Computational Analyses of YY1 and Its Target RKIP Reveal Their Diagnostic and Prognostic Roles in Lung Cancer.

Lung cancer (LC) represents a global threat, being the tumor with the highest mortality rate. Despite the introduction of novel therapies (e.g., targeted inhibitors, immune-checkpoint inhibitors), relapses are still very frequent. Accordingly, there is an urgent need for reliable predictive biomarkers and therapeutically druggable targets. Yin-Yang 1 (YY1) is a transcription factor that may work either as an oncogene or a tumor suppressor, depending on the genotype and the phenotype of the tumor. The Raf Kinase Inhibitory Protein (RKIP), is a tumor suppressor and immune enhancer often found downregulated in the majority of the examined cancers. In the present report, the role of both YY1 and RKIP in LC is thoroughly explored through the analysis of several deposited RNA and protein expression datasets. The computational analyses revealed that YY1 negatively regulates RKIP expression in LC, as corroborated by the deposited YY1-ChIP-Seq experiments and validated by their robust negative correlation. Additionally, YY1 expression is significantly higher in LC samples compared to normal matching ones, whereas RKIP expression is lower in LC and high in normal matching tissues. These observed differences, unlike many current biomarkers, bear a diagnostic significance, as proven by the ROC analyses. Finally, the survival data support the notion that both YY1 and RKIP might represent strong prognostic biomarkers. Overall, the reported findings indicate that YY1 and RKIP expression levels may play a role in LC as potential biomarkers and therapeutic targets. However, further studies will be necessary to validate the in silico results.

Lateral pharyngoplasty vs. traditional uvulopalatopharyngoplasty for patients with OSA: systematic review and meta-analysis.

OBJECTIVES: To compare the efficacy and success rates of lateral pharyngoplasty techniques (LP) vs. uvulopalatopharyngoplasty (UPPP) among adult patients surgically treated for obstructive sleep apnea. METHODS: A systematic literature review of the last 20 years' papers was conducted using PubMed/Medline, Embase, Web of Science, Scholar, and the Cochrane Library until April 2021. Only full-text English articles comparing LP and UPPP outcomes in adult patients with objective outcomes were included in the study. RESULTS: We included 9 articles for a total of 312 surgically treated patients with OSA. LP techniques for obstructive sleep apnea were used on 186 (60%) subjects, while 126 patients (40%) were treated with UPPP. Both surgical procedures resulted in significant improvements in apnea-hypopnea index (AHI), Epworth Sleepiness Scale (ESS) score, and lowest oxygen saturation (LOS) (p < 0.001 in all cases). Although better outcomes were reported with lateral pharyngoplasty, the differences were not significant compared to UPPP post-operative results (p > 0.05 in all cases). CONCLUSIONS: UPPP and LP are both effective surgical procedures in treating OSA in adults. Although not significant, LPs demonstrated improved post-operative outcomes. However, further evidence comparing the surgical effect on patients with OSA is needed to discriminate post-operative outcomes.

Quality of Life After Total LaryngectomyVoice-Related Quality of Life After Total Laryngectomy: Systematic Review and Meta-Analysis.

BACKGROUND: To compare voice rehabilitation-related quality of life among patients surgically treated for total laryngectomy and rehabilitated with esophageal (EV) and tracheoesophageal (TEV) voice. METHODS: A systematic literature review of articles from the past 20 years was conducted, and only full-text English articles comparing VTE and EV results in laryngectomized patients were included. RESULTS: We provided 15 articles for a total of 1085 laryngectomized patients undergoing voice rehabilitation, of which 869 (80.1%) were treated with voice prosthesis while 216 (19.9%) to esophageal speech. Pooled VHI outcomes showed a significantly better score for the TEV group than EV one (31.93±12.11 versus 35.39±20.6; P = 0.003), but no significant difference was recorded at VrQoL (8.27±5.98 versus 9.27±2.02; P = 0.19). CONCLUSION: TEV and EV are both effective procedures in voice rehabilitation after laryngectomy. Although TEV allows for significantly better speech performance, it does not necessarily correlate with a high VrQoL.

Effects of hormone therapy containing 2 mg drospirenone and 1 mg 17ß-estradiol on postmenopausal exacerbation of Meniere's disease: Preliminary study.

The present study aimed to evaluate the effects of 2 mg drospirenone (DRSP) and 1 mg 17ß-estradiol (E2) hormone therapy (HT) in combination with rehabilitation therapy for postmenopausal women with Meniere's disease (MD). The combined drug hormone treatment was denoted as DRSP/E2. A total of 65 postmenopausal female patients with MD and severe distress were enrolled in the present prospective study. A total of 31 women comprised the study group (group A), undergoing HT and rehabilitation therapy, whereas 34 women who opted for rehabilitation therapy alone comprised the control group (group B). Vestibular function and distress associated with MD were assessed by stabilometry and the Dizziness Handicap Inventory (DHI) questionnaire, respectively. The data were collected at baseline and during the 3- and 6-month follow-up visits. The areas of the stabilometric ellipses exhibited a higher reduction in group A compared with group B with regard to the baseline values at both follow-up assessments (P<0.001). At baseline, both groups exhibited severe self-perceived discomfort, with similar DHI scores of 72.3±3.7 (group A) and 70.6±3.9 (group B; P=0.07). At the 3-month follow-up, both groups underwent a gradual improvement, which was significantly higher in group A (47.5±3.7) compared with in group B (64.2±3.3; P<0.001). At the 6-month follow-up, the DHI scores were improved in group A (43.4±3.4) compared with in group B (58.5±3.1; P<0.001). Therefore, DRSP/E2 HT was effective in reducing the fluid overload, which is characteristic of MD. The findings of the present study demonstrated that integrated therapy based on the administration of DRSP/E2 HT and rehabilitation may be more effective compared with rehabilitation alone for the management of postmenopausal exacerbation of MD.

Droplet Digital PCR Analysis of Liquid Biopsy Samples Unveils the Diagnostic Role of hsa-miR-133a-3p and hsa-miR-375-3p in Oral Cancer.

Despite the availability of screening programs, oral cancer deaths are increasing due to the lack of diagnostic biomarkers leading to late diagnosis and a poor prognosis. Therefore, there is an urgent need to discover novel effective biomarkers for this tumor. On these bases, the aim of this study was to validate the diagnostic potential of microRNAs (miRNAs) through the analysis of liquid biopsy samples obtained from ten oral cancer patients and ten healthy controls. The expression of four selected miRNAs was evaluated by using droplet digital PCR (ddPCR) in a pilot cohort of ten oral cancer patients and ten healthy donors. Bioinformatics analyses were performed to assess the functional role of these miRNAs. The expression levels of the predicted down-regulated hsa-miR-133a-3p and hsa-miR-375-3p were significantly reduced in oral cancer patients compared to normal individuals while no significant results were obtained for the up-regulated hsa-miR-503-5p and hsa-miR-196a-5p. ROC analysis confirmed the high sensitivity and specificity of hsa-miR-375-3p and hsa-miR-133a-3p. Therefore, both miRNAs are significantly down-regulated in cancer patients and can be used as biomarkers for the early diagnosis of oral cancer. The analysis of circulating miRNAs in a larger series of patients is mandatory to confirm the results obtained in this pilot study.

Droplet digital PCR for the detection and monitoring of Legionella pneumophila.

Legionella pneumophila (L. pneumophila) is a harmful pathogen often found in water systems. In hospitals, the absence of L. pneumophila in water systems is mandatory by law, therefore, frequent and effective monitoring of water is of fundamental importance. Molecular methods based on reverse transcription­quantitative polymerase chain reaction (RT­qPCR) have been proposed for the detection of L. pneumophila, however, the sensitivity and accuracy of these methods have not been validated yet. Therefore, it is important to evaluate other strategies able to overcome the limits of culture­based and RT­qPCR methods. On these bases, we compared the sensitivity and accuracy of droplet digital PCR (ddPCR) and RT­qPCR in water samples with known concentrations of L. pneumophila and in an in vitro model of water heat treatments. ddPCR showed a higher sensitivity rate and accuracy compared to RT­qPCR in detecting low bacterial load. In addition, ddPCR is not affected by the presence of fragmented DNA and showed higher accuracy than RT­qPCR in monitoring the efficacy of heat shock treatments. In conclusion, ddPCR represents an innovative strategy to effectively detect L. pneumophila in water samples. Thanks to its high robustness, ddPCR could be applied also for the detection of L. pneumophila in patients with suspected legionellosis.

Cancer Management during COVID-19 Pandemic: Is Immune Checkpoint Inhibitors-Based Immunotherapy Harmful or Beneficial?

The coronavirus disease 2019 (COVID-19) is currently representing a global health threat especially for fragile individuals, such as cancer patients. It was demonstrated that cancer patients have an increased risk of developing a worse symptomatology upon severe acute respiratory syndrome associated coronavirus-2 (SARS-CoV-2) infection, often leading to hospitalization and intensive care. The consequences of this pandemic for oncology are really heavy, as the entire healthcare system got reorganized. Both oncologists and cancer patients are experiencing rescheduling of treatments and disruptions of appointments with a concurrent surge of fear and stress. In this review all the up-to-date findings, concerning the association between COVID-19 and cancer, are reported. A remaining very debated question regards the use of an innovative class of anti-cancer molecules, the immune checkpoint inhibitors (ICIs), given their modulating effects on the immune system. For that reason, administration of ICIs to cancer patients represents a question mark during this pandemic, as its correlation with COVID-19-associated risks is still under investigation. Based on the mechanisms of action of ICIs and the current evidence, we suggest that ICIs not only can be safely administered to cancer patients, but they might even be beneficial in COVID-19-positive cancer patients, by exerting an immune-stimulating action.

Evaluation of an improved small gauge needle for venipuncture in children with difficult venous access: Impact on sample quality, phlebotomist satisfaction and patient pain perception.

BACKGROUND: Smaller needles gauge (G) may reduce pain and improve vein access in difficult venous access (DVA). Aims were to compare the performances of two Beckton-Dickinson (BD) Vacutainer® Blood Collection Sets in a pediatric setting: UltraTouch™ Push Button (UT-PBBCS) and Safety-Lok™ (SLBCS). MATERIALS AND METHODS: Questionnaires were used to record venipuncture features, patient pain perception and phlebotomist difficulty score. Specimen quality was evaluated by hemolysis index (HI) on Roche Cobas® 6000. RESULTS: SLBCS (21/23G) or UT-PBBCS (23/25G) were used in 211 (50.2%) and 209 (49.8%) subjects. Pain was associated with age (p < 0.0001) and was lower in UT-PBBCS (p = 0.0339). Difficulty was significantly associated with age (p = 0.002), not with needle gauge (p = 0.461) and it was 0.42 points lower in UT-PBBCS. HI was not associated with blood collection set (p = 0.385). CONCLUSIONS: UT-PBBCS globally performed better than SLBCS and could enhance phlebotomy and patient comfort, without affecting sample quality in pediatric patients with DVA.

Influence of cigarette smoking on allergic rhinitis: a comparative study on smokers and non-smokers.

It has been described that exposure to tobacco smoke causes worsening of allergic rhinitis symptoms. Otherwise, some studies have demonstrated a negative association between cigarette smoke and allergic rhinitis (AR). Given this inconsistency, this study evaluated the quality of life and immuno-inflammatory parameters in current smokers and nonsmokers suffering from AR. A comparative cross-sectional study was conducted in patients who presented symptoms of AR. Patients were categorized into two groups: current smokers and non-smokers based on salivary cotinine measurements. Primary outcomes were the levels of immuno-inflammatory biomarkers (IgE, IL-4, IL-5, IL-13, IL-17, and IL-33) in serum and nasal lavage and the quality of life assessed by the Mini Rhinoconjunctivitis Quality of Life Questionnaire (MiniRQLQ). Secondary outcomes included salivary cotinine levels, and pulmonary function parameters, such as forced vital capacity (FVC), forced expiratory volume in 1s (FEV1), and FEV1/FVC ratio. Twenty-two patients per group were included in the analysis, with no significant difference regarding demographic characteristics. Statistically significant higher values in salivary cotinine levels (p<0.001) and lower lung function FEV1 (p=0.044) and FEV1/FVC (p=0.047) were found in smokers than in nonsmokers. Only serum IL-33 was significantly different in the 2 groups (p<0.001): smokers had higher values compared to non-smokers. There were no significant differences in MiniRQLQ parameters. Although cigarette smoking was not associated with more severe symptoms, smoking could be associated with increased risk of developing airway remodeling and decreased lung function in AR patients, thus appropriate treatment should be prescribed if smoke avoidance is unfeasible.

Effects of combined oral contraception containing drospirenone on premenstrual exacerbation of Meniere's disease: Preliminary study.

OBJECTIVES: Meniere's disease is caused by an augmented endolymph pressure in the inner ear; symptoms are vertigo, fluctuating hearing loss and tinnitus. Exacerbations has been noted during premenstrual phase. The study aims to evaluate the effects of a 20 µm Ethinylestradiol (EE) and 3 mg Drospirenone (DRSP) oral contraceptive (20 µmEE/3mgDRSP) in continuous regimen, associated with rehabilitation therapy on Meniere's disease. STUDY DESIGN: This non-randomized controlled study was performed from October 2015 to October 2017. Forty-two premenopausal women affected by MD with severe distress in the premenstrual phase were enrolled. Sixteen women constituted the study group (Group A), and twenty women constituted the control group (Group B). Group A underwent EE/DRSP therapy and rehabilitation and Group B underwent rehabilitation therapy alone. Stabilometry and the Dizziness Handicap Inventory questionnaire were used to measure vestibular function and distress related to the disease, respectively, at baseline (T0), 3 months (T1) and 6 months (T2). RESULTS: At T0, both groups had large, similar areas of stabilometric ellipses (p = NS) that reduced more in Group A than in Group B, at T1 and T2 (p < 0.001). High scores of the DHI (cut-off ≤54) were observed at T0 in both groups (A 66.8 ±â€¯2.8 vs B 65.5 ±â€¯3.6; p = NS). At T1, a gradual improvement in both groups was observed, manly in Group A (A 45.1 ±â€¯3.6 vs B 62.4 ±â€¯4.1; p < 0.001). At T2, the DHI scores were significantly lower in Group A (39.2 ±â€¯3.8) compared to Group B (68.8 ±â€¯3.6) (p < 0.001). CONCLUSIONS: DRSP could be effective in reducing the fluid overload typical of the premenstrual phase, improving symptoms of MD. The results support the efficacy of EE/DRSP usage associated with rehabilitation therapy on premenstrual exacerbation of MD.

Relationship between sleeping difficulties and airway symptoms severity with the health-related quality of life in patients with GERD.

BACKGROUND: Gastroesophageal reflux disease (GERD) is a chronic disease highly associated with several pulmonary disease and sleep disorders leading to a negative impact on the quality of life. Aim of this study was to investigate the correlation between these disorders and the health-related quality of life. METHODS: Patients were recruited from the Department of Medical Sciences, Surgical and Advanced Technologies, GF Ingrassia, University of Catania, from October 2015 to May 2016. Enrolled patients were classified by different symptomology severity presentation according to the Reflux, Airway and Sleep Questionnaire (RASQ). General data, modified GERD impact scores and the 36-Item Short Form (SF-36) questionnaire scores of these patients were analyzed. RESULTS: A total of 173 patients were included into the study. Patients with moderate severity of airway symptoms had a significant lower SF-36 scores than those with mild severity. Patients with advanced sleeping difficulties severity owned the lowest scores among all cases. The impact to the daily activity of each affected individuals had a positive association with the severity of airway and sleeping disorders. CONCLUSIONS: The severity of these reflux-related disorders is well correlated with a significant negative impact on the normal daily activity. The cases with advanced severity of sleeping difficulties had the worst well-being.

Analysis of STAT3 post-translational modifications (PTMs) in human prostate cancer with different Gleason Score.

Prostate Cancer (PCa) is a complex and heterogeneous disease. The androgen receptor (AR) and the signal transducer and activator of transcription 3 (STAT3) could be effective targets for PCa therapy. STAT3, a cytoplasmatic latent transcription factor, is a hub protein for several oncogenic signalling pathways and up-regulates the expression of numerous genes involved in tumor cell proliferation, angiogenesis, metastasis and cell survival. STAT3 activity can be modulated by several Post-Translational Modifications (PTMs) which reflect particular cell conditions and may be implicated in PCa development and progression. The aim of this work was to analyze STAT3 PTMs at different tumor stages and their relationship with STAT3 cellular functions. For this purpose, sixty-five prostatectomy, Formalin-fixed paraffin-embedded (FFPE) specimens, classified with different Gleason Scores, were subjected to immunoblotting, immunofluorescence staining and RT-PCR analysis. All experiments were carried out in matched non-neoplastic and neoplastic tissues. Data obtained showed different STAT3 PTMs profiles among the analyzed tumor grades which correlate with differences in the amount and distribution of specific STAT3 interactors as well as the expression of STAT3 target genes. These results highlight the importance of PTMs as an additional biomarker for the exactly evaluation of the PCa stage and the optimal treatment of this disease.

Analysis of the interaction of calcitriol with the disulfide isomerase ERp57.

Calcitriol, the active form of vitamin D3, can regulate the gene expression through the binding to the nuclear receptor VDR, but it can also display nongenomic actions, acting through a membrane-associated receptor, which has been discovered as the disulfide isomerase ERp57. The aim of our research is to identify the binding sites for calcitriol in ERp57 and to analyze their interaction. We first studied the interaction through bioinformatics and fluorimetric analyses. Subsequently, we focused on two protein mutants containing the predicted interaction domains with calcitriol: abb'-ERp57, containing the first three domains, and a'-ERp57, the fourth domain only. To consolidate the achievements we used the calorimetric approach to the whole protein and its mutants. Our results allow us to hypothesize that the interaction with the a' domain contributes to a greater extent than the other potential binding sites to the dissociation constant, calculated as a Kd of about 10-9 M.

Upregulation of TPX2 by STAT3: identification of a novel STAT3 binding site.

TPX2, a protein involved in mitosis, is considered a good marker for actively proliferating tissues, highly expressed in a number of cancer cells. We show the presence of high-affinity binding site for STAT3 in the 5'-flanking region of the Tpx2 gene, which is in vivo bound by activated STAT3. A specific STAT3 peptide inhibitor represses the expression of the Tpx2 gene and inhibits the binding of STAT3 to its consensus sequence in human cell lines where STAT3 is activated. These results indicate that activated STAT3 contributes to the over-expression of Tpx2 through the binding to an enhancer site.

The binding of silibinin to ERp57.

The flavonoid silibinin is known to intervene in many cellular processes involved in a variety of pathologies, thus appearing a promising therapeutic tool. The molecular mechanisms responsible for these activities, however, have not been clearly defined, and although some of its interactions with proteins have been identified, the relative affinities are often too low to appear relevant in vivo. Here we describe the interaction of silibinin with the protein disulfide isomerase ERp57, characterized by a submicromolar dissociation constant. This interaction enhances the formation of a ERp57/REF-1 complex, and furthermore appears to affect the intracellular distribution of ERp57. This protein is involved in signaling pathways which are also affected by silibinin. This suggests that the ERp57-silibinin interaction might explain at least some of the biological effects caused by the flavonoid.

Altered expression of the CB1 cannabinoid receptor in the triple transgenic mouse model of Alzheimer's disease.

The endocannabinoid system has gained much attention as a new potential pharmacotherapeutic target in various neurodegenerative diseases, including Alzheimer's disease (AD). However, the association between CB1 alterations and the development of AD neuropathology is unclear and often contradictory. In this study, brain CB1 mRNA and CB1 protein levels were analyzed in 3 × Tg-AD mice and compared to wild-type littermates at 2, 6 and 12 months of age, using in-situ hybridization and immunohistochemistry, respectively. Semiquantitative analysis of CB1 expression focused on the prefrontal cortex (PFC), prelimbic cortex, dorsal hippocampus (DH), basolateral amygdala complex (BLA), and ventral hippocampus (VH), all areas with high CB1 densities that are strongly affected by neuropathology in 3 × Tg-AD mice. At 2 months of age, there was no change in CB1 mRNA and protein levels in 3 × Tg-AD mice compared to Non-Tg mice in all brain areas analyzed. However, at 6 and 12 months of age, CB1 mRNA levels were significantly higher in PFC, DH, and BLA, and lower in VH in 3 × Tg-AD mice compared to wild-type littermates. CB1 immunohistochemistry revealed that CB1 protein expression was unchanged in 3 × Tg-AD at 2 and 6 months of age, while a significant decrease in CB1 receptor immunoreactivity was detected in the BLA and DH of 12-month-old 3 × Tg-AD mice, with no sign of alteration in other brain areas. The altered CB1 levels appear, rather, to be age-and/or pathology-dependent, indicating an involvement of the endocannabinoid system in AD pathology and supporting the ECS as a potential novel therapeutic target for treatment of AD.

Green tea catechins can bind and modify ERp57/PDIA3 activity.

BACKGROUND: Green tea is a rich source of polyphenols, mainly catechins (flavanols), which significantly contribute to the beneficial health effects of green tea in the prevention and treatment of various diseases. In this study the effects of four green tea catechins on protein ERp57, also known as protein disulfide isomerase isoform A3 (PDIA3), have been investigated in an in vitro model. METHODS: The interaction of catechins with ERp57 was explored by fluorescence quenching and surface plasmon resonance techniques and their effect on ERp57 activities was investigated. RESULTS: A higher affinity was observed for galloylated cathechins, which bind close to the thioredoxin-like redox-sensitive active sites of the protein, with a preference for the oxidized form. The effects of these catechins on ERp57 properties were also investigated and a moderate inhibition of the reductase activity of ERp57 was observed as well as a strong inhibition of ERp57 DNA binding activity. CONCLUSIONS: Considering the high affinity of galloylated catechins for ERp57 and their capability to inhibit ERp57 binding to other macromolecular ligands, some effects of catechins interaction with this protein on eukaryotic cells may be expected. GENERAL SIGNIFICANCE: This study provides information to better understand the molecular mechanisms underlying the biological activities of catechins and to design new polyphenol-based ERp57-specific inhibitors.

ERp57/PDIA3 binds specific DNA fragments in a melanoma cell line.

ERp57/PDIA3 is a ubiquitously expressed disulfide isomerase protein, which acts in concert with calreticulin and calnexin in the folding of glycoproteins destined to the plasma membrane or to be secreted. Its canonical compartment is the endoplasmic reticulum, where it acts as a chaperone and redox catalyst, but non canonical locations have been described as well, and ERp57 has been found associated with DNA and nuclear proteins. In previous work performed in HeLa cells, ERp57 has been demonstrated to bind specific DNA sequences involved in the stress response. The direct interaction with the DNA sequences identified as ERp57-targeted regions in HeLa cells has now been confirmed in a melanoma cell line. Furthermore, the ERp57 silencing, achieved by RNA interference, has produced a significant down-regulation of the expression of target genes. The possible involvement of other proteins in complex with ERp57 has been studied by an in vitro biotin-streptavidin based binding assay and the interacting protein APE/Ref-1 has been also assessed for its direct association with the ERp57 target regions. In conclusion, nuclear ERp57 interacts in vivo with DNA fragments in melanoma cells and is potentially involved in the transcriptional regulation of its target genes.

Detection of serum IgA to HSV1 and its diagnostic role in sudden hearing loss.

A viral etiology of sudden hearing loss has been hypothesized by many authors. HSV1 neurotropism and its involvement in sudden hearing loss has implicated HSV1 as one of the most accredited etiological agents. A non-invasive method such as the titration of HSV1-specific IgA was evaluated to determine the role of HSV1 as a possible cause sudden hearing loss. A prospective study was carried out by titration of serum IgA to HSV1 in 93 patients and in a control group of 50 healthy subjects and 35 subjects suffering from recent herpes labialis reactivation. Statistical analysis of the results disclosed that IgA titers to HSV1 higher than 1:80 are suggestive for the association of HSV1 infection and sudden hearing loss. Moreover, acyclovir therapy was effective in 81% of patients who showed high specific IgA titers. Overall, the titration of specific serum IgA to HSV1 can be a useful tool to determine the viral etiology of certain cases of sudden hearing loss. This method is simple to perform and minimally invasive. It can lead to a rapid presumptive diagnosis and to prompt specific therapy, reducing the need for corticosteroids.

Nanomolar melatonin enhances nNOS expression and controls HaCaT-cells bioenergetics.

A novel role of melatonin was unveiled, using immortalized human keratinocyte cells (HaCaT) as a model system. Within a time window compatible with its circadian rhythm, melatonin at nanomolar concentration raised both the expression level of the neuronal nitric oxide synthase mRNA and the nitric oxide oxidation products, nitrite and nitrate. On the same time scale, a depression of the mitochondrial membrane potential was detected together with a decrease of the oxidative phosphorylation efficiency, compensated by glycolysis as testified by an increased production of lactate. The melatonin concentration, ∼ nmolar, inducing the bioenergetic effects and their time dependence, both suggest that the observed nitric oxide-induced mitochondrial changes might play a role in the metabolic pathways characterizing the circadian melatonin chemistry.