Is It Chiral?towards Identifying Chirality In Unknown Samples Without Prior Spectral Assignment.

Current spectroscopic methods \footnote{Sérgio R. Domingos et al. ,Sensing Chirality with Rotational Spectroscopy Annual Review of Physical Chemistry 69:1, 499-519 (2018).}\textsuperscript{,}\footnote{ Nafie, L. A.; Dukor, R. K.; Freedman, T. B. Handbook of Vibrational Spectroscopy: Vibrational Circular Dichroism; Wiley Online Library, 2006.}can only detect and quantify chirality if the molecular composition of the sample has been determined. Therefore, before extracting enantiomeric information from a spectrum of an unknown sample, one needs to compare it to previously measured spectra, or to simulated spectra generated with previously determined rotational constants. We describe new developments towards an updated version of the microwave three-wave mixing technique (M3WM)\footnote{Patterson, D. Et al , Enantiomer-specific detection of chiral molecules via microwave spectroscopy. Nature 497, 475–477 (2013).} in a buffer gas cell, that can unambiguously identify the presence of chiral species in an unknown sample with no need for prior spectral assignment or knowledge of the sample composition. This work combines the established sensitivity of 3WM in a buffer gas cell in determining enantiomeric information with broadband microwave fields, and an optimized sample input, and it opens up new directions in chirality detection towards real-life molecular samples and complicated mixtures

Three dimensional organization mechanisms and polarized protein traffic in thyroid follicular cells

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de lectura: 19-02-2015The structural and functional unit of the thyroid gland is the thyroid follicle that consists in a single layer of follicular cells enclosing a central lumen where thyroid hormones are synthesized and stored. Follicular cells or thyrocytes are epithelial polarized cells with the apical membrane facing the lumen and the basal membrane surrounded by the basement membrane towards the stroma. We have studied the follicle formation process in three dimensional (3D) Matrigel cultures using the rat follicular cell line FRT and mouse primary thyrocytes. Our results demonstrate that follicles originated either from single or plural cells acquire apical‐basal polarity after the first cell division. Apical membrane is defined at a specific site of the cell‐cell junctions where the exocytosis of apical membrane components occurs. Lumen is formed by further apical exocytosis followed by membrane separation or coalescence between apical vacuolar compartments. Mature tight junctions separate the apical from the basolateral domains and both actin filaments and acetylated microtubules exhibit a polarized apical distribution. Through microarray‐based differential expression analysis we have identified regulators of 3D follicular organization. Among them, Pax8 transcription factor was discovered to play a critical role in polarity orientation and follicle formation. In the absence of Pax8 apical membrane orientation was inverted and lumen formation was impaired. The polarized distribution of cell cytoskeleton was altered due to the transcriptional inhibition of both cadherin‐16 and its cytoskeleton linker, αB‐crystallin. Apical transport of Rac1 through actin filaments was also blocked and thus local actin organization and apical traffic were affected. Other Pax8 transcriptional targets described to be involved in apical transport such as Rab17 and myosin Vb could also participate in follicular apical membrane formation. Thyroid function is determined by the polarized distribution of thyroid specific proteins at the apical‐basal membranes and inside the lumen. NIS, the sodium/iodide symporter, is a key protein for iodide uptake and thyroid hormone synthesis, and is localized in the basolateral plasma membrane. We have identified a clathrinmediated pathway responsible for NIS basolateral traffic, in which the clathrin adaptor complexes AP‐1A and AP‐1B sort NIS at the trans‐Golgi and the recycling endosomes. More specifically, we have demonstrated that the medium subunit of AP‐1B controls NIS basolateral sorting through common recycling endosomes. In its absence, NIS is apically missorted where remains functional. Besides, direct NIS basolateral transport from the trans‐Golgi to the basolateral membrane is mediated by AP‐1A through clathrin‐coated vesicles that also carry the transferrin receptor. In the absence of AP‐1A medium subunit, AP‐1B fully compensates its function. NIS internalization pathways were also studied and preliminary results have shown that the mechanism is clathrin‐independent and that NIS is finally degraded by lysosomes.La unidad estructural y funcional del tiroides es el folículo tiroideo que consiste en una capa de células foliculares posicionadas alrededor de un lumen central donde se sintetizan y se almacenan las hormonas tiroideas. Las células foliculares o tirocitos son células epiteliales polarizadas con la membrana apical hacia el lumen y la membrana basal rodeada de la lámina basal hacia el estroma. Hemos estudiado la formación del folículo en cultivos tridimensionales (3D) de Matrigel usando la línea tiroidea de rata FRT y cultivos primarios de tirocitos de ratón. Nuestros resultados demuestran que los folículos formados tanto a partir de una como de varias células, adquieren la polaridad ápico‐basal tras la primera división. La formación de la membrana apical se inicia entre las uniones célula‐célula en el sitio donde se exocitan los componentes de la membrana apical. El lumen se forma tras la exocitosis seguida por la separación de las membranas apicales o por coalescencia entre compartimentos apicales de vacuolas. Las uniones estrechas separan el dominio apical del dominio basolateral, y los filamentos de actina junto con los microtubulos acetilados se distribuyen apicalmente. Mediante un array de expresión hemos identificado reguladores de la organización folicular en 3D. De entre ellos el factor de transcripción Pax8 resultó tener un papel esencial en la orientación de la polaridad y en la formación del folículo. En ausencia de Pax8 la orientación de la membrana apical se invierte y se impide la formación del lumen. La distribución polarizada del citoesqueleto se altera debido a la inhibición transcripcional tanto de la cadherina‐16 como de su proteína de unión al citosqueleto, la cristalina alfa B. El transporte apical de Rac1, a través de los filamentos de actina, se inhibe y como consecuencia la organización apical de actina y el tráfico están afectados. Otras dianas transcripcionales de Pax8 que están involucradas en el transporte apical, como las proteínas Rab17 y myosina Vb, podrían estar también participando en la formación de la membrana apical. La función tiroidea depende de la localización polarizada de proteínas específicas de tiroides en la membrana ápico‐basolateral y dentro del lumen folicular. El simportador de sodio/yoduro, localizado en la membrana basolateral es una proteína clave para la captación del yoduro y la síntesis de las hormonas tiroideas. Hemos identificado una vía mediada por clatrina como responsable del tráfico basolateral de NIS, en la cual los complejos adaptadores de clatrina AP‐1A y AP‐1B segregan a NIS a nivel del trans‐Golgi y de los endosomas de reciclaje. Hemos demostrado que la subunidad mediana de AP‐1B controla el tráfico basolateral de NIS a través de los endosomas comunes de reciclaje. En su ausencia, NIS se transporta a la membrana apical donde sigue siendo funcional. AP‐1A participa en el tráfico directo de NIS del trans‐Golgi a la membrana basolateral a través de vesículas cubiertas de clatrina que cotransportan el receptor de transferrina. En ausencia de su subunidad mediana, AP‐1B compensa su función. También estudiamos las vías de internalización de NIS y resultados preliminares muestran que el mecanismo es independiente de clatrina y que NIS es finalmente degradado en los lisosomas

Comparison of receptor affinity of natSc-DOTA-TATE versus natGa-DOTA-TATE

BACKGROUND: 44Sc as a positron emitter can be an interesting alternative to 68Ga (T½ = 67.71 min) due to its longer half-life (T½ = 3.97 h). Moreover, the b– emitter 47Sc can be used for therapy when attached to the same biomolecule vectors. DOTA as a chelating agent has been proven suitable for the radiolabelling of peptides recognising tumour cell receptors in vivo with M3+ radiometals. DOTA-derivatized peptides have been successfully labelled with 90Y and 177Lu for therapy, and with 68Ga for PET imaging. However, published data on 44Sc-labelled DOTA-biomolecules as potential PET radiotracers are still very limited. The aim of this study was to compare the affinity of natGa- and natSc-labelled DOTA-TATE to somatostatin receptors subtype 2 expressed in rat pancreatic cancer cell line AR42J. MATERIAL AND METHODS: The cold complexes of DOTA-TATE with natGa and natSc were synthesized and identified by HPLC and MS analysis and evaluated in vitro for competitive binding to cancer cell line AR42J expressing somatostatin receptors subtype 2 (sstr2). RESULTS: The IC50 values calculated from the displacement curve of {125I-Tyr11}-SST-14 were: 0.20 ± 0.18, 0.70 ± 0.20, 0.64 ± 0.22 and 0.67 ± 0.12 for natGa-DOTA-TATE, natSc-DOTA-TATE, DOTA-TATE, and {Tyr11}-SST-14 complexes, respectively, with the affinity lowering in the decreasing order: natGa-DOTA-TATE > DOTA-TATE > Tyr11-SST-14 > natSc-DOTA-TATE. CONCLUSIONS: The binding affinity of natGa-DOTA-TATE appeared higher than that of natSc-DOTA-TATE. Further in vitro and in vivo studies are needed to verify the influence of the chelated metal on the affinity and uptake of the respective radiolabelled compounds. This information might be crucial when the in vivo applications of peptides labelled with 68Ga and 44Sc for PET, as well as the use of 47Sc for radiotherapy are considered. Nuclear Med Rev 2011; 14, 2: 85–8

Rigas Pheraios. Un nouveau bilan de la recherche. Jannina, 28 - 31 mai 1998.

Le congrès organisé par l’université de Jannina avait comme objectif principal, en dehors de la présentation d’études concernant la vie et l’œuvre de Rigas, tout élément nouveau dû aux recherches effectuées dans les derniers temps. C’est justement par le sous-titre du sujet du congrès « Un nouveau bilan de la recherche », que les organisateurs, membres de la faculté de Philosophie de ladite université, ont voulu souligner l’importance des éléments nouveaux qu’ils attendaient récolter au cours..

Exploring Paternal Involvement from Greek, Greek - Cypriot and Turkish Fathers’ and Mothers’ Perspectives: Cross-National Differences and Similarities

Although European fatherhood is “in the process of reconstruction and transformation” (O’Brien 2004, as cited in Lero, Ashbourne and Whitehead 2006, 5) and there is a need to create a clear picture about paternal involvement, few studies have explored perceptions on the actual father involvement as well as the factors predicting and relating to father involvement, especially in Southeastern European countries. The present study aims at exploring the role of the father and the types of paternal involvement in Greece, Cyprus and Turkey both from mother’s and fathers’ perspectives. Research results revealed statistically significant differences in terms of the role of the father and the parental styles adopted in a country level and in the way fathers are involved in a parent level. Further, results confirm the hypothesis that father involvement is a multidimensional concept since statistically significant correlations were revealed among scales and subscales

IS IT CHIRAL?TOWARDS IDENTIFYING CHIRALITY IN UNKNOWN SAMPLES WITHOUT PRIOR SPECTRAL ASSIGNMENT.

Current spectroscopic methods \footnote{Sérgio R. Domingos et al. ,Sensing Chirality with Rotational Spectroscopy Annual Review of Physical Chemistry 69:1, 499-519 (2018).}\textsuperscript{,}\footnote{ Nafie, L. A.; Dukor, R. K.; Freedman, T. B. Handbook of Vibrational Spectroscopy: Vibrational Circular Dichroism; Wiley Online Library, 2006.}can only detect and quantify chirality if the molecular composition of the sample has been determined. Therefore, before extracting enantiomeric information from a spectrum of an unknown sample, one needs to compare it to previously measured spectra, or to simulated spectra generated with previously determined rotational constants. We describe new developments towards an updated version of the microwave three-wave mixing technique (M3WM)\footnote{Patterson, D. Et al , Enantiomer-specific detection of chiral molecules via microwave spectroscopy. Nature 497, 475–477 (2013).} in a buffer gas cell, that can unambiguously identify the presence of chiral species in an unknown sample with no need for prior spectral assignment or knowledge of the sample composition. This work combines the established sensitivity of 3WM in a buffer gas cell in determining enantiomeric information with broadband microwave fields, and an optimized sample input, and it opens up new directions in chirality detection towards real-life molecular samples and complicated mixtures

Aktualne poglądy na diagnostykę i leczenie niskozróżnicowanych raków neuroendokrynnych przewodu pokarmowego

Poorly differentiated neuroendocrine carcinomas (PDNEC) are rare tumours that can originate from any site of the gastrointestinal tract exhibiting an overall aggressive behaviour that may vary between tumours according to the degree of cellular proliferation. The majority of PDNEC are locally advanced or metastatic at presentation, and are only infrequently associated with secretory hormonal syndromes. PDNEC exhibit aggressive histological features (high mitotic rate, high Ki67 labelling index and presence of necrosis) and are further subdivided into two morphological subgroups, small and large cell variants. As PDNEC express somatostatin receptors less frequently, somatostatin receptor scintigraphy is usually negative, whereas 18F-fluorodeoxyglucose positron emission tomography appears to be the best method of evaluating disease spread and guiding further treatment. PDNEC have traditionally been treated similarly to small cell lung carcinoma, although they show a number of different clinical and histopathologic features. First line systemic chemotherapy with a platinum-based agent and etoposide is used for patients with metastatic disease, leading to variable response rates that are often of relative short duration. Sequential or concurrent chemoradiation is recommended for patients with locoregional disease. In patients with localised disease, complete surgical resection should be offered followed by adjuvant treatment (chemotherapy with or without radiotherapy); the value of neoadjuvant chemotherapy has not been evaluated as yet. The role of second line therapies is evolving, with temozolomide being a promising agent. However, the majority of data regarding PDNEC is hampered by the small number of series and their retrospective nature, making it important that multicentre co-operative studies be performed.Niskozróżnicowane raki neuroendokrynne (PDNEC, poorly differentiated neuroendocrine carcinomas) to rzadkie nowotwory, które mogą wywodzić się z dowolnego miejsca w przewodzie pokarmowym, cechując się ogólnie agresywnym przebiegiem uzależnionym od stopnia nasilenia proliferacji komórek nowotworowych. Większość przypadków PDNEC w momencie rozpoznania stanowią nowotwory miejscowo zaawansowane lub przerzutowe i rzadko towarzyszą im zespoły chorobowe związane z wydzielanymi przez te nowotwory hormonami. PDNEC cechują się agresywnym obrazem histopatologicznym (duża liczba figur podziału, wysoki wskaźnik aktywności proliferacyjnej Ki67 i obecność martwicy) i wyróżnia się wśród nich dwie podgrupy morfologiczne — odmianę drobnokomórkową i wielkokomórkową. Ponieważ w PDNEC rzadziej stwierdza się ekspresję receptorów somatostatynowych, scyntygrafia receptorów somatostatynowych zwykle daje negatywne wyniki, natomiast pozytonowa tomografia emisyjna z 18F-fluorodeoksyglukozą wydaje się być najlepszą metodą do oceny rozległości choroby i pomocną przy podejmowaniu decyzji dotyczących dalszego leczenia. PDNEC zwykle leczy się podobnie do drobnokomórkowego raka płuca, choć nowotwory te wykazują szereg różnic klinicznych i histopatologicznych. U pacjentów z chorobą rozsianą stosuje się układową chemioterapię pierwszego rzutu obejmującą pochodną platyny i etopozyd. Odsetek odpowiedzi na leczenie jest różny, a sama odpowiedź utrzymuje się względnie krótko. U pacjentów z chorobą lokoregionalną zaleca się stosowanie sekwencyjnej lub jednoczasowej chemioradioterapii. U pacjentów z chorobą zlokalizowaną stosuje się radykalne leczenie chirurgiczne z chemio- lub chemioradioterapią uzupełniającą. Nie ustalono dotychczas roli chemioterapii neoadiuwantowej. Schematy leczenia drugiego rzutu na razie ewoluują; obiecujący wydaje się być temozolomid. Wartość większości danych dotyczących PDNEC jest jednak ograniczona niezbyt dużą liczbą przypadków oraz ich retrospektywnym charakterem. Dlatego też tak ważne byłoby przeprowadzenie wieloośrodkowych badań kooperacyjnych

Development of a novel bi-specific monoclonal antibody approach for tumour targeting

To overcome the disadvantages of bi-specific antibody methodologies in vivo, a novel antibody approach has been designed to improve tumour targeting and effector to target ratio. The technique involves biotinylated anti-CD3 Fab fragments and streptavidinylated anti-tumour monoclonal antibodies (mAbs) that can spontaneously form cross-links. We describe here a method for the direct cross-linking of sulphydryl-conjugated HMFG1 (anti-MUC1 mucin mAb) to streptavidin by sulphosuccinimidyl-4-(N-maleimidomethyl) cyclohexane- 1-carboxylate. Fab fragments generated by papain digestion of the 1452C11 antibody (anti-CD3 mAb without Fc to avoid peripheral activation of T-cells) were biotinylated with NHS-Iminobiotin. MUC1-transfected BALB/c breast cancer cell lines 413BCR and 425CCR and the parental cell line (410.4) were labelled with streptavidinylated mouse anti-MUC1 mucin mAb. BALB/c effector T-cells were separately labelled with biotinylated anti-CD3 Fab fragments (1452C11) and mixed with tumour cells in different effector to target ratios. Percentage of killing was assessed using the 51Cr cytotoxicity assay. Seventy per cent lysis was measured in the case of 413BCR (high MUC1 mucin expressor) and 40% in the case of 425CCR (low expressor) cell line. No lysis was apparent in the MUC1 negative cell line. These results demonstrate that the novel T-cell redirecting approach we have developed can produce effective immune lysis of target cells in vitro. © 1999 Cancer Research Campaig

A Pleomorphic Rhabdomyosarcoma Mimicking an Inguinal Hernia: A Case Report and Review of the Literature

A 59-year-old male presented with a painful right inguinal swelling and deep vein thrombosis at the ipsilateral leg. An inguinal hernia was initially diagnosed, but during surgery a large mass was found anteriorly to the peritoneal sheaths. Histology revealed a high-grade pleomorphic rhabdomyosarcoma. The mass advanced rapidly, occupying the whole right iliac fossa and metastasizing to the lung. Despite first- and second-line chemotherapy, the patient deteriorated rapidly and died. Rhabdomyosarcomas should be managed in specialized centres as they have prognostic factors and histologic features still controversial and poorly clarified

Basolateral Sorting of the Sodium/Iodide Symporter Is Mediated by Adaptor Protein 1 Clathrin Adaptor Complexes

Background: The sodium/iodide symporter (NIS) is a transmembrane protein located on the basolateral membrane of thyrocytes. Despite its physiological and clinical relevance, little is known about the mechanisms that mediate NIS subcellular sorting. In the present study, we examined NIS basolateral trafficking in vitro using non-thyroid and thyroid epithelial cells. Methods: Immunofluorescence and Western blotting were performed to analyze NIS subcellular location and function in cells grown in monolayers under unpolarized and/or polarized conditions. Strategic NIS residues were mutated, and binding of NIS to clathrin adaptor complexes was determined by immunoprecipitation. Results: We show that NIS reaches the plasma membrane (PM) through a thyrotropin-dependent mechanism 24 hours after treatment with the hormone. We demonstrate that NIS basolateral trafficking is a clathrin-mediated mechanism, in which the clathrin adaptor complexes AP-1 (A and B) sort NIS from the trans-Golgi network (TGN) and recycling endosomes (REs). Specifically, we show that the AP-1B μ1 subunit controls NIS basolateral sorting through common REs. In its absence, NIS is apically missorted but remains functional. Additionally, direct NIS basolateral transport from the TGN to the basolateral membrane is mediated by AP-1A through clathrin-coated vesicles that also carry the transferrin receptor. Loss of the μ1 subunit of AP-1A is functionally compensated by AP-1B. Furthermore, loss of both subunits diminishes NIS trafficking to the PM. Finally, we demonstrate that AP-1A binds to the L121 and LL562/563 residues on NIS, whereas AP-1B binds to L583. Conclusions: Our findings highlight the novel involvement of the clathrin-coated machinery in basolateral NIS trafficking. Given that AP-1A expression is reduced in tumors, and its expression correlates with that of NIS, these findings will help uncover new targets in thyroid cancer treatment.This work was supported by grants PID2019-105303RB I00/AEI/10.13039/501100011033 from Ministerio de Ciencia e Innovación (MICIN) and B2017/BMD-3724, Tironet2-CM from Comunidad de Madrid (Spain) to P.S.; and SAF2015-69964-R, RTI2018-099343-B-100 from the MICIN, Spain, and Fondo Europeo de Desarrollo Regional to A.D.l.V. P.S. and A.D.l.V. laboratories are supported jointly by CIBERONC CB16/12/00326 from the Instituto de Salud Carlos III (ISCIII). P.K., C.F.-M., and L.M.M. held predoctoral fellowship from Ministerio de Economía y Competitividad, Universidad Autónoma de Madrid (Spain) and CIBERONC, respectively. D.F.-D. holds a contract associated with Grant SAF2015-69964-R.S