Controlled delivery of sildenafil by ß-Cyclodextrin-decorated sulfur-doped carbon nanodots: a synergistic activation of ROS signaling in tumors overexpressing PDE-5.

Fluorescent sulfur- and nitrogen-doped carbon nanodots (CDs) are zero-dimensional nanoparticles that mediate ROS production in cancer cells, displaying inherent anticancer properties. Thus, they have been proposed as nanotheranostic tools useful in image-guided cancer therapy. Here, we try to show that cancerous cells (high PDE-5 expression) receiving sildenafil delivered by CDs-based nanostructures promote positive reinforcement of PDE-5-mediated cell death via the overexpression of genes involved in the production of ROS. We explored the regioselective Huisgen cycloaddition between azide-ß-cyclodextrin and CDs-alkyne to synthetize homogeneous nanostructures, named CDs-PEG4-ß-Cdx, consisting of CDs functionalized at the surface with ß-cyclodextrins capable of including high amount drugs such as sildenafil (>20 % w/w), and releasing them in a controlled manner. We investigated how CDs-PEG4-ß-Cdx bearing sildenafil enter cells, enhancing ROS production and cell death specifically in cancer cells overexpressing PDE-5. These nanoplatforms go beyond the bounds of EPR-based nanomedicines in which carriers are conceived as inert vehicles of toxic drugs. Our findings enable the development of clever anticancer nanoplatforms that synergistically combine nanomedicines that perturb the mitochondrial electron transport chain (ROS production) with PDE-5 inhibitors which trigger oxidative stress specifically in cancer cells regardless of their location.

Emerging Evidence on Coronary Heart Disease Screening in Kidney and Liver Transplantation Candidates: A Scientific Statement From the American Heart Association: Endorsed by the American Society of Transplantation.

Coronary heart disease is an important source of mortality and morbidity among kidney transplantation and liver transplantation candidates and recipients and is driven by traditional and nontraditional risk factors related to end-stage organ disease. In this scientific statement, we review evidence from the past decade related to coronary heart disease screening and management for kidney and liver transplantation candidates. Coronary heart disease screening in asymptomatic kidney and liver transplantation candidates has not been demonstrated to improve outcomes but is common in practice. Risk stratification algorithms based on the presence or absence of clinical risk factors and physical performance have been proposed, but a high proportion of candidates still meet criteria for screening tests. We suggest new approaches to pretransplantation evaluation grounded on the presence or absence of known coronary heart disease and cardiac symptoms and emphasize multidisciplinary engagement, including involvement of a dedicated cardiologist. Noninvasive functional screening methods such as stress echocardiography and myocardial perfusion scintigraphy have limited accuracy, and newer noninvasive modalities, especially cardiac computed tomography-based tests, are promising alternatives. Emerging evidence such as results of the 2020 International Study of Comparative Health Effectiveness With Medical and Invasive Approaches-Chronic Kidney Disease trial emphasizes the vital importance of guideline-directed medical therapy in managing diagnosed coronary heart disease and further questions the value of revascularization among asymptomatic kidney transplantation candidates. Optimizing strategies to disseminate and implement best practices for medical management in the broader end-stage organ disease population should be prioritized to improve cardiovascular outcomes in these populations.

The Repertoire of Tissue Inhibitors of Metalloproteases: Evolution, Regulation of Extracellular Matrix Proteolysis, Engineering and Therapeutic Challenges.

Tissue inhibitors of metalloproteases (TIMPs) belong to a fascinating protein family expressed in all Metazoa. They act as regulators of the turnover of the extracellular matrix, and they are consistently involved in essential processes. Herein, we recapitulate the main activities of mammalian TIMPs (TIMP1-4) in the control of extracellular-matrix degradation and pathologies associated with aberrant proteostasis. We delineate the activity of TIMPs in the control of extracellular matrix (ECM) homeostasis and discuss the diversity of TIMPs across metazoans taking into account the emergence of the components of the ECM during evolution. Thus, the TIMP repertoire herein analysed includes the homologues from cnidarians, which are coeval with the origins of ECM components; protostomes (molluscs, arthropods and nematodes); and deuterostomes (echinoderms and vertebrates). Several questions, including the maintenance of the structure despite low sequence similarity and the strategies for TIMP engineering, shed light on the possibility to use recombinant TIMPs integrating unique features and binding selectivity for therapeutic applications in the treatment of inflammatory pathologies.

Risk Factors for Low Levels of Parathyroid Hormone after Surgery for Thyroid Cancer: A Single Center Study.

BACKGROUND: Thyroidectomy is the definitive treatment for most patients with thyroid cancer. Hypoparathyroidism is the most frequent complication of thyroidectomy, and its pathogenesis is multifactorial. The aim of this study is to evaluate the patient- and surgical-related risk factors for hypoparathyroidism after surgery for thyroid cancer. METHODS: In this retrospective study, patients referred to surgery for thyroid cancer from 2016 to 2019 were enrolled. Preoperative serum calcium and parathyroid hormone (PTH) and postoperative 24 h PTH and calcium levels were evaluated. Demographic data, type of surgery, incidence of hypoparathyroidism and hypocalcemia were recorded for all the patients. Patients were divided into two groups based on post-operative PTH levels (≤12 and >12 pg/mL). RESULTS: A total of 189 patients were enrolled in this study. There were 146 women (87.3%) and 43 men (22.7%), with a mean age of 51.3 years. A total of 79 patients (41.7%) underwent a neck dissection. A total of 59 patients (31.1%) had a postoperative PTH level < 12 pg/mL. Female sex, neck dissection, the yield of lymph node dissection and incidental parathyroidectomy were significantly associated with postoperative hypoparathyroidism. Incidental parathyroidectomy was reported in 44 (23.2%) patients and was correlated with younger age (<40 years) and neck dissection. There was no difference in the rate of post-operative hypocalcemia between patients with incidental parathyroidectomy and those without. CONCLUSIONS: Young patients undergoing neck dissection and with incidental parathyroidectomy have the highest risk of postoperative hypoparathyroidism after surgery for thyroid cancer. However, a large proportion of patients without incidental parathyroidectomy may have temporary hypocalcemia, suggesting that impaired blood supply of parathyroid glands during their identification and dissection may play a relevant role.

Colorectal Cancer after Kidney Transplantation: A Screening Colonoscopy Case-Control Study.

The incidence of colorectal cancer in kidney transplant recipients has been previously reported with conflicting results. In this study, we investigated if the incidence of colorectal advanced neoplasms in kidney transplant recipients, evaluated with screening colonoscopy, was higher than in healthy individuals. One-hundred sixty kidney transplant recipients undergoing screening colonoscopy were compared with 594 age- and sex-matched healthy individuals. Advanced colorectal neoplasia was found in 22 patients (13.7%), including four patients (2.5%) with colorectal cancer. Compared with the healthy population, kidney transplant recipients did not have an increased risk of developing a colorectal cancer (OR 0.69; 95% CI 0.236-2.063, p = 0.688) although it developed at a younger age. In contrast, kidney transplant recipients had a higher risk of developing an advanced adenoma compared with the control group (OR 1.65; 95% CI 0.930-2.981, p = 0.04). In conclusion, kidney transplant recipients did not have an increased incidence of colorectal cancer compared with healthy population. However, transplant patients displayed a higher incidence of colorectal adenomas, suggesting that screening colonoscopy in kidney transplant recipients should be expanded to include even younger recipients (<50 years old).

Hyaluronic acid dressing of hydrophobic carbon nanodots: A self-assembling strategy of hybrid nanocomposites with theranostic potential.

We propose a rational design of hyaluronic acid-dressed red-emissive carbon dots (CDs), with a well-structured hydrophobic core capable of locally delivering high amount doxorubicin (Doxo) (> 9% w/w) and heat (hyperthermia) in a light stimuli sensitive fashion. We combined in a unique micelle-like superstructure the peculiar optical properties of CDs (NIR photothermal conversion and red fluorescence) with the ability of hyaluronic acid (HA) shell of stabilizing nanomedicines in aqueous environment and recognizing cancer cells overexpressing CD44 receptors on their membranes, thus giving rise to smart theranostic agents useful in cancer imaging and NIR-triggered chemo-phototherapy of solid tumors. Hydrophobic CDs, named HCDs, were used as functional beads to self-assemble amphiphilic HA derivatives carrying polylactic acid side chains (HA-g-PLA), yielding to light-sensitive and biodegradable core-shell superstructures. We explored the biocompatibility and synergistic effects of chemo-phototherapy combination, together with fluorescence imaging, showing the huge potential of the proposed engineering strategy in improving efficacy. CHEMICAL COMPOUNDS.

Carbon Nanodots for On Demand Chemophotothermal Therapy Combination to Elicit Necroptosis: Overcoming Apoptosis Resistance in Breast Cancer Cell Lines.

BACKGROUND: Engineered luminescent carbon nanodots (CDs) are appealing nanomaterials for cancer image-guided photothermal therapy combining near infrared (NIR)-triggered hyperthermia, imaging, and drug delivery in a single platform for efficient killing of cancer cells. This approach would allow eliciting synergistic regulated cell death (RCD) routes such as necroptosis, targeting breast cancer cells refractory to apoptosis, thus overcoming drug resistance. METHODS: We report the preparation of CDs bearing biotin as a targeting agent (CDs-PEG-BT), which are able to load high amounts of irinotecan (23.7%) to be released in a pulsed on-demand fashion. CDs-PEG-BT have narrow size distribution, stable red luminescence, and high photothermal conversion in the NIR region, allowing imaging of MDA-MB231 and MCF-7 cancer cells and killing them by photothermal and chemotherapeutic insults. RESULTS: Cellular uptake, viability profiles, and RCD gene expression analyses provided insights about the observed biocompatibility of CDs-PEG-BT, indicating that necroptosis can be induced on-demand after the photothermal activation. Besides, photothermal activation of drug-loaded CDs-PEG-BT implies both necroptosis and apoptosis by the TNFα and RIPK1 pathway. CONCLUSIONS: The controlled activation of necroptosis and apoptosis by combining phototherapy and on-demand release of irinotecan is the hallmark of efficient anticancer response in refractory breast cancer cell lines in view of precision medicine applications.

Characterization of Translationally Controlled Tumour Protein from the Sea Anemone Anemonia viridis and Transcriptome Wide Identification of Cnidarian Homologues.

Gene family encoding translationally controlled tumour protein (TCTP) is defined as highly conserved among organisms; however, there is limited knowledge of non-bilateria. In this study, the first TCTP homologue from anthozoan was characterised in the Mediterranean Sea anemone, Anemonia viridis. The release of the genome sequence of Acropora digitifera, Exaiptasia pallida, Nematostella vectensis and Hydra vulgaris enabled a comprehensive study of the molecular evolution of TCTP family among cnidarians. A comparison among TCTP members from Cnidaria and Bilateria showed conserved intron exon organization, evolutionary conserved TCTP signatures and 3D protein structure. The pattern of mRNA expression profile was also defined in A. viridis. These analyses revealed a constitutive mRNA expression especially in tissues with active proliferation. Additionally, the transcriptional profile of A. viridis TCTP (AvTCTP) after challenges with different abiotic/biotic stresses showed induction by extreme temperatures, heavy metals exposure and immune stimulation. These results suggest the involvement of AvTCTP in the sea anemone defensome taking part in environmental stress and immune responses.

Evolutionary conserved mechanisms pervade structure and transcriptional modulation of allograft inflammatory factor-1 from sea anemone Anemonia viridis.

Gene family encoding allograft inflammatory factor-1 (AIF-1) is well conserved among organisms; however, there is limited knowledge in lower organisms. In this study, the first AIF-1 homologue from cnidarians was identified and characterised in the sea anemone Anemonia viridis. The full-length cDNA of AvAIF-1 was of 913 bp with a 5' -untranslated region (UTR) of 148 bp, a 3'-UTR of 315 and an open reading frame (ORF) of 450 bp encoding a polypeptide with149 amino acid residues and predicted molecular weight of about 17 kDa. The predicted protein possesses evolutionary conserved EF hand Ca2+ binding motifs, post-transcriptional modification sites and a 3D structure which can be superimposed with human members of AIF-1 family. The AvAIF-1 transcript was constitutively expressed in all tested tissues of unchallenged sea anemone, suggesting that AvAIF-1 could serve as a general protective factor under normal physiological conditions. Moreover, we profiled the transcriptional activation of AvAIF-1 after challenges with different abiotic/biotic stresses showing induction by warming conditions, heavy metals exposure and immune stimulation. Thus, mechanisms associated to inflammation and immune challenges up-regulated AvAIF-1 mRNA levels. Our results suggest its involvement in the inflammatory processes and immune response of A. viridis.

Metallothionein Gene Family in the Sea Urchin Paracentrotus lividus: Gene Structure, Differential Expression and Phylogenetic Analysis.

Metallothioneins (MT) are small and cysteine-rich proteins that bind metal ions such as zinc, copper, cadmium, and nickel. In order to shed some light on MT gene structure and evolution, we cloned seven Paracentrotus lividus MT genes, comparing them to Echinodermata and Chordata genes. Moreover, we performed a phylogenetic analysis of 32 MTs from different classes of echinoderms and 13 MTs from the most ancient chordates, highlighting the relationships between them. Since MTs have multiple roles in the cells, we performed RT-qPCR and in situ hybridization experiments to understand better MT functions in sea urchin embryos. Results showed that the expression of MTs is regulated throughout development in a cell type-specific manner and in response to various metals. The MT7 transcript is expressed in all tissues, especially in the stomach and in the intestine of the larva, but it is less metal-responsive. In contrast, MT8 is ectodermic and rises only at relatively high metal doses. MT5 and MT6 expression is highly stimulated by metals in the mesenchyme cells. Our results suggest that the P. lividus MT family originated after the speciation events by gene duplications, evolving developmental and environmental sub-functionalization.

Coexposure to sulfamethoxazole and cadmium impairs development and attenuates transcriptional response in sea urchin embryo.

Among sulfonamides, sulfamethoxazole represents one of the most widely employed. A considerable amount of sulfamethoxazole is introduced into the marine environment after utilization in aquaculture. The cytotoxicity of sulfamethoxazole relies mainly on arylhydroxylamine metabolites and it is associated with the production of reactive oxygen species. Cadmium represents a metal largely employed in several anthropic activities and it is toxic for all living organisms even at low concentrations. Since it is not degraded, cadmium irreversibly accumulates into cells. In order to understand the mechanisms of response to changes in the chemical environment, we investigated by light microscopy observations and RT-qPCR assays the impact of sulfamethoxazole and cadmium in P. lividus sea urchin embryos. During development, embryos were exposed to sulfamethoxazole amount comparable to that usually used in aquaculture procedures and/or sublethal levels of cadmium chloride. Impairment of development and biomarkers for inflammation, detoxification, metal scavenging and cell death were inspected. Even though treatment with sulfamethoxazole apparently did not affect development, it stimulated a remarkable molecular response to oxidative stress. Moreover, combined exposure seriously compromised development and the defense mechanisms to cadmium were blocked. This study leads to the conclusion that coexposure to sulfamethoxazole and cadmium induces neutralizing effects on sea urchin embryos. Thus, in marine areas nearby aquaculture farms, where sulfamethoxazole discharge represents an important environmental contaminant, cadmium occurrence may alter population dynamics of P. lividus.

The nucleic acid-binding protein PcCNBP is transcriptionally regulated during the immune response in red swamp crayfish Procambarus clarkii.

Gene family encoding cellular nucleic acid binding proteins (CNBP) is well conserved among vertebrates; however, there is limited knowledge in lower organisms. In this study, a CNBP homolog from the red swamp crayfish Procambarus clarkii was characterised. The full-length cDNA of PcCNBP was of 1257 bp with a 5'-untranslated region (UTR) of 63 bp and a 3'-UTR of 331 bp with a poly (A) tail, and an open-reading frame (ORF) of 864 bp encoding a polypeptide of 287 amino acids with the predicted molecular weight of about 33 kDa. The predicted protein possesses 7 tandem repeats of 14 amino acids containing the CCHC zinc finger consensus sequence, two RGG-rich single-stranded RNA-binding domain and a nuclear localization signal, strongly suggesting that PcCNBP was a homolog of vertebrate CNBP. The PcCNBP transcript was constitutively expressed in all tested tissues of unchallenged crayfish, including hepatopancreas, gill, eyestalk, haemocytes, intestine, stomach and cuticle with highest expression in haemocytes, intestine, gills and hepatopancreas. The mRNA expression of PcCNBP in haemocytes was modulated at transcriptional level by different immune challenges, suggesting its involvement in the immune response of P. clarkii during both bacteria and viruses infection.

Effects of cadmium exposure on sea urchin development assessed by SSH and RT-qPCR: metallothionein genes and their differential induction.

In order to study the defense strategies activated by Paracentrotus lividus embryos in response to sub-lethal doses of CdCl2, we compared the induced transcripts to that of control embryos by suppression subtractive hybridization technique. We isolated five metallothionein (MT) cDNAs and other genes related to detoxification, to signaling pathway components, to oxidative, reductive and conjugative biotransformation, to RNA maturation and protein synthesis. RT-qPCR analysis revealed that two of the five P. lividus MT (PlMT7 and PlMT8) genes appeared to be constitutively expressed and upregulated following cadmium treatment, whereas the other three genes (PlMT4, PlMT5, PlMT6) are specifically switched-on in response to cadmium treatment. Moreover, we found that this transcriptional induction is concentration dependent and that the cadmium concentration threshold for the gene activation is distinct for every gene. RT-qPCR experiments showed in fact that, among induced genes, PlMT5 gene is activated at a very low cadmium concentration (0.1 µM) whereas PlMT4 and PlMT6 are activated at intermediate doses (1-10 µM). Differently, PlMT7 and PlMT8 genes increase significantly their expression only in embryos treated with the highest dose (100 µM CdCl2). We found also that, in response to a lethal dose of cadmium (1 µM), only PlMT5 and PlMT6 mRNA levels increased further. These data suggest a hierarchical and orchestrated response of the P. lividus embryo to overcome differential environmental stressors that could interfere with a normal development.

Can laparoscopic cholecystectomy be safety performed in the elderly?

AIM: To assess the suitability of laparoscopic cholecystectomy in elderly patients, although early reports have questioned the efficacy of this procedure in that patient group. MATERIAL OF STUDY: Retrospective study evaluating the medical records of the elderly patients who underwent laparoscopic cholecystectomy in our surgical unit. Data included age and gender, American Society of Anesthesiologists (ASA) score, comorbid illness, prior abdominal surgery, presentation, operative time, conversion rate, postoperative morbidity, and mortality rates and length of hospital stay. RESULTS: Fifty consecutive patients age 70 or older who underwent laparoscopic cholecystectomy were studied Postoperative complications occurred in five patients. DISCUSSION: Many Studies have shown that the incidence of complicated gallstone disease in the elderly is higher when compared with that of younger patients and gallbladder disease is particularly virulent in the elderly, with high rate of acute cholecystitis, biliary tract disease, increased morbidity, and prolonged hospital stay. This poor outcome has been attributed to the presence of severe co-morbid factors associated with the aging process. Compared to open cholecystectomy, laparoscopic cholecystectomy may cause less postoperative depression of respiratory function and cell-mediated immunity. In our study perioperative mortality rate was 0%. CONCLUSIONS: Laparoscopic cholecystectomy in elderly patients is a relatively safe procedure that can be accomplished with acceptable low morbidity. In this series of geriatric patients, there was no evidence of any increased risk for conversion to an open cholecystectomy, delayed recovery, or prolonged hospitalization.

Six novel mutations of the LDL receptor gene in FH kindred of Sicilian and Paraguayan descent.

Familial hypercholesterolemia (FH) is an autosomal dominant inherited disease caused by mutations in the gene coding for the low density lipoprotein receptor (LDL-R). It is characterized by a high concentration of low density lipoprotein (LDL), which frequently gives rise to premature coronary artery disease. We studied the probands of five FH Sicilian families with 'definite' FH and one proband of Paraguayan descent with homozygous FH who has been treated with an effective living-donor liver transplantation. In order to seek the molecular defect in these six families, we used direct sequencing to define the molecular defects of the LDL-R gene responsible for the disease. We described three novel missense mutations (C100Y, C183Y and G440C), two frameshift mutations (g.1162delC in exon 8 and g.2051delC in exon 14) and one mutation (g.2390-1Gright curved arrow A) at splicing acceptor consensus sequences located in intron 16 of the LDL-R gene; the analysis of cDNA of this splicing mutation showed the activation of a cryptic splice site in intron 16 and the binding studies showed a reduction in internalisation of LDL-DIL in the proband's cultured fibroblasts. Moreover, a g.2051delC in exon 14 was identified in the proband of Paraguayan ancestry with clinical features of homozygous FH. The mutation identified in the South American patient represents the first description of a variant in South American patients other than Brazilian FH patients. The 5 mutations identified in the Sicilian patients confirm the heterogeneity of LDL-R gene mutations in Sicily.