Inhibition of Connexin43 hemichannels impairs spatial short-term memory without affecting spatial working memory

Astrocytes are active players in higher brain function as they can release gliotransmitters, which are essential for synaptic plasticity. Various mechanisms have been proposed for gliotransmission, including vesicular mechanisms as well as non-vesicular ones, for example by passive diffusion via connexin hemichannels (HCs). We here investigated whether interfering with connexin43 (Cx43) HCs influenced hippocampal spatial memory. We made use of the peptide Gap19 that blocks HCs but not gap junction channels and is specific for Cx43. To this end, we microinfused transactivator of transcription linked Gap19 (TAT-Gap19) into the brain ventricle of male NMRI mice and assessed spatial memory in a Y maze. We found that the in vivo blockade of Cx43 HCs did not affect the locomotor activity or spatial working memory in a spontaneous alternation Y maze task. Cx43 blockade did however significantly impair the spatial short-term memory in a delayed spontaneous alternation Y maze task. These results indicate that Cx43 HCs play a role in spatial short-term memory

Inhibition of astroglial connexin43 hemichannels with TAT-Gap19 exerts anticonvulsant effects in rodents

Accumulating evidence shows a key function for astrocytic connexin43 (Cx43) signaling in epilepsy. However, the lack of experimental distinction between Cx43 gap junction channels (GJCs) and hemichannels (HCs) has impeded the identification of the exact contribution of either channel configurations to epilepsy. We therefore investigated whether TAT-Gap19, a Cx mimetic peptide that inhibits Cx43 HCs but not the corresponding Cx43 GJCs, influences experimentally induced seizures in rodents. Dye uptake experiments in acute hippocampal slices of mice demonstrated that astroglial Cx43 HCs open in response to the chemoconvulsant pilocarpine and this was inhibited by TAT-Gap19. In vivo, pilocarpine-induced seizures as well as the accompanying increase in D-serine microdialysate levels were suppressed by Cx43 HC inhibition. Moreover, the anticonvulsant action of TAT-Gap19 was reversed by exogenous D-serine administration, suggesting that Cx43 HC inhibition protects against seizures by lowering extracellular D-serine levels. The anticonvulsive properties of Cx43 HC inhibition were further confirmed in electrical seizure mouse models, i.e. an acute 6 Hertz (Hz) model of refractory seizures and a chronic 6 Hz corneal kindling model. Collectively, these results indicate that Cx43 HCs play a role in seizures and underscore their potential as a novel and druggable target in epilepsy treatment

Curcumin, Polydatin and Quercetin Synergistic Activity Protects from High-Glucose-Induced Inflammation and Oxidative Stress

none10noChronic hyperglycemia, the diagnostic biomarker of Type 2 Diabetes Mellitus (T2DM), is a condition that fosters oxidative stress and proinflammatory signals, both involved in the promotion of cellular senescence. Senescent cells acquire a proinflammatory secretory phenotype, called SASP, exacerbating and perpetuating the detrimental effects of hyperglycemia. Bioactive compounds can exert antioxidant and anti-inflammatory properties. However, the synergistic anti-inflammatory and antioxidant effects of the most extensively investigated natural compounds have not been confirmed yet in senescent cells and in hyperglycemic conditions. Here, we exposed young and replicative senescent HUVEC (yHUVEC and sHUVEC) to a high-glucose (HG) condition (45 mM) and treated them with Polydatin (POL), Curcumin (CUR) and Quercetin (QRC), alone or in combination (MIX), to mirror the anti-inflammatory component OxiDefTM contained in the novel nutraceutical GlicefenTM (Mivell, Italy). In both yHUVEC and sHUVEC, the MIX significantly decreased the expression levels of inflammatory markers, such as MCP-1, IL-1β and IL-8, and ROS production. Importantly, in sHUVEC, a synergistic effect of the MIX was observed, suggesting its senomorphic activity. Moreover, the MIX was able to reduce the expression level of RAGE, a receptor involved in the activation of proinflammatory signaling. Overall, our data suggest that the consumption of nutraceuticals containing different natural compounds could be an adjuvant supplement to counteract proinflammatory and pro-oxidative signals induced by both hyperglycemic and senescence conditions.openMatacchione, Giulia; Valli, Debora; Silvestrini, Andrea; Giuliani, Angelica; Sabbatinelli, Jacopo; Giordani, Chiara; Coppari, Sofia; Rippo, Maria Rita; Albertini, Maria Cristina; Olivieri, FabiolaMatacchione, Giulia; Valli, Debora; Silvestrini, Andrea; Giuliani, Angelica; Sabbatinelli, Jacopo; Giordani, Chiara; Coppari, Sofia; Rippo, Maria Rita; Albertini, Maria Cristina; Olivieri, Fabiol

Shifts of Faecal Microbiota during Sporadic Colorectal Carcinogenesis

Gut microbiota has been implicated in the etiopathogenesis of colorectal cancer. The development of colorectal cancer is a multistep process by which healthy epithelium slowly develops into preneoplastic lesions, which in turn progress into malignant carcinomas over time. In particular, sporadic colorectal cancers can arise from adenomas (about 85% of cases) or serrated polyps through the "adenoma-carcinoma" or the "serrated polyp-carcinoma" sequences, respectively. In this study, we performed 16 S rRNA gene sequencing of bacterial DNA extracted from faecal samples to compare the microbiota of healthy subjects and patients with different preneoplastic and neoplastic lesions. We identified putative microbial biomarkers associated with stage-specific progression of colorectal cancer. In particular, bacteria belonging to the Firmicutes and Actinobacteria phyla, as well as members of the Lachnospiraceae family, proved to be specific of the faecal microbiota of patients with preneoplastic lesions, including adenomas and hyperplastic polyps. On the other hand, two families of the Proteobacteria phylum, Alcaligeneaceae and Enterobacteriaceae, with Sutterella and Escherichia/Shigella being the most representative genera, appeared to be associated with malignancy. These findings, once confirmed on larger cohorts of patients, can represent an important step towards the development of more effective diagnostic strategies

Hormone replacement therapy enhances IGF-1 signaling in skeletal muscle by diminishing miR-182 and miR-223 expressions : a study on postmenopausal monozygotic twin pairs

MiRNAs are fine-tuning modifiers of skeletal muscle regulation, but knowledge of their hormonal control is lacking. We used a co-twin case-control study design, that is, monozygotic postmenopausal twin pairs discordant for estrogen-based hormone replacement therapy (HRT) to explore estrogen-dependent skeletal muscle regulation via miRNAs. MiRNA profiles were determined from vastus lateralis muscle of nine healthy 54-62-years-old monozygotic female twin pairs discordant for HRT (median 7 years). MCF-7 cells, human myoblast cultures and mouse muscle experiments were used to confirm estrogen's causal role on the expression of specific miRNAs, their target mRNAs and proteins and finally the activation of related signaling pathway. Of the 230 miRNAs expressed at detectable levels in muscle samples, qPCR confirmed significantly lower miR-182, miR-223 and miR-142-3p expressions in HRT using than in their nonusing co-twins. Insulin/IGF-1 signaling emerged one common pathway targeted by these miRNAs. IGF-1R and FOXO3A mRNA and protein were more abundantly expressed in muscle samples of HRT users than nonusers. In vitro assays confirmed effective targeting of miR-182 and miR-223 on IGF-1R and FOXO3A mRNA as well as a dose-dependent miR-182 and miR-223 down-regulations concomitantly with up-regulation of FOXO3A and IGF-1R expression. Novel finding is the postmenopausal HRT-reduced miRs-182, miR-223 and miR-142-3p expression in female skeletal muscle. The observed miRNA-mediated enhancement of the target genes' IGF-1R and FOXO3A expression as well as the activation of insulin/IGF-1 pathway signaling via phosphorylation of AKT and mTOR is an important mechanism for positive estrogen impact on skeletal muscle of postmenopausal women.Peer reviewe

COMPARAÇÃO ENTRE MÉTODOS ANALÍTICOS CONVENCIONAIS E MICROCONTROLADORES DE BAIXO CUSTO PARA MONITORAMENTO DE PROCESSOS BIOTECNOLÓGICOS APLICADOS À ALIMENTOS.

A kombucha é uma bebida fermentada, produzida através da fermentação alcoólica e ácida da infusão do extrato de Camellia sinensis, açúcares e uma cultura simbiótica de bactérias e leveduras microbiologicamente ativas (SCOBY) acomodada em uma matriz celulósica. Assim, para assegurar a qualidade do produto e para a adequada evolução dos microrganismos, é necessário o monitoramento do processo fermentativo, por meio do controle de parâmetros analíticos. Usualmente, são utilizados equipamentos laboratoriais convencionais. No entanto, requerem manutenções e a utilização de reagentes de alto custo, que demandam maior tempo e volume de amostras para realização das análises, inviabilizando muitas vezes o seu uso. Desse modo, o trabalho objetivou utilizar métodos convencionais de análises físico-químicas em comparação ao uso de um sistema de microcontroladores de baixo custo, o sistema Arduino. Este, consistiu de dois elementos básicos: uma placa, elemento de hardware utilizado para construção do protótipo e um programa para computador, responsável por enviar o comando desejado a placa e posteriormente receber a leitura dos dados coletados através dos sensores de pH, álcool etílico e sólidos solúveis totais. Para a execução do projeto, foi elaborada uma kombucha original em infusão de chá verde de Camellia sinensis, contendo 5% de sacarose. Na sequência, a cultura starter SCOBY foi inoculada e o sistema monitorado em estufa incubadora vertical, em temperatura de 25ºC, durante o período de 96 horas. Os microcontroladores arduino foram submersos diretamente no mosto. Estes foram previamente higienizados para evitar a contaminação do sistema. Uma alíquota da kombucha foi coletada diariamente durante o processo fermentativo para a medida dos parâmetros físico-químicos pelos métodos convencionais em triplicata. Esse processo foi realizado outras duas vezes, totalizando três rodadas para uma quantidade significativa de dados, considerando as triplicatas. Os resultados obtidos pelo uso dos sensores e pelo uso de métodos convencionais foram comparados estatisticamente, através da análise de variância (ANOVA) ao nível de 5% de significância. Foi observado com o uso do microcontrolador de pH, que não houve diferença estatística em nenhuma das três rodadas (p>0,05), o que demonstra resultados semelhantes aos obtidos pelo método convencional. Para o parâmetro de teor alcoólico não houve diferença estatística em duas de três rodadas (p>0,05), também indicando suas potencialidades. Já para o monitoramento do teor de sólidos solúveis, houveram diferenças estatísticas significativas nas três gerações (p<0,05), sendo necessário uma continuidade nos estudos de testes com os sensores, visto que na primeira rodada, o sensor apresentou resultados próximos aos obtidos pelos métodos convencionais, com possível falha durante os testes. Assim, é possível confirmar que os sensores para análises de pH e etanol apresentaram resultados promissores no monitoramento de parâmetros físico-químicos quando comparados aos seus respectivos métodos convencionais. O uso de metodologia de automação de baixo custo para a coleta de parâmetros físico-químicos, pelo uso de microcontroladores com grande volume de dados coletados de forma automatizada em comparação aos métodos convencionais, muitas vezes limitados à um número de dados reduzidos, atesta as potencialidades do sistema Arduino para monitoramento de processos biotecnológicos

A transcriptional sketch of a primary human breast cancer by 454 deep sequencing

Background: The cancer transcriptome is difficult to explore due to the heterogeneity of quantitative and qualitative changes in gene expression linked to the disease status. An increasing number of "unconventional" transcripts, such as novel isoforms, non-coding RNAs, somatic gene fusions and deletions have been associated with the tumoral state. Massively parallel sequencing techniques provide a framework for exploring the transcriptional complexity inherent to cancer with a limited laboratory and financial effort. We developed a deep sequencing and bioinformatics analysis protocol to investigate the molecular composition of a breast cancer poly(A)+ transcriptome. This method utilizes a cDNA library normalization step to diminish the representation of highly expressed transcripts and biology-oriented bioinformatic analyses to facilitate detection of rare and novel transcripts. Results: We analyzed over 132,000 Roche 454 high-confidence deep sequencing reads from a primary human lobular breast cancer tissue specimen, and detected a range of unusual transcriptional events that were subsequently validated by RT-PCR in additional eight primary human breast cancer samples. We identified and validated one deletion, two novel ncRNAs (one intergenic and one intragenic), ten previously unknown or rare transcript isoforms and a novel gene fusion specific to a single primary tissue sample. We also explored the non-protein-coding portion of the breast cancer transcriptome, identifying thousands of novel non-coding transcripts and more than three hundred reads corresponding to the non-coding RNA MALAT1, which is highly expressed in many human carcinomas. Conclusion: Our results demonstrate that combining 454 deep sequencing with a normalization step and careful bioinformatic analysis facilitates the discovery and quantification of rare transcripts or ncRNAs, and can be used as a qualitative tool to characterize transcriptome complexity, revealing many hitherto unknown transcripts, splice isoforms, gene fusion events and ncRNAs, even at a relatively low sequence sampling

Absence of system xc⁻ on immune cells invading the central nervous system alleviates experimental autoimmune encephalitis

Background: Multiple sclerosis (MS) is an autoimmune demyelinating disease that affects the central nervous system (CNS), leading to neurodegeneration and chronic disability. Accumulating evidence points to a key role for neuroinflammation, oxidative stress, and excitotoxicity in this degenerative process. System x(c)- or the cystine/glutamate antiporter could tie these pathological mechanisms together: its activity is enhanced by reactive oxygen species and inflammatory stimuli, and its enhancement might lead to the release of toxic amounts of glutamate, thereby triggering excitotoxicity and neurodegeneration. Methods: Semi-quantitative Western blotting served to study protein expression of xCT, the specific subunit of system x(c)-, as well as of regulators of xCT transcription, in the normal appearing white matter (NAWM) of MS patients and in the CNS and spleen of mice exposed to experimental autoimmune encephalomyelitis (EAE), an accepted mouse model of MS. We next compared the clinical course of the EAE disease, the extent of demyelination, the infiltration of immune cells and microglial activation in xCT-knockout (xCT(-/-)) mice and irradiated mice reconstituted in xCT(-/-) bone marrow (BM), to their proper wild type (xCT(+/+)) controls. Results: xCT protein expression levels were upregulated in the NAWM of MS patients and in the brain, spinal cord, and spleen of EAE mice. The pathways involved in this upregulation in NAWM of MS patients remain unresolved. Compared to xCT(+/+) mice, xCT(-/-) mice were equally susceptible to EAE, whereas mice transplanted with xCT(-/-) BM, and as such only exhibiting loss of xCT in their immune cells, were less susceptible to EAE. In none of the above-described conditions, demyelination, microglial activation, or infiltration of immune cells were affected. Conclusions: Our findings demonstrate enhancement of xCT protein expression in MS pathology and suggest that system x(c)- on immune cells invading the CNS participates to EAE. Since a total loss of system x(c)- had no net beneficial effects, these results have important implications for targeting system x(c)- for treatment of MS

Colorectal Cancer Stage at Diagnosis Before vs During the COVID-19 Pandemic in Italy

IMPORTANCE Delays in screening programs and the reluctance of patients to seek medical attention because of the outbreak of SARS-CoV-2 could be associated with the risk of more advanced colorectal cancers at diagnosis. OBJECTIVE To evaluate whether the SARS-CoV-2 pandemic was associated with more advanced oncologic stage and change in clinical presentation for patients with colorectal cancer. DESIGN, SETTING, AND PARTICIPANTS This retrospective, multicenter cohort study included all 17 938 adult patients who underwent surgery for colorectal cancer from March 1, 2020, to December 31, 2021 (pandemic period), and from January 1, 2018, to February 29, 2020 (prepandemic period), in 81 participating centers in Italy, including tertiary centers and community hospitals. Follow-up was 30 days from surgery. EXPOSURES Any type of surgical procedure for colorectal cancer, including explorative surgery, palliative procedures, and atypical or segmental resections. MAIN OUTCOMES AND MEASURES The primary outcome was advanced stage of colorectal cancer at diagnosis. Secondary outcomes were distant metastasis, T4 stage, aggressive biology (defined as cancer with at least 1 of the following characteristics: signet ring cells, mucinous tumor, budding, lymphovascular invasion, perineural invasion, and lymphangitis), stenotic lesion, emergency surgery, and palliative surgery. The independent association between the pandemic period and the outcomes was assessed using multivariate random-effects logistic regression, with hospital as the cluster variable. RESULTS A total of 17 938 patients (10 007 men [55.8%]; mean [SD] age, 70.6 [12.2] years) underwent surgery for colorectal cancer: 7796 (43.5%) during the pandemic period and 10 142 (56.5%) during the prepandemic period. Logistic regression indicated that the pandemic period was significantly associated with an increased rate of advanced-stage colorectal cancer (odds ratio [OR], 1.07; 95%CI, 1.01-1.13; P = .03), aggressive biology (OR, 1.32; 95%CI, 1.15-1.53; P < .001), and stenotic lesions (OR, 1.15; 95%CI, 1.01-1.31; P = .03). CONCLUSIONS AND RELEVANCE This cohort study suggests a significant association between the SARS-CoV-2 pandemic and the risk of a more advanced oncologic stage at diagnosis among patients undergoing surgery for colorectal cancer and might indicate a potential reduction of survival for these patients

Interplay of CodY and ScoC in the regulation of major extracellular protease genes of Bacillus subtilis

AprE and NprE are two major extracellular proteases in Bacillus subtilis whose expression is directly regulated by several pleiotropic transcriptional factors, including AbrB, DegU, ScoC, and SinR. In cells growing in a rich, complex medium, the aprE and nprE genes are strongly expressed only during the post-exponential growth phase; mutations in genes encoding the known regulators affect the level of post-exponential-phase gene expression but do not permit high-level expression during the exponential growth phase. Using DNA-binding assays and expression and mutational analyses, we have shown that the genes for both exoproteases are also under strong, direct, negative control by the global transcriptional regulator CodY. However, because CodY also represses scoC, little or no derepression of aprE and nprE was seen in a codY null mutant due to overexpression of scoC. Thus, CodY is also an indirect positive regulator of these genes by limiting the synthesis of a second repressor. In addition, in cells growing under conditions that activate CodY, a scoC null mutation had little effect on aprE or nprE expression; full effects of scoC or codY null mutations could be seen only in the absence of the other regulator. However, even the codY scoC double mutant did not show high levels of aprE and nprE gene expression during exponential growth phase in a rich, complex medium. Only a third mutation, in abrB, allowed such expression. Thus, three repressors can contribute to reducing exoprotease gene expression during growth in the presence of excess nutrients