showed rapid wound closure, indicating an inhibitory effect by inka2 on cell migration. Live-cell imaging of NIH3T3 cells expressing EGFP-paxillin using total internal reflection fluorescence microscopy revealed that inka2 knockdown increased the turnover rate of focal adhesions. Given that PP2A, which consists of catalytic (C), regulatory (B) and scaffolding (A) subunits, is known to regulate focal adhesions, we examined the inka2-PP2A interaction. Immunoprecipitation revealed an association between inka2 and the PP2A C subunit. Binding of Inka2 to the C subunit prevented the association between the A and C subunits, suggesting that inka2 can inhibit PP2A function. Furthermore, both inka2 expression and PP2A inhibition decreased focal adhesion kinase-paxillin interaction, resulting in reduced formation of focal adhesions. We assessed the effect of pharmacological PP2A inhibition on the inka2 knockdown-induced increase in cell migration speed and found that treatment with a PP2A inhibitor negated the accelerated migration of inka2 knockdown cells. These results suggest that inka2 knockdown exerts its effects through PP2A-dependent regulation of focal adhesions. Our findings contribute to a better understanding of the molecular mechanisms underlying cell migration.

Secretory competence in a gateway endocrine cell conferred by the nuclear receptor ßFTZ-F1 enables stage-specific ecdysone responses throughout development in Drosophila.

Hormone-induced changes in gene expression initiate periodic molts and metamorphosis during insect development. Successful execution of these developmental steps depends upon successive phases of rising and falling 20-hydroxyecdysone (20E) levels, leading to a cascade of nuclear receptor-driven transcriptional activity that enables stage- and tissue-specific responses to the steroid. Among the cellular processes associated with declining steroids is acquisition of secretory competence in endocrine Inka cells, the source of ecdysis triggering hormones (ETHs). We show here that Inka cell secretory competence is conferred by the orphan nuclear receptor ßFTZ-F1. Selective RNA silencing of ßftz-f1 in Inka cells prevents ETH release, causing developmental arrest at all stages. Affected larvae display buttoned-up, the ETH-null phenotype characterized by double mouthparts, absence of ecdysis behaviors, and failure to shed the old cuticle. During the mid-prepupal period, individuals fail to translocate the air bubble, execute head eversion and elongate incipient wings and legs. Those that escape to the adult stage are defective in wing expansion and cuticle sclerotization. Failure to release ETH in ßftz-f1 silenced animals is indicated by persistent ETH immunoreactivity in Inka cells. Arrested larvae are rescued by precisely-timed ETH injection or Inka cell-targeted ßFTZ-F1 expression. Moreover, premature ßftz-f1 expression in these cells also results in developmental arrest. The Inka cell therefore functions as a "gateway cell", whose secretion of ETH serves as a key downstream physiological output enabling stage-specific responses to 20E that are required to advance through critical developmental steps. This secretory function depends on transient and precisely timed ßFTZ-F1 expression late in the molt as steroids decline.

The Intricate Role of Ecdysis Triggering Hormone Signaling in Insect Development and Reproductive Regulation.

Insect growth is interrupted by molts, during which the insect develops a new exoskeleton. The exoskeleton confers protection and undergoes shedding between each developmental stage through an evolutionarily conserved and ordered sequence of behaviors, collectively referred to as ecdysis. Ecdysis is triggered by Ecdysis triggering hormone (ETH) synthesized and secreted from peripheral Inka cells on the tracheal surface and plays a vital role in the orchestration of ecdysis in insects and possibly in other arthropod species. ETH synthesized by Inka cells then binds to ETH receptor (ETHR) present on the peptidergic neurons in the central nervous system (CNS) to facilitate synthesis of various other neuropeptides involved in ecdysis. The mechanism of ETH function on ecdysis has been well investigated in holometabolous insects such as moths Manduca sexta and Bombyx mori, fruit fly Drosophila melanogaster, the yellow fever mosquito Aedes aegypti and beetle Tribolium castaneum etc. In contrast, very little information is available about the role of ETH in sequential and gradual growth and developmental changes associated with ecdysis in hemimetabolous insects. Recent studies have identified ETH precursors and characterized functional and biochemical features of ETH and ETHR in a hemimetabolous insect, desert locust, Schistocerca gregaria. Recently, the role of ETH in Juvenile hormone (JH) mediated courtship short-term memory (STM) retention and long-term courtship memory regulation and retention have also been investigated in adult male Drosophila. Our review provides a novel synthesis of ETH signaling cascades and responses in various insects triggering diverse functions in adults and juvenile insects including their development and reproductive regulation and might allow researchers to develop sustainable pest management strategies by identifying novel compounds and targets.

inka1b expression in the head mesoderm is dispensable for facial cartilage development.

Inka box actin regulator 1 (Inka1) is a novel protein identified in Xenopus and is found in vertebrates. While Inka1 is required for facial skeletal development in Xenopus and zebrafish, it is dispensable in mice despite its conserved expression in the cranial neural crest, indicating that Inka1 function in facial skeletal development is not conserved among vertebrates. Zebrafish bears two paralogs of inka1 (inka1a and inka1b) in the genome, with the biological roles of inka1b barely known. Here, we analyzed the expression and function of inka1b during facial skeletal development in zebrafish. inka1b was expressed sequentially in the head mesoderm adjacent to the pharyngeal pouches essential for facial skeletal development at the stage of arch segmentation. However, a loss-of-function mutation in inka1b displayed normal head development, including the pouches and facial cartilages. The normal head of inka1b mutant fish was unlikely a result of the genetic redundancy of inka1b with inka1a, given the distinct expression of inka1a and inka1b in the cranial neural crest and head mesoderm, respectively, during craniofacial development. Our findings suggest that the inka1b expression in the head mesoderm might not be essential for head development in zebrafish.

A Multidisciplinary Review of the Inka Imperial Resettlement Policy and Implications for Future Investigations.

The rulers of the Inka empire conquered approximately 2 million km2 of the South American Andes in just under 100 years from 1438-1533 CE. Inside the empire, the elite conducted a systematic resettlement of the many Indigenous peoples in the Andes that had been rapidly colonised. The nature of this resettlement phenomenon is recorded within the Spanish colonial ethnohistorical record. Here we have broadly characterised the resettlement policy, despite the often incomplete and conflicting details in the descriptions. We then review research from multiple disciplines that investigate the empirical reality of the Inka resettlement policy, including stable isotope analysis, intentional cranial deformation morphology, ceramic artefact chemical analyses and genetics. Further, we discuss the benefits and limitations of each discipline for investigating the resettlement policy and emphasise their collective value in an interdisciplinary characterisation of the resettlement policy.

The influence of delay in mononuclear cell isolation on acute myeloid leukemia phosphorylation profiles.

Mass-spectrometry (MS) based phosphoproteomics is increasingly used to explore aberrant cellular signaling and kinase driver activity, aiming to improve kinase inhibitor (KI) treatment selection in malignancies. Phosphorylation is a dynamic, highly regulated post-translational modification that may be affected by variation in pre-analytical sample handling, hampering the translational value of phosphoproteomics-based analyses. Here, we investigate the effect of delay in mononuclear cell isolation on acute myeloid leukemia (AML) phosphorylation profiles. We performed MS on immuno-precipitated phosphotyrosine (pY)-containing peptides isolated from AML samples after seven pre-defined delays before sample processing (direct processing, thirty minutes, one hour, two hours, three hours, four hours and 24 h delay). Up to four hours, pY phosphoproteomics profiles show limited variation. However, in samples processed with a delay of 24 h, we observed significant change in these phosphorylation profiles, with differential phosphorylation of 22 pY phosphopeptides (p < 0.01). This includes increased phosphorylation of pY phosphopeptides of JNK and p38 kinases indicative of stress response activation. Based on these results, we conclude that processing of AML samples should be standardized at all times and should occur within four hours after sample collection. SIGNIFICANCE: Our study provides a practical time-frame in which fresh peripheral blood samples from acute myeloid patients should be processed for phosphoproteomics, in order to warrant correct interpretation of in vivo biology. We show that up to four hours of delayed processing after sample collection, pY phosphoproteomic profiles remain stable. Extended delays are associated with perturbation of phosphorylation profiles. After a delay of 24 h, JNK activation loop phosphorylation is markedly increased and may serve as a biomarker for delayed processing. These findings are relevant for biomedical acute myeloid leukemia research, as phosphoproteomic techniques are of particular interest to investigate aberrant kinase signaling in relation to disease emergence and kinase inhibitor response. With these data, we aim to contribute to reproducible research with meaningful outcomes.

Feasibility of phosphoproteomics to uncover oncogenic signalling in secreted extracellular vesicles using glioblastoma-EGFRVIII cells as a model.

Cancer cells secrete extracellular vesicles (EVs) that contain molecular information, including proteins and RNA. Oncogenic signalling can be transferred via the cargo of EVs to recipient cells and may influence the behaviour of neighbouring cells or cells at a distance. This cargo may contain cancer drivers, such as EGFR, and also phosphorylated (activated) components of oncogenic signalling cascades. Till date, the cancer EV phosphoproteome has not been studied in great detail. In the present study, we used U87 and U87EGFRvIII cells as a model to explore EV oncogenic signalling components in comparison to the cellular profile. EVs were isolated using the VN96 ME-kit and subjected to LC-MS/MS based phosphoproteomics and dedicated bioinformatics. Expression of (phosphorylated)-EGFR was highly increased in EGFRvIII overexpressing cells and their secreted EVs. The increased phosphorylated proteins in both cells and EVs were associated with activated components of the EGFR-signalling cascade and included EGFR, AKT2, MAPK8, SMG1, MAP3K7, DYRK1A, RPS6KA3 and PAK4 kinases. In conclusion, EVs harbour oncogenic signalling networks including multiple activated kinases including EGFR, AKT and mTOR. SIGNIFICANCE: Extracellular vesicles (EVs) are biomarker treasure troves and are widely studied for their biomarker content in cancer. However, little research has been done on the phosphorylated protein profile within cancer EVs. In the current study, we demonstrate that EVs that are secreted by U87-EGFRvIII mutant glioblastoma cells contain high levels of oncogenic signalling networks. These networks contain multiple activated (phosphorylated) kinases, including EGFR, MAPK, AKT and mTOR.

Methods for Characterization of Senescent Circulating and Tumor-Infiltrating T-Cells: An Overview from Multicolor Flow Cytometry to Single-Cell RNA Sequencing.

Immunosenescence is the general term used to describe the aging-associated decline of immunological function that explains the higher susceptibility to infectious diseases and cancer, increased autoimmunity, or the reduced effectiveness of vaccinations. Senescence of CD8+ T-cells has been described in all these conditions.The most important classical markers of T senescent cells are the cell cycle inhibitors p16ink4a, p21, and p53, together with positivity for SA-ßgal expression and the acquirement of a peculiar IFNγ -based secretory phenotype commonly defined SASP (Senescence Associated Secretory Phenotype). Other surface markers are the CD28 and CD27 loss together with gain of expression of CD45RA, CD57, TIGIT, and/or KLRG1. However, this characterization could not be sufficient to distinguish from truly senescent cells and exhausted T-cells. Furthermore, more complexity is added by the wide heterogeneity of T-cells subset in aged individuals or in the tumor microenvironment. A combined analysis by multicolor flow cytometry for surface and intracellular markers integrated with gene-expression arrays and single-cell RNA sequencing is required to develop effective interventions for therapeutic modulation of specific T-cell subsets. The RNASeq offers the great possibility to reveal at single-cell resolution the exact molecular hallmarks of senescent CD8+ T-cells without the limitations of bulk analysis. Furthermore, the comprehensive integration of multidimensional approaches (genomics, epigenomics, proteomics, metabolomics) will increase our global understanding of how immunosenescence of T-cells is interlinked to human aging.

Novel biomarkers with promising benefits for diagnosis of cervical neoplasia: a systematic review.

BACKGROUND: Cervical cancer screening is slowly transitioning from Pappanicolaou cytologic screening to primary Visual Inspection with Acetic Acid (VIA) or HPV testing as an effort to enhance early detection and treatment. However, an effective triage tests needed to decide who among the VIA or HPV positive women should receive further diagnostic evaluation to avoid unnecessary colposcopy referrals is still lacking. Evidence from experimental studies have shown potential usefulness of Squamous Cell Carcinoma Antigen (SCC Ag), Macrophage Colony Stimulating Factor (M-CSF), Vascular Endothelial Growth Factor (VEGF), MicroRNA, p16INKa / ki-67, HPV E6/E7/mRNA, and DNA methylation biomarkers in detecting premalignant cervical neoplasia. Given the variation in performance, and scanty review studies in this field, this systematic review described the diagnostic performance of some selected assays to detect high-grade cervical intraepithelial neoplasia (CIN2+) with histology as gold standard. METHODS: We systematically searched articles published in English between 2012 and 2020 using key words from PubMed/Medline and SCOPUS with two reviewers assessing study eligibility, and risk of bias. We performed a descriptive presentation of the performance of each of the selected assays for the detection of CIN2 + . RESULTS: Out of 298 citations retrieved, 58 articles were included. Participants with cervical histology yielded CIN2+ proportion range of 13.7-88.4%. The diagnostic performance of the assays to detect CIN2+ was; 1) SCC-Ag: range sensitivity of 78.6-81.2%, specificity 74-100%. 2) M-CSF: sensitivity of 68-87.7%, specificity 64.7-94% 3) VEGF: sensitivity of 56-83.5%, specificity 74.6-96%. 4) MicroRNA: sensitivity of 52.9-67.3%, specificity 76.4-94.4%. 5) p16INKa / ki-67: sensitivity of 50-100%, specificity 39-90.4%. 6) HPV E6/E7/mRNA: sensitivity of 65-100%, specificity 42.7-90.2%, and 7) DNA methylation: sensitivity of 59.7-92.9%, specificity 67-98%. CONCLUSION: Overall, the reported test performance and the receiving operating characteristics curves implies that implementation of p16ink4a/ki-67 assay as a triage for HPV positive women to be used at one visit with subsequent cryotherapy treatment is feasible. For the rest of assays, more robust clinical translation studies with larger consecutive cohorts of women participants is recommended.

Breast cancer treatment and its effects on aging.

Breast cancer is the most common cancer of women in the United States. It is also proving to be one of the most treatable. Early detection, surgical intervention, therapeutic radiation, cytotoxic chemotherapies and molecularly targeted agents are transforming the lives of patients with breast cancer, markedly improving their survival. Although current breast cancer treatments are largely successful in producing cancer remission and extending lifespan, there is concern that these treatments may have long lasting detrimental effects on cancer survivors, in part, through their impact on non-tumor cells. Presently, the impact of breast cancer treatment on normal cells, its impact on cellular function and its effect on the overall function of the individual are incompletely understood. In particular, it is unclear whether breast cancer and/or its treatments are associated with an accelerated aging phenotype. In this review, we consider breast cancer survivorship from the perspective of accelerated aging, and discuss the evidence suggesting that women treated for breast cancer may suffer from an increased rate of physical and cognitive decline that likely corresponds with underlying vulnerabilities of genome instability, epigenetic changes, and cellular senescence.

Expression profiles of inka2 in the murine nervous system.

Dynamic rearrangement of the actin cytoskeleton impacts many cellular characteristics in both the developing and adult central nervous systems (CNS), including the migration and adhesion of highly motile neural progenitor cells, axon guidance of immature neurons, and reconstruction of synaptic structures in the adult brain. Inka1, a known regulator of actin cytoskeleton reconstruction, is predominantly expressed by the neural crest cell lineage and regulates the migration and differentiation of these cells. In the present study, we identified a novel gene, designated as inka2, which is related to inka1. Inka2/fam212b is an evolutionarily conserved gene found in different vertebrate species and constitutes a novel gene family together with inka1. Northern blot analysis showed that inka2 mRNA was highly enriched in the nervous system. The spatiotemporal propagation cell profiles of those cells that expressed inka2 transcripts were compatible with those of Olig2-positive oligodendrocyte progenitor cells, which originate in the ventral ventricular zone during embryogenesis. Intense expression of inka2 was also noted in the proliferative neuronal progenitors in the developing cerebellum. On the other hand, immature newborn neurons in the embryonic brain showed no expression of inka2, except for the cells residing in the marginal zone of the embryonic telencephalon, which is known to contain transient cells including the non-subplate pioneer neurons and Cajal-Retzius cells. As brain development proceeds during the postnatal stage, inka2 expression emerged in some populations of immature neurons, including the neocortical pyramidal neurons, hippocampal pyramidal neurons, and granule cells migrating in the cerebellar cortex. In the adult brain, the expression of inka2 was interestingly confined in terminally differentiated neurons in the restricted forebrain regions. Taken together, as a novel regulator of actin cytoskeletons in the CNS, inka2 may be involved in multiple actin-driven processes, including cell migration and establishment of neuronal polarity.

Correlação entre a expressão da proteína p16 INK4a e o grau de neoplasia intraepitelial anal: uma revisão sistemática / Correlation between expression of p 16 INK4A protein and degree of anal intraepithelial neoplasia (ain): a systematic review

Objetivo: identificar pela revisão sistemática a expressão da proteína p16INK4a nos diferentes graus de neoplasias intraepiteliaisanais (NIA). Método: revisão sistemática de pesquisa nas bases de dados: PubMed, MedLine Old, MedLine,LILACS, SciELO e Science Direct. Foram excluídos artigos de revisão e que não correlacionavam a expressão dap16INK4a com o grau de NIA. Resultados: foram encontrados 483 artigos, dos quais 223 na PubMed, 151 na Medline,34 na Medline Old, 3 no Lilacs e 72 na Science Direct. Apenas cinco artigos foram selecionados baseados no critériode inclusão e exclusão. Conclusão: a proteína p16INK4a utilizada como marcador mostra-se eficaz para o diagnóstico deNIA, principalmente em lesões anais de alto grau.

Objective: to identify through a systematic review the expression of p16INK4a protein in different degrees of analintraepithelial neoplasia (AIN). Method: the review was realized through research on databases PubMed, MedLineOld, MedLine, LILACS, SciELO e Science Direct. Review articles and the ones without correlation between theexpression the p16INK4a with the level of AIN were excluded. Results: it were found 483 articles, which 223 inPubMed, 151 in Medline, 34 in Medline Old, 3 in Lilacs and 72 in Science Direct. Only five articles were selectedbased on inclusion and exclusion criteria. Conclusion: the p16INK4a protein used as a marker has shown to be effectivefor diagnostic of AIN, mainly in high-grade anal lesions.