The circadian dynamics of the hippocampal transcriptome and proteome is altered in experimental temporal lobe epilepsy.

Gene and protein expressions display circadian oscillations, which can be disrupted in diseases in most body organs. Whether these oscillations occur in the healthy hippocampus and whether they are altered in epilepsy are not known. We identified more than 1200 daily oscillating transcripts in the hippocampus of control mice and 1600 in experimental epilepsy, with only one-fourth oscillating in both conditions. Comparison of gene oscillations in control and epilepsy predicted time-dependent alterations in energy metabolism, which were verified experimentally. Although aerobic glycolysis remained constant from morning to afternoon in controls, it increased in epilepsy. In contrast, oxidative phosphorylation increased in control and decreased in epilepsy. Thus, the control hippocampus shows circadian molecular remapping, which is altered in epilepsy. We suggest that the hippocampus operates in a different functioning mode in epilepsy. These alterations need to be considered when studying epilepsy mechanisms, designing drug treatments, and timing their delivery.

Robot-assisted radical cystectomy with totally intracorporeal urinary diversion: surgical and early functional outcomes through the learning curve in a single high-volume center.

The aim of the study is to report surgical and early functional outcomes of first 100 patients undergoing robot-assisted radical cystectomy (RARC) with totally intracorporeal urinary diversion (ICUD) in a single center. The main surgeon (A.P.) attended a modular training program at a referring center mentored by a worldwide-recognized robotic surgeon (P.W.). The program consisted of: (a) 10 h of theoretical lessons; (b) video session (c) step-by-step in vivo modular training. Each procedure was performed as taught, without any technique variation. Demographics, intra-operative data and post-operative complications, along with early functional outcomes, were recorded for each patient. We retrospectively evaluated the first consecutive 100 patients submitted to RARC with totally ICUD from July 2015 to December 2018. Median age at surgery was 69 years (IQR 60-74). 52 (52%), 32 (32%), and 17 (17%) patients received orthotopic neobladder, ileal conduit and uretero-cutaneostomy, respectively. Median operative time was 410 min. A median number of lymph nodes retrieved were 27 and median estimated blood loss was 240 mL with median hospitalization time of 7 days. All procedures were completed successfully without open conversion. A statistically significant improvement was found in the late (30-90 post-operative days) post-operative complications (p = 0.02) and operative time for urinary derivation. At multivariate logistic regression model ASA score ≥ 3 (OR = 4.2, p = 0.002) and number of lymph nodes retrieved (OR = 1.16, p = 0.02) were found to be predictors of 90-day complications. An adequate modular training is paramount to obtain successful results and reduce the learning curve of RARC, as demonstrated by our experience.

Coupling circadian rhythms of metabolism and chromatin remodelling.

The circadian clock controls a large variety of neuronal, endocrine, behavioural and physiological responses in mammals. This control is exerted in large part at the transcriptional level on genes expressed in a cyclic manner. A highly specialized transcriptional machinery based on clock regulatory factors organized in feedback autoregulatory loops governs a significant portion of the genome. These oscillations in gene expression are paralleled by critical events of chromatin remodelling that appear to provide plasticity to circadian regulation. Specifically, the nicotinamide adenine dinucleotide (NAD)(+) -dependent deacetylases SIRT1 and SIRT6 have been linked to circadian control of gene expression. This, and additional accumulating evidence, shows that the circadian epigenome appears to share intimate links with cellular metabolic processes and has remarkable plasticity showing reprogramming in response to nutritional challenges. In addition to SIRT1 and SIRT6, a number of chromatin remodellers have been implicated in clock control, including the histone H3K4 tri-methyltransferase MLL1. Deciphering the molecular mechanisms that link metabolism, epigenetic control and circadian responses will provide valuable insights towards innovative strategies of therapeutic intervention.

How pervasive are circadian oscillations?

Circadian oscillations play a critical role in coordinating the physiology, homeostasis, and behavior of biological systems. Once thought to only be controlled by a master clock, recent high-throughput experiments suggest many genes and metabolites in a cell are potentially capable of circadian oscillations. Each cell can reprogram itself and select a relatively small fraction of this broad repertoire for circadian oscillations, as a result of genetic, environmental, and even diet changes.

Epigenetic control and the circadian clock: linking metabolism to neuronal responses.

Experimental and epidemiological evidence reveal the profound influence that industrialized modern society has imposed on human social habits and physiology during the past 50 years. This drastic change in life-style is thought to be one of the main causes of modern diseases including obesity, type 2 diabetes, mental illness such as depression, sleep disorders, and certain types of cancer. These disorders have been associated to disruption of the circadian clock, an intrinsic time-keeper molecular system present in virtually all cells and tissues. The circadian clock is a key element in homeostatic regulation by controlling a large array of genes implicated in cellular metabolism. Importantly, intimate links between epigenetic regulation and the circadian clock exist and are likely to prominently contribute to the plasticity of the response to the environment. In this review, we summarize some experimental and epidemiological evidence showing how environmental factors such as stress, drugs of abuse and changes in circadian habits, interact through different brain areas to modulate the endogenous clock. Furthermore we point out the pivotal role of the deacetylase silent mating-type information regulation 2 homolog 1 (SIRT1) as a molecular effector of the environment in shaping the circadian epigenetic landscape.

Evolution of transmitted HIV-1 drug resistance in HIV-1-infected patients in Italy from 2000 to 2010.

Prevalence and predictors of transmitted drug resistance (TDR), defined as the presence of at least one WHO surveillance drug resistance mutation (SDRM), were investigated in antiretroviral-naïve HIV-1-infected patients, with a genotypic resistance test (GRT) performed ≤6 months before starting cART between 2000 and 2010. 3163 HIV-1 sequences were selected (69% subtype B). Overall, the prevalence of TDR was 12% (13.2% subtype B, 9% non-B). TDR significantly declined overall and for the single drug classes. Older age independently predicted increased odds of TDR, whereas a more recent GRT, a higher HIV-RNA and C vs. B subtype predicted lower odds of TDR.

Syphilis with HIV in Florence, 2003-2009: a 7-year epidemiological study.

The aims of this study were to describe the trend of acquired syphilis in the city of Florence and its province over a 7-year period, to investigate sexual behaviours in the syphilis-infected population and to analyse syphilis/HIV co-infection. A total of 259 patients were classified according to age, sex and HIV infection. We estimated that from 2004 to 2008 cases increased by 248%. Most patients with concurrent HIV infection were male (31-45 years), but 40- to 60-year-old men who had sex with men predominated in both male and HIV-positive patients. Oral sex was identified as the most significant route of transmission, although most patients did not consider it so. Late-presenters with HIV accounted for 33% of HIV-positive patients: they were unaware of their HIV status and showed syphilis lesions only. In these cases, syphilis heralded the presence of HIV infection and allowed earlier diagnosis.

Human immunodeficiency virus-1 B and non-B subtypes with the same drug resistance pattern respond similarly to antiretroviral therapy.

We analysed the 12-week virological response to protease inhibitor (PI) or non-nucleoside reverse transcriptase inhibitor (NNRTI) therapy in 1108 patients carrying B or non-B human immunodeficiency virus (HIV)-1 subtypes with matched resistance mutation patterns. Response rates were not significantly different for non-B and B subtypes stratified for treatment status (51.5% vs. 41.5% in naïve patients; 46.7% vs. 38.7% in experienced patients) or regimens (46.9% vs. 39.7% with PI; 56.7% vs. 40% with NNRTI). No difference in response was detected in patients harbouring B and non-B subtypes with any resistance profile. Further studies are advisable to fully test this approach on larger datasets.

The time of metabolism: NAD+, SIRT1, and the circadian clock.

The mammalian cell contains a molecular clock that contributes, within each organism, to circadian rhythms and variety of physiological and metabolic processes. The clock machinery is constituted by interwined transcriptional-translational feedback loops that, through the action of specific transcription factors, modulate the expression of clock-controlled genes. These oscillations in gene expression necessarily implicate events of chromatin remodeling on a relatively large, global scale, considering that as many 10% of cellular transcripts oscillate in a circadian manner. CLOCK, a transcription factor crucial for circadian function, has intrinsic histone acetyltransferase activity and operates within a large nuclear complex with other chromatin remodelers. CLOCK directs the cyclic acetylation of the histone H3 and of its own partner BMAL1. A search for the histone deacetylase (HDAC) that counterbalanced CLOCK activity revealed that SIRT1, a nicotinamide adenine dinucleotide (NAD(+))-dependent HDAC, functions in a circadian manner. Importantly, SIRT1 is a regulator of several metabolic processes and was found to interact with CLOCK and to be recruited to circadian promoters in a cyclic manner. As many transcripts that oscillate in mammalian peripheral tissues encode proteins that have central roles in metabolic processes, these findings establish a functional and molecular link among energy balance, chromatin remodeling, and circadian physiology.

Update on emergence of HIV-1 resistance to antiretroviral drug classes in an Italian national database: 2007-2009.

We analysed trends of human immunodeficiency virus type 1 (HIV-1) drug resistance during 2007-2009 in the Italian national HIV drug resistance database 'ARCA'. Prevalence of resistance in each year was examined on the basis of the presence of major International AIDS Society-2009 mutations. Predictors of resistance were analysed by multivariable logistic regression. Nine hundred and sixty-six patients were selected. Resistance to nucleoside reverse transcriptase inhibitors and protease inhibitors showed a significant decline with respect to previous surveys. Resistance to any class of drug and three drug classes remained stable. Independent predictors of three-class resistance were the number of treatment regimens experienced, prior suboptimal nucleoside reverse transcriptase inhibitor therapy and the current use of ritonavir-boosted protease inhibitors.

Effectiveness of antiretroviral regimens containing abacavir with tenofovir in treatment-experienced patients: predictors of virological response and drug resistance evolution in a multi-cohort study.

BACKGROUND: In treatment-naïve patients, a combination antiretroviral therapy (cART) containing tenofovir (TDF) and abacavir (ABC) with lamivudine leads to unacceptably high virological failure rates with frequent selection of reverse transcriptase mutations M184V and K65R. We explored the efficacy of at least 16 weeks of ABC + TDF-containing cART regimens in 307 antiretroviral-experienced HIV-1-infected individuals included in observational databases. METHODS: Virological failure was defined as an HIV RNA > 400 copies/ml after at least 16 weeks of treatment. Patients had received a median of three prior cART regimens. Of these, 76% concomitantly received a potent or high genetic barrier regimen (with at least one protease inhibitor [PI]) or non-nucleoside reverse transcriptase inhibitor or thymidine analogue) while a third non-thymidine nucleoside analogue was used in the remaining patients. RESULTS: The 1-year estimated probability of virological failure was 34% in 165 patients with HIV RNA > 400 copies/ ml at ABC + TDF regimen initiation. Independent predictors of virological failure were the absence of a potent or high genetic barrier cART, the higher number of cART regimens experienced, and the use of a new drug class. In the subset of 136 patients for whom there were genotypic resistance test results prior to ABC + TDF initiation, the virological failure (1-year estimated probability 46%) was independently predicted by the higher baseline viral load, the concomitant use of boosted PI, and the presence of reverse transcriptase mutation M41L. In 142 patients starting ABC + TDF therapy with HIV RNA pound < or =400 copies/ml, virological failure (1-year estimated probability 17%) was associated only with the transmission category. In a small subset of subjects for whom there were an available paired baseline and follow-up genotype (n = 28), the prevalence of most nucleoside analogue reverse transcriptase inhibitor resistance mutations decreased, suggesting a possible low adherence to treatment. No selection of K65R was detected. CONCLUSION: The virological response to ABC + TDF-containing regimens in this moderately-to-heavily treatment experienced cohort was good. Higher viral load and the presence of M41L at baseline were associated with worse virological responses, while the concomitant prescription of drugs enhancing the genetic barrier of the regimen conveyed a reduced risk of virological failure. The Appendix provides the names of other members of the MASTER cohort.

Prevalence of transmitted HIV-1 drug resistance in HIV-1-infected patients in Italy: evolution over 12 years and predictors.

OBJECTIVES: Transmitted HIV-1 drug resistance (TDR) can reduce the efficacy of first-line antiretroviral therapy. PATIENTS AND METHODS: A retrospective analysis was performed to assess the prevalence and correlates of TDR in Italy over time. TDR was defined as the presence of at least one of the mutations present in the surveillance drug resistance mutation (SDRM) list. RESULTS: Among 1690 antiretroviral therapy-naive patients, the most frequent HIV subtypes were B (78.8%), CRF02_AG (5.6%) and C (3.6%). Overall, TDR was 15%. TDR was 17.3% in subtype B and 7.0% in non-B carriers (P < 0.001). TDR showed a slight, although not significant, decline (from 16.3% in 1996-2001 to 13.4% in 2006-07, P = 0.15); TDR declined for nucleoside reverse transcriptase inhibitors (from 13.1% to 8.2%, P = 0.003) but remained stable for protease inhibitors (from 3.7% to 2.5%, P = 0.12) and non-nucleoside reverse transcriptase inhibitors (from 3.7% to 5.8%). TDR to any drug was stable in B subtype and showed a decline trend in non-B. In multivariable analysis, F1 subtype or any non-B subtype, compared with B subtype, and higher HIV RNA were independent predictors of reduced odds of TDR. CONCLUSIONS: Prevalence of TDR to nucleoside reverse transcriptase inhibitors seems to have declined in Italy over time. Increased prevalence of non-B subtypes partially justifies this phenomenon.

Nerve growth factor and its receptors TrkA and p75 are upregulated in the brain of mdx dystrophic mouse.

Increased angiogenesis and an altered blood-brain barrier have been reported in the brain of dystrophin-deficient mdx mouse, an experimental model of Duchenne muscular dystrophy. To further elucidate the mechanisms underlying angiogenesis in Duchenne muscular dystrophy, in this study we evaluated whether nerve growth factor (NGF) and nerve growth factor receptors (NGFRs) are involved, then correlated NGF-NGFRs expression with vascular endothelial growth factor (VEGF) and its receptor-2 (VEGFR-2) content and matrix metalloproteinases-2 and -9 (MMP-2 and -9) activity, by confocal laser microscopy and immunohistochemistry. Results showed that neurons, astrocytes and ependymal cells were strongly labeled by NGF in mdx brain, expressing NGFRs on glial and endothelial cells. In controls, NGF faintly labeled neurons and astrocytes, whereas endothelial cells were negative for NGFRs. Immunogold electron microscopy demonstrated NGFR gold particles on endothelial cells in mdx brain, while in controls few particles were recognizable only on glial end feet. Western blotting and real time polymerase chain reaction (RT-PCR) demonstrated a higher expression of NGF and NGFR mRNA and protein in mdx brain as compared to controls, and increase of VEGF-VEGFR-2 and active MMP-2 and -9 content. Overall, these data suggest that in the brain of mdx mice, an upregulation of the NGF-NGFRs system might be involved directly, or indirectly through the activation of VEGF-VEGFR-2 and MMP-2 and -9, in the angiogenic response taking place in this pathological condition.

Treatment of achalasia: lessons learned with Chagas' disease.

Chagas' disease (CD) is highly prevalent in South America. Brazilian surgeons and gastroenterologists gained valuable experience in the treatment of CD esophagopathy (chagasic achalasia) due to the high number of cases treated. The authors reviewed the lessons learned with the treatment of achalasia by different centers experienced in the treatment of Chagas' disease. Preoperative evaluation, endoscopic treatment (forceful dilatation and botulinum toxin injection), Heller's myotomy, esophagectomy, conservative techniques other than myotomy, and reoperations are discussed in the light of personal experiences and review of International and Brazilian literature. Aspects not frequently adopted by North American and European surgeons are emphasized. The review shows that nonadvanced achalasia is frequently treated by Heller's myotomy. Endoscopic treatment is reserved to limited cases. Treatment for end-stage achalasia is not unanimous. Esophagectomy was a popular treatment in advanced disease; however, the morbidity/mortality associated to the procedure made some authors seek different alternatives, such as Heller's myotomy and cardioplasties. Minimally invasive approach to esophageal resection may change this concept, although few centers perform the procedure routinely.

Regulation of human MC2-R gene expression by CREB, CREM, and ICER in the adrenocortical cell line Y1.

The MC2-Receptor (melanocortin 2 receptor, MC2-R) is a Gs-protein coupled receptor that is upregulated by its own ligand ACTH and by forskolin. The mechanisms regulating MC2-R expression are still unclear. We therefore investigated the role of the stimulatory transcription factors CREB and CREM and the inhibitory factor ICER for regulation of human MC2-R expression. We cotransfected mouse adrenocortical Y1 cells with luciferase reporter gene vectors containing full length and deleted human MC2-R promoter constructs with expression plasmids for CREB, CREBS133A, CREMtau, CREMtauS117A, or ICER. Direct protein-DNA interaction was investigated by EMSA. Wild type CREB did not significantly affect promoter activity due to high endogenous CREB activity. However, CREBS133A decreased forskolin stimulated MC2-R promoter activity by 48+/-5% (mean+/-SEM) while unstimulated values remained unchanged. CREMtau moderately increased basal and forskolin stimulated luciferase activity in a dose-dependent manner (maximum effect 252+/-24% and 186+/-13% VS. control vector, respectively). While this effect required the full length promoter, cAMP stimulation was retained in shorter constructs. ICER reduced basal luciferase activity in Y1 cells by 17+/-28%, but completely abolished forskolin stimulation. Although 5'-deletion constructs mapped the minimum promoter region required for ICER effect to the shortest -64/+40 construct, direct protein DNA interaction in this promoter region could not be identified by EMSA. Moreover, mutation of the SF-1 binding sites, which retained ICER dependent inhibition, excluded SF-1 to be required for this effect. We conclude from these data that transcription factors of the CREB/CREM/ATF family have a moderate effect on human MC2-R promoter activity, but seem to play a minor role in transmitting stimulation of the cAMP pathway to increased MC2-R expression.

Synaptic contacts impaired by styrene-7,8-oxide toxicity.

Styrene-7,8-oxide (SO), a chemical compound widely used in industrial applications, is a potential hazard for humans, particularly in occupational settings. Neurobehavioral changes are consistently observed in occupationally exposed individuals and alterations of neurotransmitters associated with neuronal loss have been reported in animal models. Although the toxic effects of styrene have been extensively documented, the molecular mechanisms responsible for SO-induced neurotoxicity are still unclear. A possible dopamine-mediated effect of styrene neurotoxicity has been previously demonstrated, since styrene oxide alters dopamine neurotransmission in the brain. Thus, the present study hypothesizes that styrene neurotoxicity may involve synaptic contacts. Primary striatal neurons were exposed to styrene oxide at different concentrations (0.1-1 mM) for different time periods (8, 16, and 24 h) to evaluate the dose able to induce synaptic impairments. The expression of proteins crucial for synaptic transmission such as Synapsin, Synaptophysin, and RAC-1 were considered. The levels of Synaptophysin and RAC-1 decreased in a dose-dependent manner. Accordingly, morphological alterations, observed at the ultrastructural level, primarily involved the pre-synaptic compartment. In SO-exposed cultures, the biochemical cascade of caspases was activated affecting the cytoskeleton components as their target. Thus the impairments in synaptic contacts observed in SO-exposed cultures might reflect a primarily morphological alteration of neuronal cytoskeleton. In addition, our data support the hypothesis developed by previous authors of reactive oxygen species (ROS) initiating events of SO cytotoxicity.