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Background: Fibromyalgia (FM) is a chronic disorder that causes damage to the neuro-muscular system and alterations in the intestinal microbiota and affects the psychological state of the patient. In our previous study, we showed that 22 women patients subjected to a specific very low-carbohydrate ketogenic therapy (VLCKD) showed an improvement in clinical scores as well as neurotransmission-related and psychological dysfunctions and intestinal dysbiosis. Furthermore, NMR metabolomic data showed that changes induced by VLCKD treatment were evident in all metabolic pathways related to fibromyalgia biomarkers. Methods : Based on this evidence, we extend our investigation into dietary interventions for fibromyalgia by evaluating the impact of transitioning from a VLCKD to a low-glycemic insulinemic (LOGI) diet over an additional 45-day period. Therefore, participants initially following a VLCKD were transitioned to the LOGI diet after 45 days to determine whether the improvements in FM symptoms and metabolic dysfunctions achieved through VLCKD could be sustained with LOGI. Results : Our findings suggested that while VLCKD serves as an effective initial intervention for correcting metabolic imbalances and alleviating FM symptoms, transitioning to a LOGI diet offers a practical and sustainable dietary strategy. This transition preserves clinical improvements and supports long-term adherence and quality of life, underscoring the importance of adaptable nutritional therapies in chronic disease management. Control patients who adhered only to the LOGI diet for 90 days showed only modest improvement in clinical and psychological conditions, but not elimination of fibromyalgia symptoms. Conclusions : In conclusion the LOGI diet is an excellent alternative to maintain the results obtained from the regime VLCKD.

Objectives : This study investigates the effectiveness of an herbal formulation, STRESSLESS (ST-65), which combines ashwagandha ( Withania somnifera ) and bacopa ( Bacopa monnieri ), on SH-SY5Y human neuroblastoma cells. Given the rising interest in natural compounds for neuroprotection and stress alleviation, we aimed to explore the cellular and molecular effects of this formulation. Methods : Utilizing a nuclear magnetic resonance (NMR) metabolomic approach and ultra-high-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS), we identified key bioactive compounds in ST-65, including withanolides from ashwagandha and bacosides from bacopa. Results : Our findings indicate that ST-65 treatment significantly alters the metabolic profile of SH-SY5Y cells. Key changes included increased levels of metabolites linked to neuroprotection, energy metabolism, and antioxidant defense. Notable enhancements were observed in specific amino acids and neuroprotective compounds, suggesting activation of neuroprotective mechanisms and mitigation of stress-induced damage. Conclusions : The study reveals a complex phyto-chemical profile of ST-65 and underscores its potential as a natural active agent for addressing stress-related neurodegenerative conditions. These insights into neuronal mechanisms provide a foundation for further exploration of herbal formulations in neuroprotection.

Fibromyalgia (FM), a chronic disease with a high incidence in women, poses a significant challenge for diagnosis and treatment, especially due to the absence of specific biomarkers and the multifaceted nature of its symptoms, which range from neuromuscular pain to mood disorders and intestinal dysbiosis. While diagnosis currently relies on rheumatological clinical evaluations and treatment options mainly focus on symptom management, FM seems to have possible links with systemic metabolic dysfunctions with a common inflammatory root. In this context, a new therapeutic avenue emerges: could a therapeutic nutritional approach be the missing piece of the puzzle? Indeed, diet therapies employed particularly for metabolic syndromes proved recently to be efficacious for correcting systemic dysmetabolism and a high number of chronic inflammation conditions. In particular, the very-low-calorie ketogenic diet (VLCKD) demonstrated therapeutic benefits in many disorders. In the present study, we aimed to investigate the specific effects of two dietary interventions, namely the oloproteic VLCKD and the low-glycemic insulinemic (LOGI) diet, on two groups of female FM patients (FM1 and FM2) over a 45-day period. Utilizing clinical and laboratory tests, as well as non-invasive NMR metabolomic analysis of serum, urine, and saliva samples, we sought to uncover how these dietary regimens impact the metabolic dysfunctions associated with FM.

Background: Limited evidence exists regarding the association between COVID-19 and Long COVID manifestations in children, particularly concerning variants of concern (VOCs). We aimed to characterize a cohort of pediatric patients hospitalized with confirmed acute SARS-CoV-2 and monitor them for Long COVID symptoms. Additionally, it seeks to explore any potential correlations between VOCs and clinical symptoms., Methods: We conducted a prospective study involving children hospitalized from November 2021 to March 2023, with confirmed acute SARS-CoV-2 infection. A telephone survey was conducted at 3-6-12 months after discharge., Results: We included 167 patients (77 F/90 M). Upon hospital admission, 95.5% of patients presented as symptomatic. Regarding patients for whom it was feasible to determine the SARS-CoV-2 variant (n = 51), the Delta variant was identified in 11 children (21.6%) and Omicron variant in the remaining 40 patients (78.4%: 27.5% BA.1 variant; 15% BA.2 variant; 57.5% BA.5 variant). 19 patients (16.5%) reported experiencing at least one symptom indicative of Long COVID (weight loss 31.6%, inappetence 26.3%, chronic cough 21.1%, fatigue 21.1%, and sleep disturbances, wheezing, abdominal pain and mood disorders 15.8%). In only 4 patients with Long COVID we could identified a specific SARS-CoV-2 variant (3 Omicron: 2 BA.1 and 1 BA.2; 1 Delta)., Conclusions: this study underscores that long COVID is a significant concern in the pediatric population. Our data reinforce the importance of continuously monitoring the impact of long-COVID in infants, children, and adolescents. A follow-up following SARS-CoV-2 infection is therefore advisable, with symptom investigation tailored to the patient's age., (© 2024. The Author(s).)

Calcium (Ca) represents about 40% of the total mineral mass, mainly in the bone, providing mechanical strength to the skeleton and teeth. An adequate Ca intake is necessary for bone growth and development in children and adolescents and for maintaining bone mineral loss in elderly age. Ca deficiency predisposes to osteopenia and osteoporosis. Healthy nutrition, including an adequate intake of Ca-rich food, is paramount to prevent and cure osteoporosis. Recently, several clinical studies have demonstrated that, in conditions of Ca dysmetabolism, Ca-rich mineral water is beneficial as a valuable source of Ca to be used as an alternative to caloric Ca-rich dairy products. Although promising, these data have been collected from small groups of participants. Moreover, they mainly regard the effect of Ca-rich mineral water on bone metabolism. In contrast, an investigation of the effect of Ca supplementation on systemic metabolism is needed to address the spreading of systemic metabolic dysfunction often associated with Ca dysmetabolism. In the present study, we analyzed urine and blood sera of 120 women in perimenopausal condition who were subjected for six months to 2l daily consumption of bicarbonate-calcium mineral water marketed under ® Lete. Remarkably, this water, in addition to being rich in calcium and bicarbonate, is also low in sodium. A complete set of laboratory tests was carried out to investigate whether the specific water composition was such to confirm the known therapeutic effects on bone metabolism. Second, but not least, urine and blood sera were analyzed using NMR-based metabolomic procedures to investigate, other than the action on Ca metabolism, potential system-wide metabolic effects. Our data show that Lete water is a valid supplement for compensating for Ca dysmetabolism and preserving bone health and integrity.

Multisystem inflammatory syndrome in children (MIS-c) is an uncommon, but serious, inflammatory response that occurs after SARS-CoV-2 infection. As time went by, MIS-c was also reported as a potential adverse event following COVID-19 vaccination. A descriptive analysis was performed of Individual Case Safety Reports (ICSRs) associated with anti COVID-19 vaccines and related to the pediatric population from 2020 to 2022. The present pharmacovigilance study aimed to describe cases of MIS-c following COVID-19 vaccination, stratified by sex, reported in the Vaccine Adverse Events Reporting System (VAERS) and meeting the Brighton Collaboration criteria for case definition. We assessed all suspected cases through the case definition and classification of the Brighton Collaboration Group, and only definitive, probable, and possible cases were included in the analysis. The Reporting Odds Ratio (ROR) with 95% Confidence Interval (CI) was computed to assess if males have a lower/higher probability of reporting ICSRs with MIS-c compared with females. Overall, we found 79 cases of potentially reported MIS-c following vaccination. This study demonstrated that MIS-c following vaccination was more commonly reported for male subjects with a median age of 10 years (IQR 10.0-11.4), especially after the first dose of anti COVID-19 vaccines with a median time to onset of 27 days. Even so, the rate of occurrence of MIS-c following anti COVID-19 vaccines is lower (0.12/100,000 vaccinated subjects; 95% CI, 0.12-0.13). Overall, all ICSRs were serious and caused or prolonged hospitalization. Finally, disproportionality analysis showed that males had a higher reporting probability of MIS-c compared with females following immunization with mRNA COVID-19 vaccines. Since only a few years of marketing are available, further data from real-life contexts are needed.

Background: The high-mobility group Hmga family of proteins are non-histone chromatin-interacting proteins which have been associated with a number of nuclear functions, including heterochromatin formation, replication, recombination, DNA repair, transcription, and formation of enhanceosomes. Due to its role based on dynamic interaction with chromatin, Hmga2 has a pathogenic role in diverse tumors and has been mainly studied in a cancer context; however, whether Hmga2 has similar physiological functions in normal cells remains less explored. Hmga2 was additionally shown to be required during the exit of embryonic stem cells (ESCs) from the ground state of pluripotency, to allow their transition into epiblast-like cells (EpiLCs), and here, we use that system to gain further understanding of normal Hmga2 function., Results: We demonstrated that Hmga2 KO pluripotent stem cells fail to develop into EpiLCs. By using this experimental system, we studied the chromatin changes that take place upon the induction of EpiLCs and we observed that the loss of Hmga2 affects the histone mark H3K27me3, whose levels are higher in Hmga2 KO cells. Accordingly, a sustained expression of polycomb repressive complex 2 (PRC2), responsible for H3K27me3 deposition, was observed in KO cells. However, gene expression differences between differentiating wt vs Hmga2 KO cells did not show any significant enrichments of PRC2 targets. Similarly, endogenous Hmga2 association to chromatin in epiblast stem cells did not show any clear relationships with gene expression modification observed in Hmga2 KO. Hmga2 ChIP-seq confirmed that this protein preferentially binds to the chromatin regions associated with nuclear lamina. Starting from this observation, we demonstrated that nuclear lamina underwent severe alterations when Hmga2 KO or KD cells were induced to exit from the naïve state and this phenomenon is accompanied by a mislocalization of the heterochromatin mark H3K9me3 within the nucleus. As nuclear lamina (NL) is involved in the organization of 3D chromatin structure, we explored the possible effects of Hmga2 loss on this phenomenon. The analysis of Hi-C data in wt and Hmga2 KO cells allowed us to observe that inter-TAD (topologically associated domains) interactions in Hmga2 KO cells are different from those observed in wt cells. These differences clearly show a peculiar compartmentalization of inter-TAD interactions in chromatin regions associated or not to nuclear lamina., Conclusions: Overall, our results indicate that Hmga2 interacts with heterochromatic lamin-associated domains, and highlight a role for Hmga2 in the crosstalk between chromatin and nuclear lamina, affecting the establishment of inter-TAD interactions., (© 2022. The Author(s).)

Background: Systemic immunosuppression characterizing cancer patients represents a concern regarding the efficacy of anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination, and real-world evidence is needed to define the efficacy and the dynamics of humoral immune response to mRNA-based anti-SARS-CoV-2 vaccines., Methods: We conducted an observational study that included patients with solid tumors who were candidates for mRNA anti-SARS-CoV-2 vaccination at the Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy. The primary objective was to monitor the immunologic response to the mRNA anti-SARS-CoV-2 vaccination in terms of anti-spike antibody levels. All the patients received two doses of the mRNA-1273 vaccine or the BNT162b2 vaccine. Healthcare workers served as a control group of healthy subjects., Results: Among the 243 patients included in the present analysis, 208 (85.60%) and 238 (97.94%) resulted seroconverted after the first and the second dose of vaccine, respectively. Only five patients (2.06%) had a negative titer after the second dose. No significant differences in the rate of seroconversion after two vaccine doses were observed in patients as compared with the control group of healthy subjects. Age and anticancer treatment class had an independent impact on the antibody titer after the second dose of vaccination. In a subgroup of 171 patients with available data about the third timepoint, patients receiving immunotherapy with immune checkpoint inhibitors seem to have a higher peak of antibodies soon after the second dose (3 weeks after), but a more pronounced decrease at a late timepoint (3 months after)., Conclusions: The systemic immunosuppression characterizing cancer patients did not seem to dramatically affect the humoral response to anti-SARS-CoV-2 mRNA vaccines in our population of patients with solid tumors. Further investigation is needed to dissect the interplay between immunotherapy and longitudinal dynamics of humoral response to mRNA vaccines, as well as to analyze the cellular response to mRNA vaccines in cancer patients., Competing Interests: Conflict of interest statement: The authors declare that there is no conflict of interest., (© The Author(s), 2022.)

Highlights: Although no definitive data exist in literature, adjuvant chemotherapy is usually recommended in patients with radically resected stage III rectal cancer treated with neo-adjuvant chemo-radiotherapy. We performed a systematic review of literature with direct and indirect comparisons to assess the role of adjuvant mono- or poli-chemotherapy in radically resected rectal cancer treated with neoadjuvant chemo-radiotherapy. Neither chemotherapy (mono-or poli-chemotherapy) nor polichemotherapy with oxaliplatin-containing regimens seems to improve Overall Survival and Disease-Free Survival in patients with radically resected rectal cancer treated with neoadjuvant chemo-adiotherapy. Neither the entire population of patients radically resected after neoadjuvant chemotherapy, nor high risk patients seem to benefit from adjuvant chemotherapy. Our data seem to suggest the need of review the actual international guidelines that suggest the need of adjuvant chemotherapy at least in high risk rectal cancer treated with surgery and neoadjuvant chemo-radiotherapy., (Copyright © 2022 Elsevier B.V. All rights reserved.)

One of the most widely conserved hallmarks of aging is a decline in functional capabilities. Mobility loss is particularly burdensome due to its association with negative health outcomes, loss of independence and disability, and the heavy impact on quality of life. Recently, a new condition, physical frailty and sarcopenia, has been proposed to define a critical stage in the disabling cascade. Physical frailty and sarcopenia are characterized by weakness, slowness, and reduced muscle mass, yet with preserved ability to move independently. One of the strategies that have shown some benefits in combatting mobility loss and its consequences for older adults is physical activity. Here, we describe the opportunities and challenges for the development of physical activity interventions in people with physical frailty and sarcopenia. The aim of this article is to review age-related physio(patho)logical changes that impact mobility in old age and to provide recommendations and procedures in accordance with the available literature., Competing Interests: All of the authors of the present work are partners of the SPRINTT consortium, which is partially funded by the European Federation of Pharmaceutical Industries and Associations (EFPIA). The authors report no potential conflicts of interest in this work., (© 2020 Billot et al.)

Background . FOLFOXIRI plus Bevacizumab is one of the most frequently used first-line treatments for patients with BRAF -mutant colorectal cancer (CRC), while second-line treatment requires extensive further research. In this pooled analysis, we evaluate the impact of anti-angiogenics in patients with pre-treated BRAF -mutant CRC. Methods. We monitored patients in randomized, controlled studies who had advanced CRC and were undergoing second-line chemotherapy in addition to utilizing Bevacizumab, Ramucirumab or Aflibercept treatments. These data were pooled together with the data and results of BRAF -mutant patients enrolled in two phase III trials (TRIBE and TRIBE-2 study), who had been treated with second-line treatment both with or without Bevacizumab. Overall survival (OS), in relation to BRAF mutational status, was the primary focus. Results . Pooled analysis included 129 patients. Anti-angiogenics were found to have a significant advantage over the placebo in terms of OS (HR 0.50, 95%CI 0.29-0.85) ( p = 0.01). Conclusions . Our pooled analysis confirms the efficacy of anti-angiogenics in pre-treated BRAF -mutant CRC, establishing the combination of chemotherapy plus Bevacizumab or Ramucirumab or Aflibercept as a valid treatment option.

Anogenital warts are a worldwide public health problem. They consist of epithelial proliferations caused by HPV, whose transmission occurs mainly through sexual intercourse. In this study, we evaluated their impact on the quality of life of adult males. We interviewed 88 men in an outpatient clinic for sexually transmitted diseases at a Brazilian public institution, using the DLQI-BRA questionnaire. Most patients (81%) presented mild or no impairment of quality of life. The main dimensions affected were sex life, symptoms, and embarrassment. The low impact on quality of life may well justify the delay in seeking medical treatment, favoring the spread of the disease.

Background: Melasma is a chronic acquired focal hypermelanosis affecting photoexposed areas, especially for women during fertile age. Several factors contribute to its development: sun exposure, sex steroids, medicines, and family history. Melanic pigmentation pathway discloses several SNPs in different populations. Here, we evaluated the association between genetic ancestry and facial melasma., Methods: A cross-sectional study involving women with melasma and an age-matched control group from outpatients at FMB-Unesp, Botucatu-SP, Brazil was performed. DNA was extracted from oral mucosa swabs and ancestry determined by studying 61 INDELs. The genetic ancestry components were adjusted by other known risk factors by multiple logistic regression., Results: We evaluated 119 women with facial melasma and 119 controls. Mean age was 39 ± 9 years. Mean age at beginning of disease was 27 ± 8 years. Pregnancy (40%), sun exposure (37%), and hormonal oral contraception (22%) were the most frequently reported melasma triggers. All subjects presented admixed ancestry, African and European genetic contributions were significantly different between cases and controls (respectively 10% vs 6%; 77% vs 82%; p < 0.05). African ancestry (OR = 1.04; 95% CI 1.01 to 1.07), first generation family history (OR = 3.04; 95% CI 1.56 to 5.94), low education level (OR = 4.04; 95% CI 1.56 to 5.94), and use of antidepressants by individuals with affected family members (OR = 6.15; 95% CI 1.13 to 33.37) were associated with melasma, independently of other known risk factors., Conclusions: Facial melasma was independently associated with African ancestry in a highly admixed population.

Ancestry information can be useful in investigations of diseases with a genetic or infectious background. As the Brazilian population is highly admixed physical traits tend to be poor indicators of ancestry. The assessment of ancestry by ancestry informative markers (AIMs) can exclude the subjectivity of self-declared ethnicity and reported family origin. We aimed to evaluate the reliability of self-reported ethnicity or reported family origin as indicators of genomic ancestry in a female population from the Southeast of Brazil. Two cohorts were included: 404 women asked to self-report their ethnicity (Pop1) and 234 women asked to report their family's origin (Pop2). Identification of AIMs was performed using a panel of 61 markers and results were plotted against parental populations-Amerindian, Western European and Sub-Saharan African-using Structure v2.3.4. In Pop1 57.4 % of women self-reported as white, 34.6 % as brown and 8.0 % as black. Median global European, Amerindian and African contributions were 66.8, 12.6 and 16.6 %. In Pop2, 66.4 % of women declared European origin, 23.9 % African origin and 26.9 % Amerindian. Median global European, Amerindian and African contributions were 80.8, 7.3 and 7.6 %, respectively. Only 31.0 and 21.0 % of the global variation in African and European contributions, respectively, could be explained by self-reported ethnicity and reported family origin only accounted for 20.0 and 5.0 % of the variations observed in African and European ancestries, respectively. Amerindian ancestry did not influence self-reported ethnicity or declared family origin. Neither self-reported ethnicity nor declared family origin are reliable indicators of genomic ancestry in these Brazilian populations.

Background: Primary cutaneous cryptococcosis is an uncommon infectious disease caused by Cryptococcus neoformans or Cryptococcus gattii affecting immunosuppressed as well as immunocompetent patients. It is often misdiagnosed as it may mimic other cutaneous diseases., Materials and Methods: We report a series of cases diagnosed from 2005 to 2010 in two general hospitals. The diagnosis in all patients was made on the basis of histopathology and culture. Phenoloxidase and canavanine-glycine-bromothymol blue tests were used in order to identify the Cryptococcus species. Systematic investigation ruled out the systemic involvement in every case., Results: Eleven patients, 81.8% male, were diagnosed during this study. The immunosuppression status was identified in 54.5% of patients, and all of them were under corticosteroid therapy due to a variable set of diseases. All patients presented with circumscribed lesions on their upper limbs. Most lesions showed an infiltrative or tumoral aspect with up to 40 cm diameter. Fluconazole, up to 400 mg/daily, was the main therapeutic regimen and proved to be efficient., Conclusions: Primary cutaneous cryptococcosis has been diagnosed in both immunosuppressed and immunocompetent patients. Its peculiar clinical aspect could facilitate early diagnosis. Culture and biochemical tests should be performed in order to define the species involved., (© 2012 The International Society of Dermatology.)

Objective: To evaluate quality of life in a population that attended a specific community event on health care education, and to investigate the association of their quality of life with the presence of cardiovascular risk factors, Introduction: Interest in health-related quality of life is growing worldwide as a consequence of increasing rates of chronic disease. However, little is known about the association between quality of life and cardiovascular risk factors., Methods: This study included 332 individuals. Demographics, blood pressure, body mass index, and casual glycemia were evaluated. The brief version of the World Health Organization Quality of Life questionnaire on quality of life was given to them. The medians of the scores obtained for the physical, psychological, emotional, and environmental domains were used as cutoffs to define "higher" and "lower" scores. A multinomial logistic regression model was used to define the parameters associated with lower scores., Results: Diabetes mellitus, dyslipidemia, and obesity were associated with lower scores in the physical domain. Dyslipidemia was also associated with lower scores in the psychological domain. Male gender and regular physical activity had protective effects on quality of life. Aging was inversely associated with decreased quality of life in the environmental domain., Conclusion: The presence of cardiovascular risk factors is related to a decreased quality of life. Conversely, male gender and regular physical activity had protective effects on quality of life. These findings suggest that exercising should be further promoted by health-related public programs, with a special focus on women.

In PC Cl3 cells, a continuous, fully differentiated rat thyroid cell line, P2Y(2) purinoceptor activation provoked a transient increase of [Ca(2+)](i), followed by a decreasing sustained phase. The alpha and beta1 protein kinase C (PKC) inhibitor Gö6976 decreased the rate of decrement to the basal [Ca(2+)](i) level and increased the peak of Ca(2+) entry of the P2Y(2)-provoked Ca(2+)transients. These effects of Gö 6976 were not caused by an increased permeability of the plasma membrane, since the Mn(2+) and Ba(2+) uptake were not changed by Gö 6976. Similarly, the Na(+)/Ca(2+) exchanger was not implicated, since the rate of decrement to the basal [Ca(2+)](i) level was equally decreased in physiological and Na(+)-free buffers, in the presence of Gö 6976. On the contrary, the activity of the sarcoplasmic-endoplasmic reticulum Ca(2+)ATPase (SERCA) 2b was profoundly affected by Gö 6976 since the drug was able to completely inhibit the stimulation of the SERCA 2b activity elicited by P2-purinergic agonists. Finally, the PKC activator phorbol myristate acetate had effects opposite to Gö 6976, in that it markedly increased the rate of decrement to the basal [Ca(2+)](i) level after P2Y(2) stimulation and also increased the activity of SERCA 2b. These results suggest that SERCA 2b plays a role in regulating the sustained phase of Ca(2+) transients caused by P2Y(2) stimulation.

Aim of the present paper was to investigate the signaling pathways of P2Y2 in rat thyroid PC Cl3 cell line and its effects on proliferation. This study demonstrates that P2Y2 activation provoked: (a) a cytosol-to-membrane translocation of PKC-alpha, -betaI and -epsilon; (b) the phosphorylation of the extra cellular signal-regulated kinases 1 and 2 (ERK1/2); (c) the expression of c-Fos protein; (d) no effects on the G1/S progression and overall cell proliferation. The P2Y2-stimulated ERK1/2 phosphorylation was: (a) completely blocked by PD098059, a mitogen-activated protein kinase (MEK) inhibitor or by W-7, a Ca2+-calmodulin (CaM) antagonist; (b) reduced by GF109203X, inhibitor of PKCs, or AG1478, inhibitor of EGFR tyrosine kinase, or LY294002/wortmannin, inhibitors of phosphoinositide 3-kinases, or cytochalasin D, inhibitor of actin microfilament bundles polymerization. The c-Fos induction was greatly diminished by Go6976 or PD098059, and completely abolished when combined. In conclusion, data indicate that the P2Y2-induced phosphorylation of ERK1/2 and the induction of c-Fos are due to the operation of CaM, with PKC, PI3K, EGFR and receptor endocytosis mechanisms endorsing the signalling. On the other hand, no mitogenic effects of P2Y2 are whatsoever noticed in PC Cl3 cells.

We have previously shown that HeLa cells express P2Y2 and P2Y6 receptors endogenously and determined the pathways by which the P2Y2 controls proliferation and Na+/K+ATPase activity. Our objective in this study was to investigate the hypothesis that P2Y6 also controls proliferation and Na+/K+ATPase activity; the pathways used in these actions were partially characterised. We found that P2Y6 activation controlled cell proliferation but not the activity of the Na+/K+ATPase. UDP activation of P2Y6 provoked: (a) an increase in free cytosolic calcium; (b) the activation of protein kinase C-alpha, -beta, -delta, -epsilon, and -zeta but not of PKC-iota and -eta; (c) the phosphorylation of the extracellular signal-regulated protein kinases 1 and 2 (ERK1/2); (d) the expression of c-Fos protein. The P2Y6 induced cell proliferation was blocked by the mitogen-activated protein kinase kinase (MAPKK) inhibitor PD098059, thereby indicating that the ERK pathway mediates the mitogenic signalling of P2Y6. PKC and phosphoinositide 3-kinase (PI3K) inhibitors were tested at two different time points of ERK1/2 phosphorylation (10 and 60 min). The results suggest that novel PKCs and PI3K initiate the response but both conventional and atypical PKCs are required for the maintenance of the UDP-induced phosphorylation of ERK1/2. The induction of c-Fos was greatly diminished by conventional or atypical PKC-zeta inhibition, suggesting that it may be due to PKC-alpha/beta and -zeta activity. These observations demonstrate that UDP acts as a proliferative agent in HeLa cells activating multiple signalling pathways involving conventional, novel, and atypical PKCs, PI3K, and ERK. Of these pathways, conventional and atypical PKCs appear responsible for the induction of c-Fos, while ERK is responsible for cell proliferation and depends upon both novel and atypical PKCs and PI3K activities., (Copyright 2004 Wiley-Liss, Inc.)

We examined the signalling pathways responsible for the Ang II induction of growth in MCF-7 human breast cancer cells. Ang II in MCF-7 cells induced: (a) the translocation from the cytosol to membrane and nucleus of atypical protein kinase C-zeta (PKC-zeta) but not of PKC-alpha, -delta, - epsilon and -eta; (b) the expression of c-fos mRNA and protein; (c) the phosphorylation of the extracellular signal-regulated protein kinases 1 and 2 (ERK1/2). All these effects were due to the activation of the Ang II type I receptor (AT1) since they were blocked by the AT1 antagonist losartan. The Ang II-stimulated ERK1/2 phosphorylation was blocked by (a) high doses of staurosporine, inhibitor of PKC-zeta, and by a synthetic myristoylated peptide with sequences based on the endogenous PKC-zeta pseudosubstrate region (zeta-PS); (b) PD098059, a mitogen-activated protein kinase kinase inhibitor (MAPKK/MEK); and, moreover, (c) the inhibitors of phosphoinositide 3-kinases (PI3K), LY294002 and wortmannin, thus indicating that PI3K may act upstream of ERK1/2. The Ang II-evoked c-fos induction was blocked only by high doses of staurosporine and by zeta-PS whilst PD098059, LY294002 and wortmannin were ineffective, thus indicating that c-fos induction is not due to ERK1/2 activity. When the epidermal growth factor-receptor (EGFR) tyrosine kinase activity was inhibited by the use of its inhibitor AG1478, Ang II was still able to induce ERK1/2 phosphorylation and c-fos expression, therefore proving that the transactivation of EGFR was not required for these Ang II effects in MCF-7 cells. The previously reported proliferation of MCF-7 cells induced by Ang II was blocked by PD098059 and by wortmannin in a dose-dependent manner, thereby indicating that in MCF-7 cells the PI3K and ERK pathways mediate the mitogenic signalling of AT1. Our results suggest that in MCF-7 cells Ang II activates multiple signalling pathways involving PKC-zeta, PI3K and MAPK; of these pathways only PKC-zeta appears responsible for the induction of c-fos., (Copyright 2003 Wiley-Liss, Inc.)

The effects of P2Y2 purinoceptor activation on c-Fos expression and the signaling pathways evoked by extracellular ATP/UTP in HeLa cells were investigated. We found that P2Y2 activation induced c-Fos protein and phosphorylated the extracellular signal-regulated kinases 1 and 2 (ERK1/2). The P2Y2-stimulated c-Fos induction was partly blocked (a) by U73122, a phospholipase C inhibitor, (b) by Gö6976, a conventional PKC inhibitor, (c) by PD098059, a mitogen-activated protein kinase kinase inhibitor, and, moreover, (d) by the inhibitors of phosphoinositide 3-kinases (PI3K), LY294002 and wortmannin. When Gö6976 and PD098059, or Gö6976 and wortmannin, were combined there was a totally inhibition of P2Y2-induced c-Fos increase. Either U73122 or Gö6976 did not inhibit ERK1/2 phosphorylation induced by ATP/UTP, while it was inhibited by LY294002 (or wortmannin) and by staurosporine. Additionally, wortmannin inhibited the cytosol-to-membrane translocation of PKC- epsilon induced by ATP/UTP. These data indicated that agonist-induced PI3K and downstream PKC- epsilon activation mediated the effect of ATP/UTP on ERK1/2 activation. To test the biological consequences of ERK1/2 activation, the effect of P2Y2 on cell functions were examined. P2Y2 stimulation increased cell proliferation and this effect was attenuated by PD098059 in a dose-dependent manner, thereby indicating that the ERK pathway mediates mitogenic signaling by P2Y2. In conclusion, the activation of conventional PKCs through P2Y2 receptor acts in concert with ERK and PI3K/PKC- epsilon pathways to induce c-Fos protein and HeLa cell proliferation., (Copyright 2003 Wiley-Liss, Inc.)

It was previously shown that in rat thyroid PC-Cl3 cell line, a purinergic P2Y receptor increases the concentration of free cytosolic Ca(2+) ([Ca(2+)](i)) via phospholipase C activation. We here studied whether in a transformed cell line (PC-E1Araf) derived from parental PC-Cl3 cells, ATP is still able to transduce the [Ca(2+)](i)-based intracellular signal.We demonstrate the expression of mRNA for P2Y2 in both PC-Cl3 and PC-E1Araf cells; mRNAs for P2Y1, P2Y4, P2Y6 and P2Y11 were absent. In both cell lines activation of P2Y2 receptor provokes a transient increase in [Ca(2+)](i) followed by a lower sustained phase persisting for over 5min in PC-Cl3 and only 1.5 min in PC-E1Araf cells. In both cell lines the [Ca(2+)](i) reached a plateau level significantly higher than the basal [Ca(2+)](i) level persisting for over 10 min. Removal of extracellular Ca(2+) reduced the initial transient response to ATP in PC-Cl3, but not in PC-E1Araf cells, and completely abolished the plateau phase in both cell lines. In the presence of extracellular Ca(2+) thapsigargin (TG) caused a rise in [Ca(2+)](i) significantly higher in PC-Cl3 than transformed PC-E1Araf cells, while in Ca(2+)-free medium the effect of TG was similar in both cell lines. The capacitative Ca(2+)-entry in PC-Cl3 resulted significantly higher than in PC-E1Araf cells. Further studies were performed in order to investigate whether the different effects of ATP on [Ca(2+)](i) was due to variation in divalent cation plasma membrane permeability. PC-E1Araf cells showed a much lower permeability to Ca(2+), Ba(2+), Sr(2+), Mn(2+), and Co(2+) that may be responsible for the differences in purinergic Ca(2+) signaling pathway with respect to parental PC-Cl3 cells.

The role of ATP on regulation of the Na(+)/K(+)-ATPase activity in the human cancerous HeLa cells was investigated. HeLa cells stimulated with increasing ATP concentrations showed a dose-dependent inhibition of the Na(+)/K(+)-ATPase activity. These effects were also obtained by UTP. ATP and UTP provoked a rise in intracellular calcium concentration ([Ca(2+)](i)) persisting for at least 4 min. The inhibitor of phospholipase C, U73122, blocked the elevation of [Ca(2+)](i) provoked by ATP/UTP. The expression of mRNA for P2Y2 and P2Y6 receptors was demonstrated by RT-PCR. ATP/UTP activated PKC-alpha, -betaI and -epsilon isoforms, but not PKC-delta and -zeta. The inhibition of the Na(+)/K(+)-ATPase activity by ATP/UTP was blocked by Gö6976, a specific inhibitor of the calcium-dependent PKCs. In conclusion, our results suggest that ATP/UTP modulate Na(+)/K(+)-ATPase activity in HeLa cells through the P2Y2 purinoceptor via calcium mobilisation and activation of calcium-dependent PKCs., (Copyright 2002 Elsevier Science Inc.)

In PC-Cl3 rat thyroid cell line, ATP and UTP provoked a transient increase in [Ca(2+)](i), followed by a lower sustained phase. Removal of extracellular Ca(2+) reduced the initial transient response and completely abolished the plateau phase. Thapsigargin (TG) caused a rapid rise in [Ca(2+)](i) and subsequent addition of ATP was without effect. The transitory activation of [Ca(2+)](i) was dose-dependently attenuated in cells pretreated with the specific inhibitor of phospholipase C (PLC), U73122. These data suggest that the ATP-stimulated increment of [Ca(2+)](i) required InsP(3) formation and binding to its specific receptors in Ca(2+) stores. Desensitisation was demonstrated with respect to the calcium response to ATP and UTP in Fura 2-loaded cells. Further studies were performed to investigate whether the effect of ATP on Ca(2+) entry into PC-Cl3 cells was via L-type voltage-dependent Ca(2+) channels (L-VDCC) and/or by the capacitative pathway. Nifedipine decreased ATP-induced increase on [Ca(2+)](i). Addition of 2 mM Ca(2+) induced a [Ca(2+)](i) rise after pretreatment of the cells with TG or with 100 microM ATP in Ca(2+)-free medium. These data indicate that Ca(2+) entry into PC-Cl3 stimulated with ATP occurs through both an L-VDCC and through a capacitative pathway. Using buffers with differing Na(+) concentrations, we found that the effects of ATP were dependent of extracellular Na(+), suggesting that a Na(+)/Ca(2+) exchange mechanism is also operative. These data suggest the existence, in PC-Cl3 cell line, of a P2Y purinergic receptor able to increase the [Ca(2+)](i) via PLC activation, Ca(2+) store depletion, capacitative Ca(2+) entry and L-VDCC activation.