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Repetitive and restricted behaviors and interests are a major feature of Autism Spectrum Disorder (ASD). Insistence on sameness, rigid adherence to rituals and routines and resistance to change, and related symptoms can have a highly disruptive impact on an individual's ability to transition from one task to the next and progress through the different steps within a task. Difficulties with transitions can represent a major barrier in treatment and care, impede learning, and contribute to significant distress in individuals with an ASD. A number of behavioral approaches to facilitating transitions in persons with an ASD have been investigated. The current paper reviews the available research on strategies to support individuals in transitions between daily activities and tasks. Current trends and future directions are discussed.

Three of the Grand Challenges identified by the social work profession are specifically related to health care: healthy youth development, closing the health care gap, and advancing long and productive lives. Idaho State University (ISU) initiated a collaborative interprofessional health education project (IET) over 30 years ago. This collaborative includes ten health care disciplines across five colleges and provides in-depth assessment and referral to three families annually. This study describes the development of a collaborative, high impact, teaching exemplar, and examines the knowledge, perceptions, and competency of participating students using the framework of the IPEC competencies and the CSWE practice competencies for social work education. Results are derived from a pre-post survey administered during the previous two years. The project includes two groups of students: those that engaged in a hands-on experience with a client and those that only observed clients. The Observation-Only group completed the SPICE-R2 and the IEPS. Students that had direct interaction with clients completed the IEPS (pre-post engagement with clients) as well as the ICCAS. Attitudes, perceptions, and perceived competency improved amongst students participating in the IET course from pre-assessment to post-assessment with moderate to large effects being observed. [ABSTRACT FROM AUTHOR]

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• Southern pine beetle and mountain pine beetle are significant forest pests of North and Central America; both with expanding geographic ranges associated with climate change. • RNA interference (RNAi) is a species-specific approach to pest management. • Southern pine beetle and mountain pine beetle exhibit reciprocal effects of their congener's dsRNA. • There is considerable potential for cross-species effects within the genus Dendroctonus. • Congeneric effects could benefit management strategies if invasion fronts converge. Losses associated with forest pest outbreaks are increasing at an alarming rate, exacerbated by fluctuating climate patterns, and unimpeded by traditional management approaches that fail to keep pace with growing pressures. Future forest pest management strategies demand augmentation with next generation approaches. RNA interference, or RNAi, is a naturally occurring eukaryotic immune response to viral infection that works by disrupting the translation of mRNA into protein and can be manipulated to cause insect mortality through the introduction of carefully designed dsRNAs. The effective induction of the RNAi pathway depends on the exact match of a sequence at least sixteen nucleotides in length, making non-target effects unlikely. Thus, RNAi is increasingly praised as a species-specific pest management approach. The southern pine beetle (SPB), Dendroctonus frontalis , and its congener, the mountain pine beetle (MPB), D. ponderosae , are highly destructive forest pests endemic to North and Central America experiencing unprecedented range expansions, with extensive impacts on affected ecosystems. We have demonstrated that oral ingestion of specific dsRNAs induces gene silencing via RNAi and causes rapid and significant mortality in both SPB and MPB. Here we evaluate the ability of dsRNAs targeting SPB to affect gene expression in MPB, and the ability of dsRNAs targeting MPB to affect gene expression in SPB. We found that species-specific dsRNAs designed for gene silencing in one species can silence genes in the reciprocal species, offering the potential for development of RNAi as a Dendroctonus -specific management tool against rapidly expanding bark beetle populations. [ABSTRACT FROM AUTHOR]

• Healthy Trees – Healthy People engages urban dwellers with nature. • Provides "eyes on the ground" for exotic tree pest detection. • Creates unique opportunities for community engagement focused on nature. • Improvements in mindfulness, tree ID, and exotic pest awareness reported. Healthy Trees – Healthy People (HT-HP) is an outreach and education program created to enlist and train participants in exotic pest detection while providing them with structured opportunities to connect with urban nature and increase their physical and emotional health. HT- HP creates infrastructure to increase engagement by the urban populace in the urban tree canopy. The program solicits participants to engage in an 8-week intervention designed to expand capacity to detect non-native insect pests and pathogens, while increasing physical activity, raising awareness of healthy lifestyle choices, and improving the health of participants. Program participants were trained in tree and tree pest identification to provide 'eyes on the ground' that can contribute to exotic pest detection, reducing the risk of pest establishment, and increasing the sustainability of urban tree canopies. Participants in this pilot program reported significant improvements in their knowledge of tree identification and tree care, an awareness of, and ability to identify selected exotic invasive tree pests, and improved mindfulness. Healthy Trees – Healthy People is nimble, easily adaptable, and serves as a unique model that could include tailored tree identification and pest detection. The program can engage citizen scientists in exotic pest detection while increasing usage of urban parks. [ABSTRACT FROM AUTHOR]

• RNA interference (RNAi) in MPB can be activated with exogenous dsRNAs. • When silenced, essential MPB genes cause beetle mortality. • Oral ingestion and dermal absorption of dsRNAs creates management potential. • The hsp gene is expressed at higher levels in females, regardless of dsRNA exposure. • Differential gene expression is a factor when considering RNAi for pest management. The mountain pine beetle (MPB), Dendroctonus ponderosae , is an eruptive endemic forest pest that is undergoing substantial range expansion in response to recent climatic changes, breaching geographic barriers, exploiting novel hosts, and affecting millions of hectares of conifer forests in western North America. Current management approaches have been unable to keep pace with MPB population outbreaks, and novel and aggressive management responses are required as MPB's range expansion progresses. Gene silencing through RNA interference (RNAi) is an emerging pest management approach that is being developed for agricultural pests, and has also been shown to be effective against some xylophagous forest pests, including the southern pine beetle (SPB), D. frontalis. When essential genes are targeted, RNAi can cause rapid insect mortality; here we focus on evaluating its effectiveness in MPB. We identified reference genes for quantitative real-time PCR (qPCR) and validated RNAi responses in MPB by analyzing gene expression and beetle survival. Using an adult bioassay that combined oral ingestion and dermal absorption of dsRNAs targeting three genes (hsp, iap, and shi), we measure gene expression and demonstrate silencing, as well as insect mortality, following dsRNA exposure. All three genes were silenced and all treatment beetles died within 7 d. This validates reference genes for expression analyses and demonstrates that MPB, similar to the congeneric SPB, has a highly sensitive RNAi response. Additionally, we document sex-specific differences in gene expression for one of the three target genes, hsp ; any differences in gene expression and subsequent mortality based on sex must be considered as this technology progresses as a pest management tool. RNAi causes rapid insect mortality when essential genes are targeted, is highly specific to the target pest, and has no environmental contamination risks, making it an attractive approach for further development in forest pest suppression. [ABSTRACT FROM AUTHOR]

Background: Small-molecule compounds that even partially restore the GTPase activity of RAS G12V can be used in anticancer therapy. Until now, attempts to obtain such compounds have failed. Compounds with this ability have been defined in our research., Methods: The compounds were initially identified through virtual screening, and their optimal binding conformation in the RAS SW-II pocket was determined using the flexible docking technique. Efficacy was verified based on the IC50 determination, GTPase activity, as well as the AKT and ERK phospho WB assays., Results: The IC50 of the tested compounds was significantly lower against cells with the RAS G12V mutation than against selected types of normal cells. The molecular mechanism of action of these compounds was proposed - minimization of the negative impact of the V12 sidechain on GTP hydrolysis of RAS G12V . The work also indicates that the model of action of RAS mutants in cell lines is incomplete. The analysed cell line (SW-480) with RAS mutations does not always show increased ERK and AKT activity., Conclusions: We have demonstrated molecules that partially restore the GTPase activity of RAS G12V . Their mechanism of action is well explained based on current RAS mutant conformation and mechanistic models. These molecules inhibit the RAS-AKT pathway and show higher cytotoxicity against cancer cells with the RAS G12V mutation (SW-480 cell line). However, SW-480 cells can switch into the subline proliferating independently of AKT phosphorylation and show partial resistance to the molecules described in this article., Competing Interests: Declarations. Ethics approval and consent to participate: Not applicable. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Effective transgene expression is critical for genetically engineered cell therapy. Therefore, one of CAR-T cell therapy's critical areas of interest, both in registered products and next-generation approaches is the expression of transgenes. It turns out that various constitutive promoters used in clinical products may influence CAR-T cell antitumor effectiveness and impact the manufacturing process. Furthermore, next-generation CAR-T starts to install remotely controlled inducible promoters or even autonomous expression systems, opening new ways of priming, boosting, and increasing the safety of CAR-T. In this article, a wide range of constitutive and inducible promoters has been grouped and structured, making it possible to compare their pros and cons as well as clinical usage. Finally, logic gates based on Synthetic Notch have been elaborated, demonstrating the coupling of desired external signals with genetically engineered cellular responses., Competing Interests: The authors declare no competing interests., (© 2024 The Author(s).)

Cell therapy offers hope, but it also presents challenges, most particularly the limited ability of human organs and tissues to regenerate. Since many diseases are associated with irreversible pathophysiological or traumatic changes, stem cells and their derivatives are unable to secure healing. Although regenerative medicine offers chances for improvements in many diseases, such as type one diabetes and Parkinson's disease, it cannot eliminate the primary cause of many of them. While successes can be expected for diseases such as sickle cell disease, this is not the case for hereditary diseases with varied mutation types or for ciliopathies, which start in embryogenesis. In this complicated medical environment, synthetic biology offers some solutions, but their implementation will take many years. Still, positive examples such as CAR-T therapy offer hope., (© 2024 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

The emergence of therapies such as CAR-T has created a need for reliable, validated methods for detecting EGFRvIII in patient tumor cells. Particularly so since previous studies have already suggested that some anti-EGFRvIII antibodies may be non-specific. The present paper evaluates the use of the L8A4 antibody in the immunohistochemical (IHC) and immunocytochemical (ICC) detection of EGFRvIII in 30 glioblastoma specimens, and compares it with other methods such as RT-PCR, MLPA, and FISH. The results indicate that Real-time PCR appears to be a very specific and sensitive method of EGFRvIII detection. ICC analysis with L8A4 also appears specific but requires cell culture. IHC analyses of EGFRvIII returned a number of false positives when using L8A4. Due to the growing need for an effective diagnostic tool before starting immunotherapy methods, such as the CAR-T anti-EGFRvIII or SynNotch CAR-T recognizing EGFRvIII, it is necessary to identify a more reliable and simple method of EGFRvIII detection or improve the specificity of the anti-EGFRvIII antibody, until then, immunocytochemistry may temporarily replace immunohistochemistry., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The number of glioblastoma (GB) cases is increasing every year, and the currently available therapies remain ineffective. A prospective antigen for GB therapy is EGFRvIII, an EGFR deletion mutant containing a unique epitope that is recognized by the L8A4 antibody used in CAR-T (chimeric antigen receptor T cell) therapy. In this study, we observed that the concomitant use of L8A4 with particular tyrosine kinase inhibitors (TKIs) does not impede the interaction between L8A4 and EGFRvIII; moreover, in this case, the stabilization of formed dimers results in increased epitope display. Unlike in wild-type EGFR, a free cysteine at position 16 (C16) is exposed in the extracellular structure of EGFRvIII monomers, leading to covalent dimer formation in the region of L8A4-EGFRvIII mutual interaction. Following in silico analysis of cysteines possibly involved in covalent homodimerization, we prepared constructs containing cysteine-serine substitutions of EGFRvIII in adjacent regions. We found that the extracellular part of EGFRvIII possesses plasticity in the formation of disulfide bridges within EGFRvIII monomers and dimers due to the engagement of cysteines other than C16. Our results suggest that the EGFRvIII-specific L8A4 antibody recognizes both EGFRvIII monomers and covalent dimers, regardless of the cysteine bridging structure. To summarize, immunotherapy based on the L8A4 antibody, including CAR-T combined with TKIs, can potentially increase the chances of success in anti-GB therapy.

Alström syndrome (ALMS) and Bardet-Biedl syndrome (BBS) are rare genetic diseases with a number of common clinical features ranging from early-childhood obesity and retinal degeneration. ALMS and BBS belong to the ciliopathies, which are known to have the expression products of genes, encoding them as cilia-localized proteins in multiple target organs. The aim of this study was to perform transcriptomic and proteomic analysis on cellular models of ALMS and BBS syndromes to identify common and distinct pathological mechanisms present in both syndromes. For this purpose, epithelial cells were isolated from the urine of patients and healthy subjects, which were then cultured and reprogrammed into induced pluripotent stem (iPS) cells. The pathways of genes associated with the metabolism of lipids and glycosaminoglycan and the transport of small molecules were found to be concomitantly downregulated in both diseases, while transcripts related to signal transduction, the immune system, cell cycle control and DNA replication and repair were upregulated. Furthermore, protein pathways associated with autophagy, apoptosis, cilium assembly and Gli1 protein were upregulated in both ciliopathies. These results provide new insights into the common and divergent pathogenic pathways between two similar genetic syndromes, particularly in relation to primary cilium function and abnormalities in cell differentiation.

Background: The biological role of EGFRvIII (epidermal growth factor receptor variant three) remains unclear., Methods: Three glioblastoma DK-MG sublines were tested with EGF (epidermal growth factor) and TGFβ (transforming growth factor β). Sublines were characterized by an increased percentage of EGFRvIII-positive cells and doubling time (DK-MG low to DK-MG extra-high ), number of amplicons, and EGFRvIII mRNA expression. The influence of the growth factors on primary EGFRvIII positive glioblastomas was assessed., Results: The overexpression of exoEGFRvIII in DK-MG high did not convert them into DK-MG extra-high , and this overexpression did not change DK-MG low to DK-MG high ; however, the overexpression of RAS G12V increased the proliferation of DK-MG low . Moreover, the highest EGFRvIII phosphorylation in DK-MG extra-high did not cause relevant AKT (known as protein kinase B) and ERK (extracellular signal-regulated kinase) activation. Further analyses indicate that TGFβ is able to induce apoptosis of DK-MG high cells. This subline was able to convert to DK-MG extra-high , which appeared resistant to this proapoptotic effect. EGF acted as a pro-survival factor and stimulated proliferation; however, simultaneous senescence induction in DK-MG extra-high cells was ambiguous. Primary EGFRvIII positive (and SOX2 (SRY-Box Transcription Factor 2) positive or SOX2 negative) glioblastoma cells differentially responded to EGF and TGFβ., Conclusions: The roles of TGFβ and EGF in the EGFRvIII context remain unclear. EGFRvIII appears as a weak oncogene and not a marker of GSC (glioma stem cells). Hence, it may not be a proper target for CAR-T (chimeric antigen receptor T cells).

Among the many oncology therapies, few have generated as much excitement as CAR-T. The success of CAR therapy would not have been possible without the many discoveries that preceded it, most notably, the Nobel Prize-winning breakthroughs in cellular immunity. However, despite the fact that CAR-T already offers not only hope for development, but measurable results in the treatment of hematological malignancies, CAR-T still cannot be safely applied to solid tumors. The reason for this is, among other things, the lack of tumor-specific antigens which, in therapy, threatens to cause a lethal attack of lymphocytes on healthy cells. In the case of hematological malignancies, dangerous complications such as cytokine release syndrome may occur. Scientists have responded to these clinical challenges with molecular switches. They make it possible to remotely control CAR lymphocytes after they have already been administered to the patient. Moreover, they offer many additional capabilities. For example, they can be used to switch CAR antigenic specificity, create logic gates, or produce local activation under heat or light. They can also be coupled with costimulatory domains, used for the regulation of interleukin secretion, or to prevent CAR exhaustion. More complex modifications will probably require a combination of reprogramming (iPSc) technology with genome editing (CRISPR) and allogenic (off the shelf) CAR-T production.

Although the role of senescence in many physiological and pathological processes is becoming more identifiable, many aspects of senescence are still enigmatic. A special attention is paid to the role of this phenomenon in tumor development and therapy. This review mainly deals with a large spectrum of oncological issues, beginning with therapy-induced senescence and ending with oncogene-induced senescence. Moreover, the role of senescence in experimental approaches, such as primary cancer cell culture or reprogramming into stem cells, is also beginning to receive further consideration. Additional focus is made on senescence resulting from mitotic catastrophe processes triggered by events occurring during mitosis and jeopardizing chromosomal stability. It has to be also realized that based on recent findings, the basics of senescent cell property interpretation, such as irreversibility of proliferation blockade, can be undermined. It shows that the definition of senescence probably requires updating. Finally, the role of senescence is lately more understandable in the immune system, especially since senescence can diminish the effectiveness of the chimeric antigen receptor T-cell (CAR-T) therapy. In this review, we summarize the current knowledge regarding all these issues., Competing Interests: The authors have declared that there are no conflicts of interest regarding the publication of this article., (Copyright © 2022 Ewelina Stoczynska-Fidelus et al.)

Bronchial epithelial cells and fibroblasts play an essential role in airway remodelling, due to their protective and secretory functions. There are many studies proving that infection caused by human rhinovirus may contribute to the process of airway remodelling. The beneficial properties of curcumin, the basic ingredient of turmeric, have been proved in many studies. Therefore, the aim of this study was the evaluation of curcumin immunomodulatory properties in development of airway remodelling. Fibroblasts (WI-38 and HFL1) and epithelial cells (NHBE) were incubated with curcumin. Additionally, remodelling conditions were induced with rhinovirus (HRV). Airway remodelling genes were determined by qPCR and immunoblotting. Moreover, NF-κB, c-Myc and STAT3 were silenced to analyse the pathways involved in airway remodelling. Curcumin reduced the expression of the genes analysed, especially MMP-9, TGF-β and collagen I. Moreover, curcumin inhibited the HRV-induced expression of MMP-9, TGF-β, collagen I and LTC4S (p < 0.05). NF-κB, c-Myc and STAT3 changed their course of expression. Concluding, our study shows that curcumin significantly downregulated gene expression related to the remodelling process, which is dependent on NF-κB and, partially, on c-Myc and STAT3. The results suggest that the remodelling process may be limited and possibly prevented, however this issue requires further research., (© 2021 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Mutation in isocitrate dehydrogenase 1 (IDH1R132H) occurs in various types of cancer, including low and high grade gliomas. Despite high incidence indicating its central role in tumor initiation and progression there are no targeted therapies directed against this oncogene available in the clinic. This is due to the limited understanding of the role of IDH1R132H in carcinogenesis, which is further propagated by the lack of appropriate experimental models. Moreover, proper in vitro models for analysis of gliomagenesis are required. In this study, we employed a Tet On system to generate human induced neural stem cells with doxycycline-inducible IDH1R132H. Equivalent expression of both forms of IDH1 in the presented model remains similar to that described in tumor cells. Additional biochemical analyses further confirmed tightly controlled gene regulation at protein level. Formation of a functional mutant IDH1 enzyme was supported by the production of D-2-hydroxyglutarate (D2HG). All samples tested for MGMT promoter methylation status, including parental cells, proved to be partially methylated. Analysis of biological effect of IDH1R132H revealed that cells positive for oncogene showed reduced differentation efficiency and viability. Inhibition of mutant IDH1 with selective inhibitor efficiently suppressed D2HG production as well as reversed the effect of mutant IDH1 protein on cell viability. In summary, our model constitutes a valuable platform for studies on the molecular basis and the cell of origin of IDH-mutant glioma (e.g. by editing P53 in these cells and their derivatives), as well as a reliable experimental model for drug testing., Competing Interests: The authors have declared that no competing interests exist.

Cellular origin of glioblastoma (GB) is constantly discussed and remains a controversial subject. Unfortunately, neurobiologists are not consistent in defining neural stem cells (NSC) complicating this issue even further. Nevertheless, some suggestions referring to GB origin can be proposed based on comparing GB to central nervous system (CNS) cells. Firstly, GB cells show in vitro differentiation pattern similar to GFAP positive neural cells, rather than classical (GFAP negative) NSC. GB cells in primary cultures become senescent in vitro , similar to GFAP positive neural progenitors, whereas classical NSC proliferate in vitro infinitely. Classical NSC apoptosis triggered by introduction of IDH1R132H undermines hypothesis stating that IDH-mutant (secondary) GB origins from these NSC. Analysis of biological role of typical IDH-wildtype (primary) GB oncogene such as EGFRvIII also favors GFAP positive cells rather than classical NSC as source of GB. Single-cell NGS and single-cell transcriptomics also suggest that GFAP positive cells are GB origin. Considering the above-mentioned and other discussed in articles data, we suggest that GFAP positive cells (astrocytes, radial glia, or GFAP positive neural progenitors) are more likely to be source of GB than classical GFAP negative NSC, and further in vitro assays should be focused on these cells. It is highly possible that several populations of tumor initiating cells (TIC) exist within GB, adjusting their phenotype and even genotype to various environmental conditions including applied therapy and periodically going through different TIC states as well as non-TIC state. This adjustment is driven by changes in number and types of amplicons. The existence of various populations of TIC would enable creating neoplastic foci in different environments and increase tumor aggressiveness., Competing Interests: The authors have declared that there are no conflicts of interest regarding the publication of this article., (Copyright © 2020 Aneta Wlodarczyk et al.)

Epidermal growth factor receptor variant III (EGFR vIII ) seems to constitute the perfect therapeutic target for glioblastoma (GB), as it is specifically present on up to 28-30% of GB cells. In case of other tumor types, expression and possible role of this oncogene still remain controversial. In spite of EGFR vIII mechanism of action being crucial for the design of small active anticancer molecules and immunotherapies, i.e., CAR-T technology, it is yet to be precisely defined. EGFR vIII is known to be resistant to degradation, but it is still unclear whether it heterodimerizes with EGF-activated wild-type EGFR (EGFR WT ) or homodimerizes (including covalent homodimerization). Constitutive kinase activity of this mutated receptor is relatively low, and some researchers even claim that a nuclear, but not a membrane function, is crucial for its activity. Based on the analyses of recurrent tumors that are often lacking EGFR vIII expression despite its initial presence in corresponding primary foci, this oncogene is suggested to play a marginal role during later stages of carcinogenesis, while even in primary tumors EGFR vIII expression is detected only in a small percentage of tumor cells, undermining the rationality of EGFR vIII -targeting therapies. On the other hand, EGFR vIII -positive cells are resistant to apoptosis, more invasive, and characterized with enhanced proliferation rate. Moreover, expression of this oncogenic receptor was also postulated to be a marker of cancer stem cells. Opinions regarding the role that EGFR vIII plays in tumorigenesis and for tumor aggressiveness are clearly contradictory and, therefore, it is crucial not only to determine its mechanism of action, but also to unambiguously define its role at early and advanced cancer stages., Competing Interests: The authors declare that there are no conflicts of interest regarding the publication of this paper., (Copyright © 2019 Adrianna Rutkowska et al.)

Glioblastoma, the most malignant astrocytic tumour, is associated with limited survival and thus rare metastases. We analysed a particularly interesting case - a 51-year-old male diagnosed within 2 years with primary and recurrent glioblastoma, isocitrate dehydrogenase (IDH)-wild type, as well as with numerous extra-central nervous system (CNS) metastatic foci. Genetic material obtained from primary and recurrent tumours, as well as from pulmonary metastasis was analysed and compared at a molecular level. Next generation sequencing (NGS) analysis revealed BRAFV600E mutation, detected only in 2-5% of glioblastomas, in both the primary tumour and pulmonary metastases. Importantly, this mutation provides a possible therapeutic option as it constitutes a target for clinically approved inhibitors. This case study not only demonstrates a molecular comparison of primary, recurrent and metastatic glioblastoma, but also emphasizes the need for precise molecular diagnostics, which may facilitate treatment choice, especially in tumours currently lacking efficient treatment.

Despite intensive research no therapies targeted against the oncogenic EGFRvIII are present in the clinic. One of the reasons is the elusive nature of the molecular structure and activity of the truncated receptor. The recent publications indicate the EGF-bound wild-type EGFR to trans -phosphorylate the EGFRvIII initiating aberrant signaling cascade. The elevated stability of the mutant receptor contributes towards oncogenic potential, preventing termination of signaling by receptor degradation. Here, we show that inhibition of phosphatases leads to a marked increase in phosphorylation of wild-type EGFR and EGFRvIII, indicating that both undergo cyclic rounds of phosphorylation and dephosphorylation on all investigated tyrosine residues, including Tyr1045. Still, we observe elevated stability of the mutant receptor, suggesting phosphorylation as insufficient to cause degradation. Hyperphosphorylation of EGFRvIII was hindered only by EGFR tyrosine kinase inhibitors. Co-immunoprecipitation as well as semi-native Western blotting structural analyses together with functional investigation of EGFRvIII's phosphorylation following depletion of wild-type EGFR by shRNA or EGF-mediated degradation indicated homodimerization as the predominant quaternary structure of the mutant receptor. Dimers were observed only under non-reducing conditions, suggesting that homodimerization is mediated by covalent bonds. Previous reports indicated cysteine at position 16 to mediate covalent homodimerization. Upon its substitution to serine, we have observed impaired formation of dimers and lower phosphorylation levels of the mutated oncogene. Based on the obtained results we propose that EGFRvIII is predominantly regulated dynamically by phosphatases that counteract the process of trans -phosphorylation occurring within the homodimers., Competing Interests: CONFLICTS OF INTEREST There are no conflicts of interest to declare.

Background: The presence as well as the potential role of EGFR vIII in tumors other than glioblastoma still remains a controversial subject with many contradictory data published. Previous analyses, however, did not consider the level of EGFR vIII mRNA expression in different tumor types. Methods: Appropriately designed protocol for Real-time quantitative reverse-transcription PCR (Real-time qRT-PCR) was applied to analyze EGFR vIII and EGFR WT mRNA expression in 155 tumor specimens. Additionally, Western Blot (WB) analysis was performed for selected samples. Stable cell lines showing EGFR vIII expression (CAS-1 and DK-MG) were analyzed by means of WB, immunocytochemistry (ICC) and fluorescence in situ hybridization (FISH). Results: Our analyses revealed EGFR vIII expression in 27.59% of glioblastomas (8/29), 8.11% of colorectal cancers (3/37), 6.52% of prostate cancers (3/46) and none of breast cancers (0/43). Despite the average relative expression of EGFR vIII varying greatly among tumors of different tissues (approximately 800-fold) or even within the same tissue group (up to 8000-fold for GB), even the marginal expression of EGFR vIII mRNA can be detrimental to cancer progression, as determined by the analysis of stable cell lines endogenously expressing the oncogene. Conclusion: EGFR vIII plays an unquestionable role in glioblastomas with high expression of this oncogene. Our data suggests that EGFR vIII importance should not be underestimated even in tumors with relatively low expression of this oncogene., Competing Interests: The authors have declared that no competing interest exists.

Background: Induced pluripotent stem cells (iPSC) possess an enormous potential as both, scientific and therapeutic tools. Their application in the regenerative medicine provides new treatment opportunities for numerous diseases, including type 1 diabetes. In this work we aimed to derive insulin producing cells (IPC) from iPS cells established in defined conditions., Methods: We optimized iPSC generation protocol and created pluripotent cell lines with stably integrated PDX1 and NKX6.1 transgenes under the transcriptional control of doxycycline-inducible promoter. These cells were differentiated using small chemical molecules and recombinant Activin A in the sequential process through the definitive endoderm, pancreatic progenitor cells and insulin producing cells. Efficiency of the procedure was assessed by quantitative gene expression measurements, immunocytochemical stainings and functional assays for insulin secretion., Results: Generated cells displayed molecular markers characteristic for respective steps of the differentiation. The obtained IPC secreted insulin and produced C-peptide with significantly higher hormone release level in case of the combined expression of PDX1 and NKX6.1 induced at the last stage of the differentiation., Conclusions: Efficiency of differentiation of iPSC to IPC can be increased by concurrent expression of PDX1 and NKX6.1 during progenitor cells maturation. Protocols established in our study allow for iPSC generation and derivation of IPC in chemically defined conditions free from animal-derived components, which is of the utmost importance in the light of their prospective applications in the field of regenerative medicine.