Development and validation of an alternative procedure for quantitative quality control analysis of 99mTc-radiopharmaceuticals using a Geiger Müller counter.

BACKGROUND AND OBJECTIVES: In a hospital radiopharmacy with 2a operational level, including the preparation of radiopharmaceuticals from prepared and approved reagent kits, it is common to have a single activimeter or dose calibrator for labeling and fractionation, and to perform the quality controls of the 99mTc-radiopharmaceuticals. In certain cases, the accumulation of radioactive material or accidental contamination of the work area causes the background to exceed the limits to carry out the radiochemical purity analyses and it is necessary to look for viable alternatives. In this work, a Geiger Müller detector (equipped with a probe for measuring surface contamination) frequently used for radioprotection purposes, was validated as an alternative and its performance was compared against the activimeter for 99mTc-radiopharmaceuticals. MATERIALS AND METHODS: Using [99mTc]pertechnetate, systematic studies of error analyses and detector response to activity concentration, activity and measurement time were carried out in liquid matrices and in paper. The results were compared against an activimeter calibrated for [99mTc]Tc. RESULTS: The developed method was used to determine the radiochemical purity of the compounds [99mTc]Tc-MDP and [99mTc]Tc-MIBI by ascending paper chromatography tests, obtaining comparable values to those measured with an activimeter in the same system (within 1% uncertainty) and using the method of vial partitioning in a dedicated equipment. CONCLUSIONS: This work demonstrates that a Geiger Müller detector with a probe for measuring surface contamination can be adequately used to replace other equipment in the control of radiochemical purity in the hospital radiopharmacy.

MicroRNA expression profiling of RAS-mutant thyroid tumors with follicular architecture: microRNA signatures to discriminate benign from malignant lesions.

PURPOSE: RAS mutations represent common driver alterations in thyroid cancer. They can be found in benign, low-risk and malignant thyroid tumors with follicular architecture, which are often diagnosed as indeterminate nodules on preoperative cytology. Therefore, the detection of RAS mutations in preoperative setting has a suboptimal predictive value for malignancy. In this study, we investigated differentially expressed microRNA (miRNA) in benign and malignant thyroid tumors with follicular architecture carrying mutations in RAS genes. METHODS: Total RNA was purified from 60 RAS-mutant follicular-patterned thyroid tumors, including follicular adenoma (FA), noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP), papillary and follicular thyroid carcinoma cases (PTC, FTC); 22 RAS-negative FAs were used as controls. The expression analysis of 798 miRNAs was performed by digital counting (nCounter nanoString platform). RESULTS: Comparing RAS-mutant and RAS-negative FAs, 12 miRNAs showed significant deregulation, which was likely related to the oncogenic effects of RAS mutations. Twenty-two miRNAs were differentially expressed in RAS-mutant benign versus malignant tumors. Considering the tumor type, 24 miRNAs were deregulated in PTC, 19 in NIFTP, and seven in FTC and compared to FA group; among these, miR-146b-5p, miR-144-3p, and miR-451a showed consistent deregulation in all the comparisons with the highest fold change. CONCLUSIONS: The miRNA expression analysis of follicular-patterned thyroid tumors demonstrated that RAS mutations influences miRNA profile in benign tumors. In addition, several miRNAs showed a histotype-specific deregulation and could discriminate between RAS-mutant benign and RAS-mutant malignant thyroid lesions, thus deserving further investigation as potential diagnostic markers.

Adipose tissue in COVID-19: detection of SARS-CoV-2 in adipocytes and activation of the interferon-alpha response.

OBJECTIVE: Obesity is a recognized risk factor for the progression to severe forms of COVID-19, yet the mechanisms of the association are unclear. METHODS: Subcutaneous abdominal adipose tissue specimens of subjects deceased from COVID-19 (n = 23) were compared to those of controls dying abruptly from causes other than infectious (accidental trauma, sudden cardiac death). Alterations of lung parenchyma consistent with moderate to severe disease were detected in all COVID-19 cases, not in controls. Investigations included: histopathologic features, detection of virus antigens and genome, characterization of infiltrating leukocytes, transcription levels of immune-related genes. RESULTS: By RT-PCR, the SARS-CoV-2 genome was detected in the adipose tissue of 13/23 (56%) cases of the COVID-19 cohort. The virus nucleocapsid antigen was detected in the cytoplasm of 1-5% adipocytes in 12/12 COVID-19 cases that were virus-positive by PCR in the adipose tissue (one case could not be assessed due insufficient tissue). The adipose tissue of COVID-19 cases showed leukocyte infiltrates and upregulation of the interferon-alpha pathway. After adjusting for age and sex, the activation score of IFN-alpha was directly related with transcription levels of the ACE2 gene, a key entry factor of SARS-CoV-2. CONCLUSIONS: In lethal COVID-19 cases, the SARS-CoV-2 nucleocapsid antigen has been detected in a sizeable proportion of adipocytes, showing that the virus may directly infect the parenchymal cells of subcutaneous fat. Infection appears to activate the IFN alpha pathway and to attract infiltrating leukocytes. Due to the huge numbers of adipocytes in adults, the adipose tissue represents a significant reservoir for SARS-CoV-2 and an important source of inflammatory mediators.

Histological pattern and gene expression profiling of thyroid tissue in subjects with obesity.

PURPOSE: Subjects with obesity may exhibit an increase in serum TSH concentrations. Several mechanisms have been proposed to explain this association, including the presence of a compensatory mechanism to counterbalance an accelerated turnover of thyroid hormones in subjects with obesity. This study aimed at evaluating whether the thyroids of subjects with obesity differs from those of normal-weight individuals regarding histology and gene expression profiling. METHODS: Ninety-eight patients were selected among those scheduled for thyroidectomy. At histology, thyroid tissue samples were investigated for the presence of adipocytes and/or lymphocyte infiltration. In a subset of patients, the expression at mRNA level of several genes involved in metabolic pathways and immune cell-related mechanisms was quantified by NanoString Technology. RESULTS: The presence of adipose cells was documented in thyroid specimens from 40% normal weight, 52.9% overweight and 73.5% patients with obesity. The number of infiltrating adipocytes was greater in specimens of patients with overweight or obesity compared to normal weight. The lymphocytes common antigen (CD45) and mast cell (MC) scores, and the number of CD3+ and CD8+ lymphocytes were higher in patients with overweight and obesity than in normal-weight subjects. Several genes involved in metabolic pathways were differently expressed in patients with overweight or obesity compared to normal weight, with upregulation of Leptin receptor and downregulation of Fatty Acid-Binding Protein 5. CONCLUSIONS: Increased BMI is associated with adipocyte and lymphocyte infiltration of the thyroid, not related to an autoimmune process, which might affect thyroid function in subjects with obesity. A differential gene expression profiling of metabolic and immune pathways in thyroid tissues of patients with obesity was also observed.

COVID-19 autopsy cases: detection of virus in endocrine tissues.

PURPOSE: The SARS-CoV-2 genome has been detected in a variety of human samples including blood, urine, semen, and faeces. However, evidence of virus presence in tissues other than lung are limited. METHODS: We investigated whether SARS-CoV-2 could be detected in 50 autoptic specimens of endocrine organs from 29 patients who died of COVID-19. RESULTS: The virus was detected in 25 specimens including ten abdominal subcutaneous adipose tissue samples (62%), six testes (67%), and nine thyroid (36%) samples. The analysis of multiple endocrine organ samples obtained from the same patients showed that, in virus-positive cases, the viral genome was consistently detected in all but two matched specimens. CONCLUSION: Our findings show that the virus spread into endocrine organs is a common event in severe cases. Further studies should assess the rate of the phenomenon in clinically mild cases. The potential long-term effects of COVID-19 on endocrine functions should be taken into consideration.

Cyclometalated Pt(ii) complexes with a bidentate Schiff-base ligand displaying unexpected cis/trans isomerism: synthesis, structures and electronic properties.

Square planar platinum complexes are an important class of compounds used in (nano)technology, optoelectronics, medicinal chemistry and catalysis. The major research interests in cyclometalated Pt(ii) complexes focus on the selective modulation of their electronic properties and the control of the (cis/trans) geometry. For the first time, we unveil and demonstrate that cis-trans isomers of Pt(ii) complexes can be obtained in a derivative carrying the 1-phenyl-pirazolate (Hppz) and 2-hydroxy-1-naphtyl-(N-phenyl)imine ligands. The two isomers display significant differences in both optical and electronic properties. While luminescence is quenched in solution, they are brightly emissive in the PMMA matrix at room temperature and in the 2MeTHF rigid matrix at 77 K. The phosphorescent emission of the cis isomer, blue-shifted compared to that of the trans one, results from the significantly different trans influence of the ppz ligand. Theoretical investigation highlights the almost isoenergetic potential energy of the two isomers therefore explaining their formation and evidences a large geometry distortion of their triplet state, which should be responsible for the observed luminescence efficiency.

Improvement of DNA recognition through molecular imprinting: hybrid oligomer imprinted polymeric nanoparticles (oligoMIP NPs).

High affinity and specific binding are cardinal properties of nucleic acids in relation to their biological function and their role in biotechnology. To this end, structural preorganization of oligonucleotides can significantly improve their binding performance, and numerous examples of this can be found in Nature as well as in artificial systems. Here we describe the production and characterization of hybrid DNA-polymer nanoparticles (oligoMIP NPs) as a system in which we have preorganized the oligonucleotide binding by molecular imprinting technology. Molecularly imprinted polymers (MIPs) are cost-effective "smart" polymeric materials capable of antibody-like detection, but characterized by superior robustness and the ability to work in extreme environmental conditions. Especially in the nanoparticle format, MIPs are dubbed as one of the most suitable alternatives to biological antibodies due to their selective molecular recognition properties, improved binding kinetics as well as size and dispersibility. Nonetheless, there have been very few attempts at DNA imprinting in the past due to structural complexity associated with these templates. By introducing modified thymine bases into the oligonucleotide sequences, which allow establishing covalent bonds between the DNA and the polymer, we demonstrate that such hybrid oligoMIP NPs specifically recognize their target DNA, and that the unique strategy of incorporating the complementary DNA strands as "preorganized selective monomers" improves the recognition properties without affecting the NPs physical properties such as size, shape or dispersibility.

Contact X-ray microscopy of living cells by using LiF crystal as imaging detector.

In this paper, the use of lithium fluoride (LiF) as imaging radiation detector to analyse living cells by single-shot soft X-ray contact microscopy is presented. High resolved X-ray images on LiF of cyanobacterium Leptolyngbya VRUC135, two unicellular microalgae of the genus Chlamydomonas and mouse macrophage cells (line RAW 264.7) have been obtained utilizing X-ray radiation in the water window energy range from a laser plasma source. The used method is based on loading of the samples, the cell suspension, in a special holder where they are in close contact with a LiF crystal solid-state X-ray imaging detector. After exposure and sample removal, the images stored in LiF by the soft X-ray contact microscopy technique are read by an optical microscope in fluorescence mode. The clear image of the mucilaginous sheath the structure of the filamentous Leptolyngbya and the visible nucleolus in the macrophage cells image, are noteworthiness results. The peculiarities of the used X-ray radiation and of the LiF imaging detector allow obtaining images in absorption contrast revealing the internal structures of the investigated samples at high spatial resolution. Moreover, the wide dynamic range of the LiF imaging detector contributes to obtain high-quality images. In particular, we demonstrate that this peculiar characteristic of LiF detector allows enhancing the contrast and reveal details even when they were obscured by a nonuniform stray light.

Flake size-dependent cyto and genotoxic evaluation of graphene oxide on in vitro A549, CaCo2 and vero cell lines.

This study was carried out by varying both graphene oxide (GO) concentration (10 μg/mL, 50 μg/mL, 100 μg/mL) and flakes sizes of 1320 nm and 130 nm. Characterization by scanning electron microscopy and Raman spectroscopy demonstrate that the area of GO flakes varies of one order of magnitude but their chemical structure remains unmodified. A 24-h cytotoxicity test showed, for A549, a loss in the viability, while the test exhibits overall a positive increase in the viability for CaCo2 and Vero. A 24-h comet assay shows a marked GO genotoxicity: for micrometer-sized GO flakes the genotoxicity is in positive correlation with the concentration, while for nanometer-sized GO flakes there was a high degree of genotoxicity at the lowest concentration tested.

Besides the HLA loci: SNPs and non-HLA gene products as immunoregulatory factors in the onset of psoriatic arthritis subtypes.

Psoriatic arthritis (PsA) is described as a multifactorial autoimmune rheumatic disease although its development is surely linked to some specific HLA genes (especially the HLA-Cw*06:02 allele). To date, its complex immunopathogenetic mechanism is not well clarified. Actually, increasing evidence suggest that qualitative and quantitative interplays between some PsA-susceptibility HLA alleles and other genetic, regulatory and environmental factors, may develop distinct subphenotypes of PsA. We first provide a brief summary of current knowledge about the various PsA conditions. Then, we consider the reasons why further analysis of the clinical course of patients affected by distinct PsA subsets, and who receive different therapeutic treatments, should be carried out in conjunction with deeper investigations about the identification of key genes and immunoregulatory agents by applying the most recent advances in biotechnology. This approach could better explain the molecular mechanisms responsible for both the onset and progression of this multi-faceted pathology with the purpose of significantly improving the management of PsA patients.

Immunogenetics and HPLC analyses contribute to understanding the etiopathology of rheumatoid arthritis through studies on ancient human remains.

Genetic investigations on ancient human remains affected by rheumatological pathologies are a research field of particular interest for identifying origins and the etiopathology of diseases, especially those having an autoimmune background such as rheumatoid arthritis (RA). We wish to demonstrate how reliable studies concerning this topic require collaboration between multiple disciplines, usually starting from paleopathologic observations up to immunogenetic screening, even involving analytical chemistry. Here, we focused our investigation on the skeleton of Cardinal Carlo de'Medici (1595-1666) for whom RA and psoriatic arthritis (PsA) were postulated after paleopathologic examination. RA susceptibility is linked to specific HLA alleles belonging to DRB1 04 locus, such as DRB1 0401, while Cw 0602 and DRB1 07 predispose to PsA. Thus, we genotyped the Cardinal?s remains to search for RA or PsA risk genes. Ancient DNA is often subjected to hydrolysis followed by fragmentation. For this reason, all immunogenetic tests were preceded by an original RP-HPLC-FL method able to inform on the ancient DNA preservation and the extent of contamination, with the purpose of avoiding the risk of false positive results. After DNA isolation from a piece of bone from the Cardinal, PCR-SSP and reverse-SSO hybridization assays were applied to perform genomic HLA-typing. RP-HPLC-FL analysis revealed a good preservation of DNA without contamination by exogenous genomes. Molecular tests assigned to the Cardinal the genotype DRB1 0401/1102 for HLA-DRB locus and Cw 04/ 12 for HLA-C locus, data that support a genetic predisposition for RA but not for PsA. This multidisciplinary study has allowed us: (i) to ascertain that the remains undoubtedly belonged to the specific subject, Cardinal Carlo de?Medici; (ii) to sustain that the subject suffered from RA rather then that PsA, and (iii) to state that RA was already widespread in Europe at the Renaissance age, despite some authors claiming that the disease was introduced to the Old Continent from America after colonization during the 18th century.

Adaptive resolution simulation of liquid para-hydrogen: testing the robustness of the quantum-classical adaptive coupling.

Adaptive resolution simulations for classical systems are currently made within a reasonably consistent theoretical framework. Recently we have extended this approach to the quantum-classical coupling by mapping the quantum nature of an atom onto a classical polymer ring representation within the path integral approach [Poma & Delle Site, Phys. Rev. Lett., 2010, 104, 250201]. In this way the process of interfacing adaptively a quantum representation to a classical one corresponds to the problem of interfacing two regions with a different number of effective "classical" degrees of freedom; thus the classical formulation of the adaptive algorithm applies straightforwardly to the quantum-classical problem. In this work we show the robustness of such an approach for a liquid of para-hydrogen at low temperature. This system represents a highly challenging conceptual and technical test for the adaptive approach due to the extreme thermodynamical conditions where quantum effects play a central role.

Classical to path-integral adaptive resolution in molecular simulation: towards a smooth quantum-classical coupling.

Simulations that couple different molecular models in an adaptive way by changing resolution on the fly allow us to identify the relevant degrees of freedom of a system. This, in turn, leads to a detailed understanding of the essential physics which characterizes a system. While the delicate process of transition from one model to another is well understood for the adaptivity between classical molecular models the same cannot be said for the quantum-classical adaptivity. The main reason for this is the difficulty in describing a continuous transition between two different kinds of physical principles: probabilistic for the quantum and deterministic for the classical. Here we report the basic principles of an algorithm that allows for a continuous and smooth transition by employing the path integral description of atoms.

[Update on the laparoscopic adrenal surgery in the second decade of the century: "doubts no more?]. / Update sulla chirurgia laparoscopica del surrene nel secondo decennio del secolo: "doubts no more?"

Laparoscopic adrenal surgery has significantly improved during the last years. Thus at the moment it is possible to define such technique as the therapeutic "Gold Standard" option in the treatment of the adrenal tumors. However, some doubts are still remaining concerning the feasibility of laparoscopic adrenalectomy in case of malignant adrenal tumors, hyper-vascular tumors (pheochromocytoma) and indeterminate incidentaloma. This study aimed to review all the literature of the last three years (only article with abstracts) using the criteria of selection of the Cochrane Library, in order to find class I and class II-III studies which are able to surely or, respectively, probably respond to the various questions yet to be answered Two hundred and twelve papers have been selected. The class I studies stated the following evidences: laparoscopic treatment of pheochromocytoma is the Gold Standard too, independently from the dimension; the short term results of laparoscopic anterior and posterior approach are equivalent to those of the lateral one. Doubts still remain concerning the role of laparoscopy in the treatment of metastases, big pheochromocytomas, small size incidentalomas (risk of over-treatement). Nearly no respond has been given to others issues such as "the single port techniques" in laparoscopic adrenalectomy, the role of radiofrequency laparoscopic ablation of the adrenal tumor, the kind of treatment of stadium I and II adrenocortical carcinoma and big size (> 8 cm) tumors, the management of non-functioning incidentaloma of 4-6 cm, the role of the robot, and, finally, the approach of the bilateral tumors. We conclude that, despite many issues on the feasibility and safety of laparoscopy in the adrenal surgery have been definitely clarified, so that such technique has been declared the "Gold Standard" method in the treatment of the adrenal tumor, doubts still remain in some aspects of this method. However, since the researches in this field are proceeding with high evolution velocity, in the next future most of the questions that are still present should be definitively adressed.

Saponaria officinalis karyology and karyotype by means of image analyzer and atomic force microscopy.

The aim of this work was to offer a contribution to the characterization of taxonomic entity of Saponaria officinalis (2n = 28; an herbaceous perennial species; saporin, a type 1 Ribosome Inactivating Protein, is present in leaves and seeds) by a cytogenetic and karyomorphological approach. We investigated the karyotype's morphometry correlated with Stebbin's symmetric index; the same information has been used for computing the indices resemblance between chromosomes (REC), symmetric indices (SYI), and total form (TF%) which allow the comparison between species and evaluation of karyological evolution. Fluorescence intensities of the stained nuclei were measured by a flow cytometer and, for the first time, values for nuclear DNA content were estimated by comparing nuclei fluorescence intensities of the test population with those of appropriate internal DNA standards. Our study is also aimed to introduce chromosomal volumes, which were determined by atomic force microscopy (AFM), as novel karyomorphological parameter which could allow for chromosome discrimination especially when tiny ones are present.

Mapping the intake of different elements in vegetal tissues by dual-energy X-ray imaging at DaPhine synchrotron light source.

This article reports on the first utilization of the soft X-ray beamline at the DaPhine synchrotron light source for mapping the intake of different elements in plant tissues. As a test, the method of dual-energy X-ray microradiography was applied to the investigation of the natural sulfur content in dried leaf and root samples. Our ultimate goal was to monitor the pollutant lead and its intake, which was added in controlled doses to the hydroponic medium of laboratory-controlled samples of vegetal species. The results obtained by the nondestructive X-ray radiographic analysis are compared to the values of concentrations determined by a standard chemical analysis utilizing atomic absorption spectroscopy. From this comparison the validity of the X-ray detection of heavy metals in biological samples has been confirmed. The superposition of the dual energy results on the simple planar radiography shows the representation of the pollutant intake directly on the sample structures. It should be pointed out that this method, developed here for plant root and leaves could be applied to any biological sample of interest, but the preparation and observation conditions necessitate different strategies according to the type of sample under analysis.

Monitoring of the heavy-metal hyperaccumulation in vegetal tissues by X-ray radiography and by femto-second laser induced breakdown spectroscopy.

This article reports on the utilization of X-ray microradiography and laser induced breakdown spectroscopy (LIBS) techniques for investigation of the metal accumulation in different part of leaf samples. The potential of the LIBS-analysis for finding the proper plant species for phytoremediation is compared with the results of microradiography measurements at the HERCULES source at ENEA, Rome (Italy) and X-ray microradiography experiments at the ELETTRA Synchrotron, Trieste (Italy).

Interactions between saporin, a ribosome-inactivating protein, and DNA: a study by atomic force microscopy.

Saporins are enzymes belonging to the PNAG class (polynucleotide: adenosine glycosidase), plant enzymes commonly known as ribosome-inactivating proteins (RIP), as a result of their property of irreversibly damaging eukaryotic ribosomes. Direct imaging with tapping-mode atomic force microscopy (AFM) has been used to study pGEM-4Z plasmid DNA binding to the saporin-SO6 (isoform from Saponaria officinalis seeds). Saporin wrapped the plasmidic DNA, and distribution of the enzyme molecules along the DNA chain was markedly variable; plasmid digested with saporin-SO6 appeared fragmented or topologically modified. The supercoiled DNA strands were cleaved, giving rise to a linearized form and to relaxed forms. Electrophoretic analysis of the effect of standard preparations of saporin-SO6 on pGEM-4S confirmed the presence of DNA strand-cleaving activity.

Raltitrexed plus oxaliplatin as first-line chemotherapy in metastatic colorectal carcinoma: a multicentric phase II trial.

For advanced colorectal carcinoma, two new drugs, raltitrexed (TOM) and oxaliplatin (L-OHP), have recently shown interesting results. Preclinical and clinical studies suggest that this combination, because of its favorable toxicity profile, high response rate and convenient schedule of administration, can be administered successfully in this disease. In our phase II study, 37 non pre-treated patients with metastatic colorectal carcinoma were treated with TOM (3 mg/m(2)) and L-OHP (130 mg/m(2)) every 3 weeks. In total, 222 cycles were administered; all patients received at least 2 cycles (median 6, range 2-8). There were two complete and 14 partial responses for an overall response rate of 43% (95% CI 27-69%). The median time to response was 2.5 months (range 2-4) and the median duration was 10.3 months (range 5-18). Twelve of the 23 (52%) patients with symptomatic colorectal cancer were classified as clinical benefit responders for at least 4 weeks during the study period. Treatment was well tolerated, and both acute, essentially hematologic, and cumulative hepatic and neurologic toxicities were manageable and reversible. Response rate and toxic effects observed during this study warrant additional studies comparing this TOM-L-OHP regimen with CPT-11 and/or capacitebine-containing regimens in metastatic colorectal carcinoma.

Nuclear damage induced by liposomes containing fitc-labelled saporin on human melanoma cells in vitro.

Ribosome-inactivating proteins are enzymes of plant origin which de-adenilate the major ribosomal RNA, making it unable to bind the elongation factor and thus arresting protein synthesis. Recently the N-glycosidase activity of these enzymes has been extended also to deoxyribonucleotides substrates. In the present study we report the successful entrapment of the type 1 ribosome-inactivating protein saporin, covalently labelled with fluorescein isothiocyanate (FITC) into L-alpha lecitin/cholesterol liposomes and describe its delivery to human melanoma cells in vitro. The fluorescein reacted toxin maintained its enzymatic activity, although to a reduced extent; its interaction with liposomes resulted in the entry of the protein through the lipid bilayers. The resulting vesicles are carriers that can deliver the toxin inside cells; as a consequence the cytotoxic effects of the encapsulated enzyme were evident at a concentration two order of magnitude lower than that of the native one. In particular the nuclear damage, as revealed by micronuclei formation, was evident within 44 hr. The intracellular dynamics of the enzyme, as analyzed by confocal microscopy, point to an endocytic pathway of vesicles entry.