DNA-directed immobilization fluorescent immunoarray for multiplexed antibiotic residue determination in milk.

The presence of antibiotic residues in cow's milk entails high risk for consumers, the dairy industry, and the environment. Therefore, the development of highly specific and sensitive screening tools for the rapid and cost-effective identification of traces of these compounds is urgently needed. A multiplexed screening platform utilizing DNA-directed immobilization (DDI) was developed aiming to detect three classes of antibiotic residues (fluoroquinolones, sulfonamides, and tylosin) prevalently found in milk. Throughout this work, each oligonucleotide sequence was conjugated to a different hapten molecule, while the three complementary strands were immobilized in 24 independent microarray chips on a single glass slide. First, the array was incubated with the pool of hapten-oligonucleotide conjugate site encoded the signal through DNA hybridization. Next, commercial milk samples were incubated with the cocktail of monoclonal antibodies following a secondary fluorophore-labeled antibody which was required for fluorescent readout. Direct sample detection was achieved in milk diluting 20 times in assay buffer. The limits of detection (LODs) reached were 1.43 µg kg-1, 1.67 µg kg-1, and 0.89 µg kg-1 for TYLA, STZ, and CIP, respectively, which represented in raw milk 7.15 µg kg-1, 8.35 µg kg-1, and 4.45 µg kg-1 for TYLA, STZ, and CIP, respectively, that are below the EU regulatory limits. Cross-reactivity profiles were evaluated against the family of structurally related antibiotics in order to demonstrate the capability to detect antibiotics from the same family of compounds. A pre-validation study was performed by spiking 20 blind samples above and below the maximum residue limits established by the EU guidelines. The system was successfully implemented towards randomized sample classification as compliant or non-compliant. The proposed DDI-based immunoarray provides a fast and cost-effective alternative to obtain semi-quantitative information about the presence of three veterinary residues simultaneously in milk samples.

Family Pediatrician and Public Health collaboration, an alliance to increase vaccination coverage: an experience with MenB vaccination in Italy.

Background: Invasive Meningococcal Disease is a severe disease mainly affecting infants and young children. Most infections are caused by serogroups A, B, C, W, X, and Y. In the last 10 years, serogroup B has been the main cause of Invasive Meningococcal Disease in Europe. Recent data resulting from an observational study conducted in Italy show a significant reduction in the number of Invasive Meningococcal Disease cases due to Neisseria meningitidis B after the introduction of vaccine 4CMenB. Thus, the Naples Team of Federation of Italian Primary Care Pediatricians and the Public Health Department started an active collaboration focused on vaccination process management (named "Progetto Via") with the aim of increasing Meningococcal B vaccination coverage. Study design: Source of data is the regional platform "GE.VA.". Every Primary care Pediatrician uses daily to record vaccination activity. This platform is integrated with data entered by operators of the District/Vaccination Center. Methods: Time: January 2019 - December 2019. The Federation of Italian Primary Care Pediatricians/Naples organized a meeting to identify six coordinators. The pediatricians could choose to counsel in their own offices and send children to the vaccination center or to counsel and vaccinate directly in their own clinics. Results: A total of 78 pediatricians took part in the project: 46 did only counseling and 32 did both counseling and vaccination in their medical clinic. Data obtained show an overall average vaccination coverage growth of about 13% in the first 4 months of the survey, and a further growth of about 11% in the following seven months, with a total growth in the entire period of 24%. The pediatricians' counseling is essential to recover non-compliant subjects, considering both the relationship of trust with the families and the visits already scheduled as an ideal moment for vaccinations' status check. Conclusions: The project highlights how an effective collaboration between family pediatricians and the Local Health Authority becomes valuable in getting closer to reach the Ministerial goal of 95%. Vaccination coverage increased significantly when family pediatricians supported the activity of vaccine centers in distress in many regional situations. The trust relationship, the hourly availability and the capillary network of family pediatricians' clinics were key elements for the success of this project and were also recognized by parents.

Efficacy and safety of treatment simplification to atazanavir/ritonavir + lamivudine in HIV-infected patients with virological suppression: 144 week follow-up of the AtLaS pilot study.

OBJECTIVES: AtLaS was a single-arm pilot study that demonstrated promising efficacy and safety of treatment simplification to a dual regimen with atazanavir/ritonavir + lamivudine in virologically suppressed HIV-positive patients. Here, we report data from the 144 week follow-up. METHODS: At baseline, patients treated with a three-drug atazanavir/ritonavir-based regimen were switched to 300/100 mg of atazanavir/ritonavir plus 300 mg of lamivudine once daily. Major clinical events, laboratory parameters, neurocognitive performance, bone composition and body fat distribution were monitored. Treatment failure was defined as a discontinuation/switch of the regimen or virological failure (HIV-RNA >50 copies/mL in two consecutive determinations or a single level above 1000 copies/mL). RESULTS: After 144 weeks, 9/40 (22.5%) treatment failures occurred, including two virological failures (Weeks 48 and 53, without resistance). A significant increase in the CD4 count was observed at Week 96 (+124 cells/mm(3); P = 0.002) and Week 144 (+94 cells/mm(3); P = 0.008). After 144 weeks, a significant increase in total cholesterol (+25 mg/dL; P = 0.001), HDL cholesterol (+6 mg/dL; P = 0.024) and LDL cholesterol (+12 mg/dL; P = 0.008) was observed, without any change in triglyceride levels, total cholesterol/HDL ratio or LDL/HDL ratio. A significant increase in the estimated glomerular filtration rate (+25 mL/min/1.73 m(2); P < 0.001) and lumbar spine T-score and Z-score (+0.2, P = 0.011; and +0.35, P = 0.001, respectively) and a decrease in trunk fat (-1.898 g; P = 0.005) were also observed. Neurocognitive function did not decline over time. Concerning safety, 10 moderate to severe adverse events were recorded in eight patients; overall seven cases of renal colic (possibly treatment related) were observed, leading to a discontinuation of treatment in two patients. CONCLUSIONS: Data from the 144 week follow-up suggested good long-term efficacy of the simplification strategy that was investigated, with rare virological failure and a potential for improvement of the CD4 count, renal function and bone mineral density. This strategy warrants further investigation in a randomized trial.

Sensitive and label-free biosensing of RNA with predicted secondary structures by a triplex affinity capture method.

A novel biosensing approach for the label-free detection of nucleic acid sequences of short and large lengths has been implemented, with special emphasis on targeting RNA sequences with secondary structures. The approach is based on selecting 8-aminoadenine-modified parallel-stranded DNA tail-clamps as affinity bioreceptors. These receptors have the ability of creating a stable triplex-stranded helix at neutral pH upon hybridization with the nucleic acid target. A surface plasmon resonance biosensor has been used for the detection. With this strategy, we have detected short DNA sequences (32-mer) and purified RNA (103-mer) at the femtomol level in a few minutes in an easy and level-free way. This approach is particularly suitable for the detection of RNA molecules with predicted secondary structures, reaching a limit of detection of 50 fmol without any label or amplification steps. Our methodology has shown a marked enhancement for the detection (18% for short DNA and 54% for RNA), when compared with the conventional duplex approach, highlighting the large difficulty of the duplex approach to detect nucleic acid sequences, especially those exhibiting stable secondary structures. We believe that our strategy could be of great interest to the RNA field.

Colorimetric analysis platform based on thin layer chromatography for monitoring gluten cross-contamination in food industry.

Monitoring of the accidental presence of gluten (Glu), resulting from cross-contamination, is imperative in different industries, in particular food industry. The objective of this study was the development of an analytical platform utilizing thin-layer chromatography (TLC) with colorimetric read-out for making binary (yes/no) decisions on surfaces and/or point of these industries. The composition of the extractive phase was optimized with commercial products used in cleaning processing lines. Subsequently, an exploration of TLC separation and detection was undertaken. CN-modified nanosilica plates and 30:70 acetonitrile:water were used to achieve a selective signal for Glu residues. The study of the detection performance showed that both spectroscopic measurement and image analysis were resulted in satisfactory results for quantitate analysis (RSD = 5 %, LOD = 0.12 mg). The practical application of the proposed methodology on surfaces of the food processing lines. This work demonstrated the operational feasibility in detecting gluten cross-contaminations within the food processing industry.

Study of alkaloid berberine and its interaction with the human telomeric i-motif DNA structure.

The alkaloid berberine presents many biological activities related to its potential to bind DNA structures, such as duplex or G-quadruplex. Recently, it has been proposed that berberine may interact with i-motif structures formed from the folding of cytosine-rich sequences. In the present work, the interaction of this alkaloid with the i-motif formed by the human telomere cytosine-rich sequence, as well as with several positive and negative controls, has been studied. Molecular fluorescence and circular dichroism spectroscopies, as well as nuclear magnetic resonance spectrometry and competitive dialysis, have been used with this purpose. The results shown here reveal that the interaction of berberine with this i-motif is weak, mostly electrostatics in nature and takes place with bases not involved in C·C+ base pairs. Moreover, this ligand is not selective for i-motif structures, as binds equally to both, folded structure, and unfolded strand, without producing any stabilization of the i-motif. As a conclusion, the development of analytical methods based on the interaction of fluorescent ligands, such as berberine, with i-motif structures should consider the thermodynamic aspects related with the interaction, as well as the selectivity of the proposed ligands with different DNA structures, including unfolded strands.

Sequence analysis and genetic variability of stearoyl CoA desaturase (SCD) gene in the Italian Mediterranean river buffalo.

Stearoyl-CoA desaturase (SCD) plays a key metabolic role by changing the saturated FA content of ruminant milk and meat. In this study we characterized for the first time the stearoyl-CoA desaturase (SCD) gene in river buffalo (Bubalus bubalis) and investigated its genetic variability. On a total of 78 buffaloes, 15 SNPs were detected and 6 of them were preliminarily genotyped. In particular, the g.133A>C SNP was found to create a new consensus site for the SP1 binding site, thus generating a new tandem repeat in the promoter region. A preliminary association study with the milk fatty acid content showed that the C allele significantly affects the total desaturation index (P<0.01). Linkage disequilibrium analysis allowed identification of 7 haplotypes and 4 tag SNPs. Such polymorphisms could represent useful genetic markers for association studies with fatty acid composition, but further studies are needed to evaluate their potential use to improve the nutritional quality of the dairy products.

Evaluation of the effect of polymorphism on G-quadruplex-ligand interaction by means of spectroscopic and chromatographic techniques.

Guanine-rich sequences may fold into highly ordered structures known as G-quadruplexes. Apart from the monomeric G-quadruplex, these sequences may form multimeric structures that are not usually considered when studying interaction with ligands. This work studies the interaction of a ligand, crystal violet, with three guanine-rich DNA sequences with the capacity to form multimeric structures. These sequences correspond to short stretches found near the promoter regions of c-kit and SMARCA4 genes. Instrumental techniques (circular dichroism, molecular fluorescence, size-exclusion chromatography and electrospray ionization mass spectrometry) and multivariate data analysis were used for this purpose. The polymorphism of G-quadruplexes was characterized prior to the interaction studies. The ligand was shown to interact preferentially with the monomeric G-quadruplex; the binding stoichiometry was 1:1 and the binding constant was in the order of 105M-1 for all three sequences. The results highlight the importance of DNA treatment prior to interaction studies.

DNA-based nanoscaffolds as vehicles for 5-fluoro-2'-deoxyuridine oligomers in colorectal cancer therapy.

Fluoropyrimidines, such as 5-fluorouracil (5-FU) and related prodrugs, are considered one of the most successful agents in the treatment of colorectal cancer, yet poor specificity and tumor cell resistance remain the major limiting bottlenecks. Here, we exploited for the first time the ability of two DNA nanoscaffolds, a DNA tetrahedron (Td) and rectangle DNA origami, to incorporate 5-fluoro-2'-deoxyuridine (FdUn) oligomers. In addition, cholesterol moieties were synthetically attached to Td and DNA origami staples to enhance cellular uptake. DNA nanostructures functionalized with FdUn exhibited an enhanced cytotoxicity and higher ability to trigger apoptosis in colorectal cancer cells relative to conventional 5-FU and FdU, especially having cholesterol as an internalization helper. The cholesterol content mostly correlates with the increase of the FdUn nanostructure cytotoxicity. DNA nanoscaffolds bearing FdUn were able to circumvent the low sensitivity of colorectal cancer cells towards 5-FU. Both DNA nanostructures attained a comparable cytotoxic effect yet Td displays higher antiproliferative action. The ability to reduce the proliferation of cancer cells is mainly related to the concentration of DNA nanostructures. The present work suggests that self-assembled DNA nanoparticles are privileged vehicles for delivering fluoropyrimidines, opening new avenues to the development of promising therapeutics for cancer treatment.

Cell-cycle molecules in mesothelioma: an overview.

Cell cycle progression is mediated by a group of proteins named cyclins that activate a highly conserved family of protein kinases, the cyclin-dependent kinases (CDKs). CDKs are also regulated by related proteins called cdk inhibitors, grouped into two families: the INK4 inhibitors (p16, p15, p19 and p18) and the Cip/Kip inhibitors (p21, p27). Moreover, several tumour suppressor genes (such as Retinoblastoma gene and p53 gene) are implicated in the regulation of the molecular mechanism of cell division. Several studies report the importance of cell cycle regulator proteins in the pathogenesis and the prognosis of mesothelioma. This article will review the most recent data from the literature about the expression and the diagnostic and prognostic significance of cell cycle molecules in mesothelioma.

Biphasic pulses enhance bleomycin efficacy in a spontaneous canine perianal tumors model.

Perianal tumors (adenoma and carcinoma of the hepatoid glands) are frequently reported in veterinary literature. They are locally aggressive tumors with a low tendency to metastatic spread. An hormonal ethiology has been identified for the development of perianal adenomas in male dogs, while the carcinomas are free from hormonal influence. Standard treatments include surgery, cryotherapy or, in selected cases, radiation therapy. In this article we describe the outcome of a small cohort of canine patients with perianal tumors treated with bleomycin selectively driven by trains of biphasic pulses (electrochemotherapy). Twelve canine patients, eight with adenoma and four with carcinoma of the perianal glands, entered the study and received two sessions of ECT under sedation. The pets had local injection ofbleomycin at the concentration of 1.5 mg/mg and five minutes after the chemotherapy, trains of 8 biphasic electric pulses lasting 50 + 50 micros each, with 1 ms interpulse intervals, were delivered by means of modified caliper and needle array electrodes or, for difficult districts, through paired needle electrode. The overall response rate was 91% with a 83% of complete response (10/12); one dog had a PR that lasted 12 months and another had progressive disease. Electrochemotherapy appears as a safe and efficacious modality for the treatment of perianal tumors and warrants further investigations.

Vasoactive intestinal polypeptide secreting islet cell tumors: a 15-year experience and review of the literature.

BACKGROUND: Vasoactive intestinal polypeptide (VIP)-secreting tumors of the pancreas represent a rare subtype of pancreatic islet cell tumors with an estimated incidence of 0.2 to 0.5 per million per year. We provide data on a relatively large series of patients with VIP-secreting tumors and review current literature regarding this specific entity. METHODS: A retrospective review was performed of all patients with VIP-secreting tumors of the pancreas treated from 1977 to 1992 at our institution. Presenting signs, symptoms, mode of diagnosis, extent of disease, surgical resectability, tumor size, treatments, hormone levels, and survival were assessed. RESULTS: Eighteen patients were identified, 9 male and 9 female. Ages ranged from 23 to 74 years (mean 51 years). Secretory diarrhea was the most common symptom, occurring in 16 of 18 patients (89%). The most common tumor location was the tail of the pancreas (9 patients). Fourteen patients (78%) had liver metastasis at diagnosis. Curative resections were attempted in only 5 patients (28%). The mean survival was 3.6 years with the longest disease-free survival being 15 years and longest overall survival 15 years. CONCLUSIONS: VIP-secreting tumors are extremely rare entities and usually metastatic at the time of diagnosis. Despite advanced disease, these patients can have extended survival.

Effect of a topical vasoconstrictor on computed tomography of paranasal sinus disease.

Apparent ethmoid inflammation which resolved with alternation of the nasal cycle or following application of topical vasoconstrictors has been observed with magnetic resonance imaging. A similar phenomenon might occur to a lesser degree with computed tomography (CT), leading to overdiagnosis of limited sinus disease. The degree to which ostiomeatal complex disease is reversible by topical vasoconstrictors was investigated. Ten patients with histories of chronic or recurrent sinusitis underwent coronal CT studies of the paranasal sinuses before and after the application of a topical vasoconstrictor. Mucosal volume or thickness measurements were obtained from the turbinates, infundibulum, ethmoidal cells, and antrum. Vasoconstrictor application markedly reduced turbinate size and appeared to reduce mucosal thickening in the ethmoidal infundibulum. Minimal mucosal changes identified in the paranasal sinuses by computed tomography were not reversed by vasoconstrictors and therefore are likely to be pathologic.

Properties of triple helices formed by oligonucleotides containing 8-aminopurines.

The synthesis of parallel hairpins carrying 8-aminopurines is described. These hairpins have a high affinity for specific polypyrimidine sequences resulting in the formation of very stable triplexes.

[Diagnosis and management of acute pharyngotonsillitis in the primary care pediatrician's office]. / Diagnosi e gestione della faringotonsillite acuta nell'ambulatorio del pediatra di famiglia.

Acute pharyngotonsillitis is one of the most frequent causes of visits in the primary care pediatrician'office. Group A b-hemolytic streptococci (GABHS) or Streptococcus pyogenes causes 15-30% of cases of acute pharyngotonsillitis in pediatric age. Children with pharyngotonsillitis due to GABHS commonly present sore throat, fever more than 38 °C, tonsillar exudate, and tender cervical adenopathy, but the severity of illness ranges from mild throat pain to classic exudative tonsillitis with high fever. The McIsaac criteria is a clinical scoring system to predict the likelihood of streptococcal infection among children. This score is based on 5 clinical criteria: age 3-14 years, fever more than 38°C, tonsillar swelling or exudate, tender and enlarged anterior cervical lymph nodes, and absence of cough, but none of these findings is specific for GABHS pharyngotonsillitis. Culture of a throat swab on a blood agar plate (BAP) remains the gold standard for the diagnosis of acute streptococcal pharyngotonsillitis. Because of the major disadvantage of culturing throat swabs on BAP culture is the delay in obtaining the results (at least 1 day), in the past decades rapid antigen detection test (RAD) were introduced for the rapid identification of GABHS directly from throat swabs. Accurate diagnosis and treatment of GABHS pharyngotonsillitis provides positive benefits, including prevention of complications, such as acute rheumatic fever and peritonsillar abscess and reduce the acute morbidity associated with the illness. Conversely, improper diagnosis may result in negative consequences, including unnecessary antibiotic prescriptions that confer increased health care costs and contibute to the development of bacterial resistance.

Does prosthetic covering of nitinol stents alter healing characteristics or hemodynamics?

PURPOSE: To evaluate placement of polyester (Dacron) coverings on nitinol stents implanted in the canine aorta to determine the effect on cross-sectional lumen area, development of intimal hyperplasia, device endothelialization, and flow hemodynamics. METHODS: Ten polyester-covered and 10 uncovered nitinol stents (60-mm length, 10- or 12-mm diameter) were deployed percutaneously in the normal infrarenal aorta of 20 adult mongrel dogs using random assignment. Angiography, intravascular ultrasound (IVUS), and duplex ultrasound performed at device deployment and before explantation at 6 weeks were used to measure aorta/device diameter and cross-sectional area. Pressure-perfusion-fixed aortic segments were compared for surface endothelialization (CD31 staining) and for thickness of neointimal formation. RESULTS: All 20 endoluminal devices were accurately positioned in the infrarenal aorta without early or delayed evidence of device thrombosis, significant lumen narrowing, or device deformity. IVUS and duplex scanning identified no anatomical stenosis in either the covered or the bare devices by duplex ultrasound; peak systolic velocity measurements were similar (106+/-25 cm/s in the covered stent versus 96+/-25 cm/s for bare stents, p > 0.05). Mean neointimal thickness was significantly greater (p < 0.005) in the covered (326+/-145 microm) compared with the bare (219+/-62 microm) stents. Intima-to-media height ratios were greater in the covered stents (3.0+/-1.1 compared with 1.1+/-0.2, p < 0.003). Mean surface area endothelialization in the proximal, middle, and distal sections of each device was similar (p > 0.05) in covered (59%, 56%, and 69%) and bare (59%, 65%, and 53%) stents. CONCLUSIONS: Deployment and balloon dilation of a covered nitinol stent in a nondiseased canine aorta increased neointimal development compared with an uncovered stent, but overall lumen cross-sectional area was preserved. No differences in device patency, intradevice thrombus formation, flow hemodynamics, or luminal endothelialization were demonstrated, despite a thicker intradevice neointima induced by the polyester covering.

A simple method for N-15 labelling of exocyclic amino groups in synthetic oligodeoxynucleotides.

The use of the ammonia deprotection step to introduce (15)N labels at specific exocyclic amino positions of adenine, cytosine, guanine or 2-aminopurine of oligodeoxynucleotides is described.

Duplex features of vein graft stenosis and the success of percutaneous transluminal angioplasty.

PURPOSE: To determine if criteria exist that are correlated to a successful outcome after balloon angioplasty for vein graft stenosis. METHODS: During a 5-year period, duplex surveillance of 380 infrainguinal vein bypasses identified 76 hemodynamically failing grafts (87 stenoses) requiring intervention. Percutaneous transluminal angioplasty (PTA) was selected over surgical repair based on 3 criteria: time interval from primary grafting procedure, vein graft diameter, and stenosis length. The 28 (32%) stenoses (20 grafts) treated by PTA were used in a retrospective analysis to test if any variables favored a successful outcome. Patient and lesion characteristics, graft patency, and restenosis following PTA were correlated with duplex features of the stenosis recorded prior to, immediately after, and at 3- to 6-month intervals postprocedurally. RESULTS: Lesion characteristics that correlated with a successful outcome were vein size > or = 3.5 mm, lesion length < 2 cm, and appearance > 3 months after surgery. Conduit type, PTA site, patient demographics, and indication for bypass did not correlate with PTA durability. Nineteen lesions in 13 grafts met these criteria (group 1), while 9 stenoses in 7 grafts did not (group 2). Lesion severity based on duplex velocity measurements were similar in both groups before (p = 0.40) and after (p = 0.32) treatment. During the mean 21-month follow-up, group 1 grafts required less intervention (p = 0.035). At last follow-up, hemodynamic changes were durable in group 1 (p = 0.0068) but not in group 2 (p = 0.39). CONCLUSIONS: Selection of vein graft stenoses for treatment by PTA can be based on temporal and duplex data. PTA of short (< 2 cm) stenoses in good caliber veins (> or = 3.5 mm) appearing > 3 months after bypass placement was durable with a late intervention rate of approximately 10%. Direct surgical repair or replacement is recommended for early (< 3 months) and/or long segment stenoses that develop in small caliber conduits.