Lifetime prevalence and associated factors of itch with skin conditions: Atopic dermatitis, psoriasis and dry skin in individuals aged over 50.

BACKGROUND: Itch, common in dermatological conditions, is often accompanied by psychological distress and reduced quality of life. However, research on the prevalence and associated factors of itch with skin conditions in general populations is limited. OBJECTIVES: This cross-sectional study aimed to determine the lifetime prevalence of itch with skin conditions and identify its associated factors in middle-aged and elderly individuals. METHODS: Participants from the Rotterdam Study, a population-based cohort, were interviewed to assess whether they had ever had an itchy skin condition, defining lifetime itch with skin conditions. Over 20 demographic, lifestyle, dermatological, and non-dermatological factors were collected. Multivariable logistic regression analysis explored associations between these factors and itch with skin conditions, reported as odds ratios (ORs) with 95% confidence intervals (CIs). RESULTS: 5,246 eligible participants (age range: 51-100, median age: 67, female: 56.0%) revealed a lifetime prevalence of 33.7% for itch with skin conditions. Female sex (OR (95% CI): 1.26 (1.11-1.43)), body mass index (1.02 (1.01-1.03)), self-reported and presence of atopic dermatitis (4.29 (3.74-4.92), and 1.97 (1.60-2.43)), self-reported and presence of psoriasis (2.31 (1.77-3.01), and 2.11 (1.55-2.87)), self-reported dry skin (1.95 (1.73-2.29)), self-reported asthma (1.40 (1.08-1.83)), renal impairment (1.45 (1.17-1.79)), and clinically relevant depressive and anxiety symptoms (1.85 (1.52-2.25), and 1.36 (1.11-1.66)) were significantly associated with it. CONCLUSIONS: This study reveals a substantial one-third lifetime prevalence of itch with skin conditions in individuals aged over 50. Significant associations with diverse lifestyle, demographic, dermatological and, intriguingly, non-dermatological factors including renal impairment, imply additional contributors to itch induction or persistence in individuals with skin conditions.

Hearing disorders, genetic predisposition, and risk of new-onset atrial fibrillation: A prospective cohort study in the UK biobank.

BACKGROUND: Atrial fibrillation (AF) is the most common sustained arrhythmia and is associated with significant morbidity and mortality. Hearing impairment has been linked to several cardiovascular diseases. However, the association between hearing disorders, genetic predisposition, and new-onset AF remains largely unknown. METHODS: A total of 476,773 participants (mean age 56.5 years) free of AF at baseline (from 2006 to 2010) were included from the UK Biobank study. The presence of hearing disorders including hearing difficulty and tinnitus was self-reported through the touchscreen questionnaire. AF was defined using ICD-10 code: I48 and was followed till February 1st. 2022. The Cox model was used to calculate hazard ratios (HRs) and 95% confidence intervals (95% CI). RESULTS: During a median follow-up of 13.0 years, the AF incidence rate was 2.9 per 1000 person-years. After adjustments for potential confounders, the presence of hearing difficulty (HR, 1.35; 95% CI: 1.32-1.39) and the use of hearing aid (1.45; 1.37-1.53) were significantly associated with risk of new-onset AF. Compared to individuals without tinnitus, the AF risk increased by 17% among those who experienced tinnitus occasionally (1.17; 1.09-1.25), 23% among those who experienced tinnitus frequently (1.23; 1.10-1.39), and 32% among those who experienced tinnitus consistently (1.32; 1.22-1.42). No significant difference was observed across different groups of genetic risk score for AF onset. CONCLUSIONS: Our study provides evidence regarding significant associations of hearing difficulty, use of hearing aid, and tinnitus with risk of incident AF. Findings highlight the potential that screening hearing disorders can benefit AF prevention.

High-Precision Microscale Particulate Matter Prediction in Diverse Environments Using a Long Short-Term Memory Neural Network and Street View Imagery.

In this study, we propose a novel long short-term memory (LSTM) neural network model that leverages color features (HSV: hue, saturation, value) extracted from street images to estimate air quality with particulate matter (PM) in four typical European environments: urban, suburban, villages, and the harbor. To evaluate its performance, we utilize concentration data for eight parameters of ambient PM (PM1.0, PM2.5, and PM10, particle number concentration, lung-deposited surface area, equivalent mass concentrations of ultraviolet PM, black carbon, and brown carbon) collected from a mobile monitoring platform during the nonheating season in downtown Augsburg, Germany, along with synchronized street view images. Experimental comparisons were conducted between the LSTM model and other deep learning models (recurrent neural network and gated recurrent unit). The results clearly demonstrate a better performance of the LSTM model compared with other statistically based models. The LSTM-HSV model achieved impressive interpretability rates above 80%, for the eight PM metrics mentioned above, indicating the expected performance of the proposed model. Moreover, the successful application of the LSTM-HSV model in other seasons of Augsburg city and various environments (suburbs, villages, and harbor cities) demonstrates its satisfactory generalization capabilities in both temporal and spatial dimensions. The successful application of the LSTM-HSV model underscores its potential as a versatile tool for the estimation of air pollution after presampling of the studied area, with broad implications for urban planning and public health initiatives.

Access to Amides and Lactams via Pyridotriazole as a Transformable Directing Group.

Amide and lactam frameworks were synthesized via an efficient two-step strategy. In this protocol, pyridotriazoles were first treated with isocyanates to form the corresponding amides, which were found to be sufficiently reactive to undergo subsequent intramolecular N-H insertion in the absence of any additional reagents or catalysts.

The optimal healthy ranges of thyroid function defined by the risk of cardiovascular disease and mortality: systematic review and individual participant data meta-analysis.

BACKGROUND: Reference intervals of thyroid-stimulating hormone (TSH) and free thyroxine (FT4) are statistically defined by the 2·5-97·5th percentiles, without accounting for potential risk of clinical outcomes. We aimed to define the optimal healthy ranges of TSH and FT4 based on the risk of cardiovascular disease and mortality. METHODS: This systematic review and individual participant data (IPD) meta-analysis identified eligible prospective cohorts through the Thyroid Studies Collaboration, supplemented with a systematic search via Embase, MEDLINE (Ovid), Web of science, the Cochrane Central Register of Controlled Trials, and Google Scholar from Jan 1, 2011, to Feb 12, 2017 with an updated search to Oct 13, 2022 (cohorts found in the second search were not included in the IPD). We included cohorts that collected TSH or FT4, and cardiovascular outcomes or mortality for adults (aged ≥18 years). We excluded cohorts that included solely pregnant women, individuals with overt thyroid diseases, and individuals with cardiovascular disease. We contacted the study investigators of eligible cohorts to provide IPD on demographics, TSH, FT4, thyroid peroxidase antibodies, history of cardiovascular disease and risk factors, medication use, cardiovascular disease events, cardiovascular disease mortality, and all-cause mortality. The primary outcome was a composite outcome including cardiovascular disease events (coronary heart disease, stroke, and heart failure) and all-cause mortality. Secondary outcomes were the separate assessment of cardiovascular disease events, all-cause mortality, and cardiovascular disease mortality. We performed one-step (cohort-stratified Cox models) and two-step (random-effects models) meta-analyses adjusting for age, sex, smoking, systolic blood pressure, diabetes, and total cholesterol. The study was registered with PROSPERO, CRD42017057576. FINDINGS: We identified 3935 studies, of which 53 cohorts fulfilled the inclusion criteria and 26 cohorts agreed to participate. We included IPD on 134 346 participants with a median age of 59 years (range 18-106) at baseline. There was a J-shaped association of FT4 with the composite outcome and secondary outcomes, with the 20th (median 13·5 pmol/L [IQR 11·2-13·9]) to 40th percentiles (median 14·8 pmol/L [12·3-15·0]) conveying the lowest risk. Compared with the 20-40th percentiles, the age-adjusted and sex-adjusted hazard ratio (HR) for FT4 in the 80-100th percentiles was 1·20 (95% CI 1·11-1·31) for the composite outcome, 1·34 (1·20-1·49) for all-cause mortality, 1·57 (1·31-1·89) for cardiovascular disease mortality, and 1·22 (1·11-1·33) for cardiovascular disease events. In individuals aged 70 years and older, the 10-year absolute risk of composite outcome increased over 5% for women with FT4 greater than the 85th percentile (median 17·6 pmol/L [IQR 15·0-18·3]), and men with FT4 greater than the 75th percentile (16·7 pmol/L [14·0-17·4]). Non-linear associations were identified for TSH, with the 60th (median 1·90 mIU/L [IQR 1·68-2·25]) to 80th percentiles (2·90 mIU/L [2·41-3·32]) associated with the lowest risk of cardiovascular disease and mortality. Compared with the 60-80th percentiles, the age-adjusted and sex-adjusted HR of TSH in the 0-20th percentiles was 1·07 (95% CI 1·02-1·12) for the composite outcome, 1·09 (1·05-1·14) for all-cause mortality, and 1·07 (0·99-1·16) for cardiovascular disease mortality. INTERPRETATION: There was a J-shaped association of FT4 with cardiovascular disease and mortality. Low concentrations of TSH were associated with a higher risk of all-cause mortality and cardiovascular disease mortality. The 20-40th percentiles of FT4 and the 60-80th percentiles of TSH could represent the optimal healthy ranges of thyroid function based on the risk of cardiovascular disease and mortality, with more than 5% increase of 10-year composite risk identified for FT4 greater than the 85th percentile in women and men older than 70 years. We propose a feasible approach to establish the optimal healthy ranges of thyroid function, allowing for better identification of individuals with a higher risk of thyroid-related outcomes. FUNDING: None.

Neural Temporal Denoising for Indirect Illumination.

Various temporal denoising methods have been proposed to clean up the noise for real-time ray tracing (RTRT). These methods rely on the temporal correspondences of pixels between the current and previous frames, i.e. per-pixel screen-space motion vectors. However, the state-of-the-art temporal reuse methods with traditional motion vectors cause artifacts in motion occlusions. We accordingly propose a novel neural temporal denoising method for indirect illumination of Monte Carlo (MC) ray tracing at 1 sample per pixel. Based on end-to-end multi-scale kernel-based reconstruction, we apply temporally reliable dual motion vectors to facilitate better reconstruction of the occlusions, and also introduce additional motion occlusion loss to reduce ghosting artifacts. Experiments show that our method significantly reduces the over-blurring and ghosting artifacts while generating high-quality images at real-time rates.

Levels and drivers of urban black carbon and health risk assessment during pre- and COVID19 lockdown in Augsburg, Germany.

This study aimed to evaluate the levels and phenomenology of equivalent black carbon (eBC) at the city center of Augsburg, Germany (01/2018 to 12/2020). Furthermore, the potential health risk of eBC based on equivalent numbers of passively smoked cigarettes (PSC) was also evaluated, with special emphasis on the impact caused by the COVID19 lockdown restriction measures. As it could be expected, peak concentrations of eBC were commonly recorded in morning (06:00-8:00 LT) and night (19:00-22:00 LT) in all seasons, coinciding with traffic rush hours and atmospheric stagnation. The variability of eBC was highly influenced by diurnal variations in traffic and meteorology (air temperature (T), mixing-layer height (MLH), wind speed (WS)) across days and seasons. Furthermore, a marked "weekend effect" was evidenced, with an average eBC decrease of ∼35% due to lower traffic flow. During the COVID19 lockdown period, an average ∼60% reduction of the traffic flow resulted in ∼30% eBC decrease, as the health risks of eBC exposure was markedly reduced during this period. The implementation of a multilinear regression analysis allowed to explain for 53% of the variability in measured eBC, indicating that the several factors (e.g., traffic and meteorology) may contribute simultaneously to this proportion. Overall, this study will provide valuable input to the policy makers to mitigate eBC pollutant and its adverse effect on environment and human health.

Up-regulation of Dsg2 confered stem cells with malignancy through wnt/ß-catenin signaling pathway.

In the previous study, we originally developed cancer stem cells (CSCs) models from mouse induced pluripotent stem cells (miPSCs) by culturing miPSCs in the conditioned medium of cancer cell lines, which mimiced as carcinoma microenvironment. However, the molecular mechanism of conversion in detail remains to be uncovered. Microarray analysis of the CSCs models in this study revealed Dsg2, one of the members of the desmosomal cadherin family, was up-regulated when compared with the original miPSCs. Moreover, the expression of key factors in Wnt/ß-catenin signaling pathway were also found up-regulated in one of the CSCs models, named miPS-LLCcm. An autocrine loop was implied between Dsg2 and Wnt/ß-catenin signaling pathway when miPSCs were treated with Wnt/ß-catenin signaling pathway activators, Wnt3a and CHIR99021, and when the CSCs model were treated with inhibitors, IWR-1 and IWP-2. Furthermore, the ability of proliferation and self-renewal in the CSCs model was markedly decreased in vitro and in vivo when Dsg2 gene was knocked down by shRNA. Our results showed that the Wnt/ß-catenin signaling pathway is activated by the up-regulation of Dsg2 expresssion during the conversion of miPSCs into CSCs implying a potential mechanism of the tranformation of stem cells into malignant phenotype.

Ulinastatin ameliorates preeclampsia induced by N(gamma)-nitro-l-arginine methyl ester in a rat model via inhibition of the systemic and placental inflammatory response.

BACKGROUND: The pathogenesis of preeclampsia (PE) is associated with inflammation and endothelial damage. Ulinastatin (UTI) mainly inhibits proteolytic activity and significantly reduces the release of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) from macrophages. It also ameliorates vascular endothelial damage in pathological conditions. Hence, we investigated the effects of UTI in a rat model of PE induced using N(gamma)-nitro-l-arginine methyl ester (L-NAME). METHODS: Although inducing PE in a rat model, 5000 U/kg of UTI were injected daily. Systolic blood pressure (SBP) and protein levels in the urine were measured. Renal function, and serum concentrations of TNF-α, IL-6, placental growth factor (PLGF), and von Willebrand factor (vWF) were evaluated. The number and weight of live fetuses as well as the weight of placentas were measured. Placentas were collected for western blot and pathological analysis. RESULTS: UTI slightly ameliorated proteinuria and the increases in SBP, blood urea nitrogen (BUN), and serum creatinine. Furthermore, UTI improved serum and placental protein expression levels of TNF-α, IL-6, vWF, and PLGF. Pathological analysis revealed that vascular density and blood flow perfusion was enhanced, vessel wall thickening and neutrophil infiltration were diminished, and the weight and number of live fetuses as well as the weight of the placentas were improved with UTI. CONCLUSION: Preventive use of UTI in the PE rat model induced by L-NAME partially alleviated hypertension, proteinuria, and impaired renal function; improved fetal growth restriction; diminished vascular endothelial injury; and ameliorated placental vasculogenesis abnormality and malperfusion by inhibiting the systemic and placental inflammatory response, suggesting that UTI is a potential drug for PE prevention or treatment.

Investigation of COVID-19-related lockdowns on the air pollution changes in augsburg in 2020, Germany.

The COVID-19 pandemic in Germany in 2020 brought many regulations to impede its transmission such as lockdown. Hence, in this study, we compared the annual air pollutants (CO, NO, NO2, O3, PM10, PM2.5, and BC) in Augsburg in 2020 to the record data in 2010-2019. The annual air pollutants in 2020 were significantly (p < 0.001) lower than that in 2010-2019 except O3, which was significantly (p = 0.02) higher than that in 2010-2019. In a depth perspective, we explored how lockdown impacted air pollutants in Augsburg. We simulated air pollutants based on the meteorological data, traffic density, and weekday and weekend/holiday by using four different models (i.e. Random Forest, K-nearest Neighbors, Linear Regression, and Lasso Regression). According to the best fitting effects, Random Forest was used to predict air pollutants during two lockdown periods (16/03/2020-19/04/2020, 1st lockdown and 02/11/2020-31/12/2020, 2nd lockdown) to explore how lockdown measures impacted air pollutants. Compared to the predicted values, the measured CO, NO2, and BC significantly reduced 18.21%, 21.75%, and 48.92% in the 1st lockdown as well as 7.67%, 32.28%, and 79.08% in the 2nd lockdown. It could be owing to the reduction of traffic and industrial activities. O3 significantly increased 15.62% in the 1st lockdown but decreased 40.39% in the 2nd lockdown, which may have relations with the fluctuations the NO titration effect and photochemistry effect. PM10 and PM2.5 were significantly increased 18.23% an 10.06% in the 1st lockdown but reduced 34.37% and 30.62% in the 2nd lockdown, which could be owing to their complex generation mechanisms.

Optimization of production and characterization of a recombinant soluble human Cripto-1 protein inhibiting self-renewal of cancer stem cells.

Human Cripto-1 is a member of the epidermal growth factor (EGF)-Cripto-FRL-1-Cryptic (CFC) family family and performs critical roles in cancer and various pathological and developmental processes. Recently we demonstrated that a soluble form of Cripto-1 suppresses the self-renewal and enhances the differentiation of cancer stem cells (CSCs). A functional form of soluble Cripto-1 was found to be difficult to obtain because of the 12 cysteine residues in the protein which impairs the folding process. Here, we optimized the protocol for a T7 expression system, purification from inclusion bodies under denatured conditions refolding of a His-tagged Cripto-1 protein. A concentrations of 0.2-0.4 mM isopropyl ß-D-1-thiogalactopyranoside (IPTG) at 37°C was found to be the optimal concentration for Cripto-1 expression while imidazole at 0.5 M was the optimum concentration to elute the Cripto-1 protein from a Ni-column in the smallest volume. Cation exchange column chromatography of the Cripto-1 protein in the presence of 8 M urea exhibited sufficient elution profile at pH 5, which was more efficient at recovery. The recovery of the protein reached to more than 26.6% after refolding with arginine. The purified Cripto-1 exhibited high affinity to the anti-ALK-4 antibody and suppressed sphere forming ability of CSCs at high dose and induced cell differentiation.

Diphenyleneiodonium efficiently inhibits the characteristics of a cancer stem cell model derived from induced pluripotent stem cells.

Diphenyleneiodonium (DPI) has long been evaluated as an anticancer drug inhibiting NADPH oxidase, the IC50 in several cancer cell lines was reported 10 µM, which is too high for efficacy. In this study, we employed miPS-Huh7cmP cells, which we previously established as a cancer stem cell (CSC) model from induced pluripotent stem cells, to reevaluate the efficacy of DPI because CSCs are currently one of the main foci of therapeutic strategy to treat cancer, but generally considered resistant to chemotherapy. As a result, the conventional assay for the cell growth inhibition by DPI accounted for an IC50 at 712 nM that was not enough to define the effectiveness as an anticancer drug. Simultaneously, the wound-healing assay revealed an IC50 of approximately 500 nM. Comparatively, the IC50 values shown on sphere formation, colony formation, and tube formation assays were 5.52, 12, and 8.7 nM, respectively. However, these inhibitory effects were not observed by VAS2780, also a reputed NADPH oxidase inhibitor. It is noteworthy that these three assays are evaluating the characteristic of CSCs and are designed in the three-dimensional (3D) culture methods. We concluded that DPI could be a suitable candidate to target mitochondrial respiration in CSCs. We propose that the 3D culture assays are more efficient to screen anti-CSC drug candidates and better mimic tumor microenvironment when compared to the adherent monolayer of 2D culture system used for a conventional assay, such as cell growth inhibition and wound-healing assays.

The efficacy of PI3Kγ and EGFR inhibitors on the suppression of the characteristics of cancer stem cells.

Cancer stem cells (CSCs) are capable of continuous proliferation, self-renewal and are proposed to play significant roles in oncogenesis, tumor growth, metastasis and cancer recurrence. We have established a model of CSCs that was originally developed from mouse induced pluripotent stem cells (miPSCs) by proposing miPSCs to the conditioned medium (CM) of cancer derived cells, which is a mimic of carcinoma microenvironment. Further research found that not only PI3K-Akt but also EGFR signaling pathway was activated during converting miPSCs into CSCs. In this study, we tried to observe both of PI3Kγ inhibitor Eganelisib and EGFR inhibitor Gefitinib antitumor effects on the models of CSCs derived from miPSCs (miPS-CSC) in vitro and in vivo. As the results, targeting these two pathways exhibited significant inhibition of cell proliferation, self-renewal, migration and invasion abilities in vitro. Both Eganelisib and Gefitinib showed antitumor effects in vivo while Eganelisib displayed more significant therapeutic efficacy and less side effects than Gefitinib on all miPS-CSC models. Thus, these data suggest that the inhibitiors of PI3K and EGFR, especially PI3Kγ, might be a promising therapeutic strategy against CSCs defeating cancer in the near future.

Differentiation of cancer stem cells into erythroblasts in the presence of CoCl2.

Cancer stem cells (CSCs) are subpopulations in the malignant tumors that show self-renewal and multilineage differentiation into tumor microenvironment components that drive tumor growth and heterogeneity. In previous studies, our group succeeded in producing a CSC model by treating mouse induced pluripotent stem cells. In the current study, we investigated the potential of CSC differentiation into blood cells under chemical hypoxic conditions using CoCl2. CSCs and miPS-LLCcm cells were cultured for 1 to 7 days in the presence of CoCl2, and the expression of VEGFR1/2, Runx1, c-kit, CD31, CD34, and TER-119 was assessed by RT-qPCR, Western blotting and flow cytometry together with Wright-Giemsa staining and immunocytochemistry. CoCl2 induced significant accumulation of HIF-1α changing the morphology of miPS-LLCcm cells while the morphological change was apparently not related to differentiation. The expression of VEGFR2 and CD31 was suppressed while Runx1 expression was upregulated. The population with hematopoietic markers CD34+ and c-kit+ was immunologically detected in the presence of CoCl2. Additionally, high expression of CD34 and, a marker for erythroblasts, TER-119, was observed. Therefore, CSCs were suggested to differentiate into erythroblasts and erythrocytes under hypoxia. This differentiation potential of CSCs could provide new insight into the tumor microenvironment elucidating tumor heterogenicity.

Hypermethylation of DLG3 Promoter Upregulates RAC1 and Activates the PI3K/AKT Signaling Pathway to Promote Breast Cancer Progression.

OBJECTIVE: This investigation aimed to figure out the relation between discs large homolog 3 (DLG3) expression and the progression and prognosis of breast cancer (BC). METHODS: qRT-PCR was utilized for confirming DLG3 expression and RAC1 mRNA expression in BC tissues and cells. Subsequently, after overexpression or interference of DLG3, the changes of the biological activities of BC cells, including cell proliferation, migration, invasion, and apoptosis, were detected through CCK-8, colony formation assay, wound healing assay, transwell assay, and flow cytometry, respectively. Furthermore, western blotting was utilized to measure the protein expression of DLG3 and RAC1, as well as related proteins of epithelial-mesenchymal transition (EMT) and the PI3K/AKT signaling pathway. RESULTS: At both cellular and tissue level in BC, DLG3 was downregulated and methylation level was upregulated; RAC1 showed an opposite change and was of a negative correlation with DLG3. In MCF-7 and HCC1937, we found that the upregulation of DLG3 could inhibit RAC1 expression as well as cell proliferation, invasion, migration, and EMT, while promoting apoptosis. Also, DLG3 inhibited the activation of the P13K/AKT pathway. CONCLUSION: Hypermethylation of DLG3 promoter upregulates RAC1 and activates the PI3K/AKT pathway, thus promoting BC progression. This conclusion provides ideas and experimental basis for improving and treating BC patients.

Cytological features of ovarian or tubal high-grade serous carcinoma: A retrospective study of 12 cases with abnormal cervical liquid-based smear.

BACKGROUND: High-grade serous carcinoma (HGSC) is the most common epithelial carcinoma of the tubo-ovarian region, with a poor prognosis, which presents with an advanced stage at the time of diagnosis. This study summarizes the cytological features of cervical liquid-based cytological examination in order to diagnose ovarian or tubal HGSC at an early stage by cervical cytology smear. METHODS: A total of 12 patients who were diagnosed with atypical glandular cell (AGC) and above lesions by cervical cytological examination and ovarian or tubal HGSC by histopathology were enrolled in this study. The cytological characteristics, including the arrangement and shape of the neoplastic cells, nuclear and cytoplasmic features, and the presence of tumor diathesis were reviewed. RESULTS: Nine cases were determined to be AGC, and three cases were determined to be AGC favor neoplastic (AGC-FN) in the 12 cervical cytological diagnoses. The glandular cells showed significant atypia and proliferated actively with a three-dimensional structure. Increased nuclear-to-cytoplasmic ratios, prominent nucleoli, and hyperchromasia were common. Vacuole-like changes were observed in the cytoplasm. Tumor diathesis was observed in only one case (1/12, 8.30%). CONCLUSION: Ovarian or tubal HGSC can occasionally be detected using cervical liquid-based cytology. It has distinct cytological characteristics. Primary ovarian or tubal HGSC with positive cervical cytology was accompanied by tumor cells in ascites, which suggested that the patient had progressed to an advanced stage.

Signaling Inhibitors Accelerate the Conversion of mouse iPS Cells into Cancer Stem Cells in the Tumor Microenvironment.

Cancer stem cells (CSCs) are a class of cancer cells characterized by self-renewal, differentiation and tumorigenic potential. We previously established a model of CSCs by culturing mouse induced pluripotent stem cells (miPSCs) for four weeks in the presence of a conditioned medium (CM) of cancer cell lines, which functioned as the tumor microenvironment. Based on this methodology of developing CSCs from miPSCs, we assessed the risk of 110 non-mutagenic chemical compounds, most of which are known as inhibitors of cytoplasmic signaling pathways, as potential carcinogens. We treated miPSCs with each compound for one week in the presence of a CM of Lewis lung carcinoma (LLC) cells. However, one-week period was too short for the CM to convert miPSCs into CSCs. Consequently, PDO325901 (MEK inhibitor), CHIR99021 (GSK-3ß inhibitor) and Dasatinib (Abl, Src and c-Kit inhibitor) were found to confer miPSCs with the CSC phenotype in one week. The tumor cells that survived exhibited stemness markers, spheroid formation and tumorigenesis in Balb/c nude mice. Hence, we concluded that the three signal inhibitors accelerated the conversion of miPSCs into CSCs. Similarly to our previous study, we found that the PI3K-Akt signaling pathway was upregulated in the CSCs. Herein, we focused on the expression of relative genes after the treatment with these three inhibitors. Our results demonstrated an increased expression of pik3ca, pik3cb, pik3r5 and pik3r1 genes indicating class IA PI3K as the responsible signaling pathway. Hence, AKT phosphorylation was found to be up-regulated in the obtained CSCs. Inhibition of Erk1/2, tyrosine kinase, and/or GSK-3ß was implied to be involved in the enhancement of the PI3K-AKT signaling pathway in the undifferentiated cells, resulting in the sustained stemness, and subsequent conversion of miPSCs into CSCs in the tumor microenvironment.

Deletion of the middle region of the transcription factor ClrB in Penicillium oxalicum enables cellulase production in the presence of glucose.

Enzymes that degrade lignocellulose to simple sugars are of great interest in research and for biotechnology because of their role in converting plant biomass to fuels and chemicals. The synthesis of cellulolytic enzymes in filamentous fungi is tightly regulated at the transcriptional level, with the transcriptional activator ClrB/CLR-2 playing a critical role in many species. In Penicillium oxalicum, clrB overexpression could not relieve the dependence of cellulase expression on cellulose as an inducer, suggesting that clrB is controlled post-transcriptionally. In this study, using a reporter gene system in yeast, we identified the C-terminal region of ClrB/CLR-2 as a transcriptional activation domain. Expression of clrBID , encoding a ClrB derivative in which the DNA-binding and transcriptional activation domains are fused together to remove the middle region, led to cellulase production in the absence of cellulose in P. oxalicum Strikingly, the clrBID -expressing strain produced cellulase on carbon sources that normally repress cellulase expression, including glucose and glycerol. Results from deletion of the carbon catabolite repressor gene creA in the clrBID -expressing strain suggested that the effect of clrBID is independent of CreA's repressive function. A similar modification of clrB in Aspergillus niger resulted in the production of a mannanase in glucose medium. Taken together, these results indicate that ClrB suppression under noninducing conditions involves its middle region, suggesting a potential strategy to engineer fungal strains for improved cellulase production on commonly used carbon sources.

Introduction of heterologous transcription factors and their target genes into Penicillium oxalicum leads to increased lignocellulolytic enzyme production.

Genetic engineering of transcription factors is an efficient strategy to improve lignocellulolytic enzyme production in fungi. In this study, the xylanase transcriptional regulators of Trichoderma reesei (Xyr1) and Neurospora crassa (XLR-1), as well as their constitutively active mutants (Xyr1A824V and XLR-1A828V), were heterologously expressed in Penicillium oxalicum. The two heterologous regulators were identified to be able to activate lignocellulolytic enzyme gene expression in P. oxalicum. Particularly, expression of T. reesei Xyr1 resulted in a higher cellulase production level compared with the expression of native xylanase transcriptional regulator XlnR using the same promoter. Xyr1A824V and XLR-1A828V were found to be able to confer P. oxalicum more enhanced lignocellulolytic abilities than wild-type regulators Xyr1 and XLR-1. Furthermore, introduction of regulatory modules containing Xyr1A824V/XLR-1A828V and their target cellulase genes resulted in greater increases in cellulase production than alone expression of transcriptional regulators. Through the cumulative introduction of three regulatory modules containing regulator mutants and their corresponding target cellulase genes from P. oxalicum, T. reesei, and N. crassa, a 2.8-fold increase in cellulase production was achieved in P. oxalicum.

Mutation of a Conserved Alanine Residue in Transcription Factor AraR Leads to Hyperproduction of α-l-Arabinofuranosidases in Penicillium oxalicum.

α-l-Arabinofuranosidases are important in the degradation of plant polysaccharides and are used in several industrial processes. Although the use of filamentous fungi for the production of α-l-arabinofuranosidases is widely reported, there are few reports on strain engineering for enhanced production of these enzymes by fungi. In this study, the function of transcription factor AraR in l-arabinose release and catabolism by the fungus Penicillium oxalicum (P. oxalicum) is investigated. Also, a mutant of AraR, AraRA731V , is constructed to improve the production of α-l-arabinofuranosidases on the basis of the sequence homology between AraR and the xylanolytic gene activator XlnR. The AraRA731V -overexpressing strain can synthesize α-l-arabinofuranosidase in the absence of an inducer and shows a 54.1-fold increase in α-l-arabinofuranosidase production and a 7.4-fold increase in α-galactosidase production in the medium containing wheat bran. Determination of the transcript abundances of lignocellulolytic enzyme genes reveals significant upregulation of multiple α-l-arabinofuranosidase genes and downregulation of some cellulolytic and xylanolytic enzyme genes in the engineered strain relative to its parent. Taken together, the results suggest the conserved regulatory function of AraR in the family Trichocomaceae and provide a strategy for engineering fungal strains for enhanced α- l-arabinofuranosidase production.