Fluorescently Labeled Ceramides and 1-Deoxyceramides: Synthesis, Characterization, and Cellular Distribution Studies.

Ceramides (Cer) are bioactive sphingolipids that have been proposed as potential disease biomarkers since they are involved in several cellular stress responses, including apoptosis and senescence. 1-Deoxyceramides (1-deoxyCer), a particular subtype of noncanonical sphingolipids, have been linked to the pathogenesis of type II diabetes. To investigate the metabolism of these bioactive lipids, as well as to have a better understanding of the signaling processes where they participate, it is essential to expand the toolbox of fluorescent sphingolipid probes exhibiting complementary subcellular localization. Herein, we describe a series of new sphingolipid probes tagged with two different organic fluorophores, a far-red/NIR-emitting coumarin derivative (COUPY) and a green-emitting BODIPY. The assembly of the probes involved a combination of olefin cross metathesis and click chemistry reactions as key steps, and these fluorescent ceramide analogues exhibited excellent emission quantum yields, being the Stokes' shifts of the COUPY derivatives much higher than those of the BODIPY counterparts. Confocal microscopy studies in HeLa cells confirmed an excellent cellular permeability for these sphingolipid probes and revealed that most of the vesicles stained by COUPY probes were either lysosomes or endosomes, whereas BODIPY probes accumulated either in Golgi apparatus or in nonlysosomal intracellular vesicles. The fact that the two sets of fluorescent Cer probes have such different staining patterns indicates that their subcellular distribution is not entirely defined by the sphingolipid moiety but rather influenced by the fluorophore.

A Nanoencapsulated Ir(III)-Phthalocyanine Conjugate as a Promising Photodynamic Therapy Anticancer Agent.

Despite the potential of photodynamic therapy (PDT) in cancer treatment, the development of efficient and photostable photosensitizing molecules that operate at long wavelengths of light has become a major hurdle. Here, we report for the first time an Ir(III)-phthalocyanine conjugate (Ir-ZnPc) as a novel photosensitizer for high-efficiency synergistic PDT treatment that takes advantage of the long-wavelength excitation and near infrared (NIR) emission of the phthalocyanine scaffold and the known photostability and high phototoxicity of cyclometalated Ir(III) complexes. In order to increase water solubility and cell membrane permeability, the conjugate and parent zinc phthalocyanine (ZnPc) were encapsulated in amphoteric redox-responsive polyurethane-polyurea hybrid nanocapsules (Ir-ZnPc-NCs and ZnPc-NCs, respectively). Photobiological evaluations revealed that the encapsulated Ir-ZnPc conjugate achieved high photocytotoxicity in both normoxic and hypoxic conditions under 630 nm light irradiation, which can be attributed to dual Type I and Type II reactive oxygen species (ROS) photogeneration. Interestingly, PDT treatments with Ir-ZnPc-NCs and ZnPc-NCs significantly inhibited the growth of three-dimensional (3D) multicellular tumor spheroids. Overall, the nanoencapsulation of Zn phthalocyanines conjugated to cyclometalated Ir(III) complexes provides a new strategy for obtaining photostable and biocompatible red-light-activated nano-PDT agents with efficient performance under challenging hypoxic environments, thus offering new therapeutic opportunities for cancer treatment.

Exploring Structure-Activity Relationships in Photodynamic Therapy Anticancer Agents Based on Ir(III)-COUPY Conjugates.

Photodynamic therapy holds great promise as a non-invasive anticancer tool against drug-resistant cancers. However, highly effective, non-toxic, and reliable photosensitizers with operability under hypoxic conditions remain to be developed. Herein, we took the advantageous properties of COUPY fluorophores and cyclometalated Ir(III) complexes to develop novel PDT agents based on Ir(III)-COUPY conjugates with the aim of exploring structure-activity relationships. The structural modifications carried out within the coumarin scaffold had a strong impact on the photophysical properties and cellular uptake of the conjugates. All Ir(III)-COUPY conjugates exhibited high phototoxicity under green light irradiation, which was attributed to the photogeneration of ROS, while remaining non-toxic in the dark. Among them, two hit conjugates showed excellent phototherapeutic indexes in cisplatin-resistant A2780cis cancer cells, both in normoxia and in hypoxia, suggesting that photoactive therapy approaches based on the conjugation of far-red/NIR-emitting COUPY dyes and transition metal complexes could effectively tackle in vitro acquired resistance to cisplatin.

Halting ErbB-2 isoforms retrograde transport to the nucleus as a new theragnostic approach for triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is clinically defined by the absence of estrogen and progesterone receptors and the lack of membrane overexpression or gene amplification of receptor tyrosine kinase ErbB-2/HER2. Due to TNBC heterogeneity, clinical biomarkers and targeted therapies for this disease remain elusive. We demonstrated that ErbB-2 is localized in the nucleus (NErbB-2) of TNBC cells and primary tumors, from where it drives growth. We also discovered that TNBC expresses both wild-type ErbB-2 (WTErbB-2) and alternative ErbB-2 isoform c (ErbB-2c). Here, we revealed that the inhibitors of the retrograde transport Retro-2 and its cyclic derivative Retro-2.1 evict both WTErbB-2 and ErbB-2c from the nucleus of BC cells and tumors. Using BC cells from several molecular subtypes, as well as normal breast cells, we demonstrated that Retro-2 specifically blocks proliferation of BC cells expressing NErbB-2. Importantly, Retro-2 eviction of both ErbB-2 isoforms from the nucleus resulted in a striking growth abrogation in multiple TNBC preclinical models, including tumor explants and xenografts. Our mechanistic studies in TNBC cells revealed that Retro-2 induces a differential accumulation of WTErbB-2 at the early endosomes and the plasma membrane, and of ErbB-2c at the Golgi, shedding new light both on Retro-2 action on endogenous protein cargoes undergoing retrograde transport, and on the biology of ErbB-2 splicing variants. In addition, we revealed that the presence of a functional signal peptide and a nuclear export signal (NES), both located at the N-terminus of WTErbB-2, and absent in ErbB-2c, accounts for the differential subcellular distribution of ErbB-2 isoforms upon Retro-2 treatment. Our present discoveries provide evidence for the rational repurposing of Retro-2 as a novel therapeutic agent for TNBC.

First Report and Comparative Genomics Analysis of a blaOXA-244-Harboring Escherichia coli Isolate Recovered in the American Continent.

The carbapenemase OXA-244 is a derivate of OXA-48, and its detection is very difficult in laboratories. Here, we report the identification and genomic analysis of an Escherichia coli isolate (28Eco12) harboring the blaOXA-244 gene identified in Colombia, South America. The 28Eco12 isolate was identified during a retrospective study, and it was recovered from a patient treated in Colombia. The complete nucleotide sequence was established using the PacBio platform. A comparative genomics analysis with other blaOXA-244-harboring Escherichia coli strains was performed. The 28Eco12 isolate belonged to sequence type (ST) 38, and its genome was composed of two molecules, a chromosome of 5,343,367 bp and a plasmid of 92,027 bp, which belonged to the incompatibility group IncY and did not harbor resistance genes. The blaOXA-244 gene was chromosomally encoded and mobilized by an ISR1-related Tn6237 composite transposon. Notably, this transposon was inserted and located within a new genomic island. To our knowledge, this is the first report of a blaOXA-244-harboring Escherichia coli isolate in America. Our results suggest that the introduction of the OXA-244-producing E. coli isolate was through clonal expansion of the ST38 pandemic clone. Other isolates producing OXA-244 could be circulating silently in America.

Europatitan eastwoodi, a new sauropod from the lower Cretaceous of Iberia in the initial radiation of somphospondylans in Laurasia.

The sauropod of El Oterillo II is a specimen that was excavated from the Castrillo de la Reina Formation (Burgos, Spain), late Barremian-early Aptian, in the 2000s but initially remained undescribed. A tooth and elements of the axial skeleton, and the scapular and pelvic girdle, represent it. It is one of the most complete titanosauriform sauropods from the Early Cretaceous of Europe and presents an opportunity to deepen our understanding of the radiation of this clade in the Early Cretaceous and study the paleobiogeographical relationships of Iberia with Gondwana and with other parts of Laurasia. The late Barremian-early Aptian is the time interval in the Cretaceous with the greatest diversity of sauropod taxa described in Iberia: two titanosauriforms, Tastavinsaurus and Europatitan; and a rebbachisaurid, Demandasaurus. The new sauropod Europatitan eastwoodi n. gen. n. sp. presents a series of autapomorphic characters in the presacral vertebrae and scapula that distinguish it from the other sauropods of the Early Cretaceous of Iberia. Our phylogenetic study locates Europatitan as the basalmost member of the Somphospondyli, clearly differentiated from other clades such as Brachiosauridae and Titanosauria, and distantly related to the contemporaneous Tastavinsaurus. Europatitan could be a representative of a Eurogondwanan fauna like Demandasaurus, the other sauropod described from the Castrillo de la Reina Formation. The presence of a sauropod fauna with marked Gondwananan affinities in the Aptian of Iberia reinforces the idea of faunal exchanges between this continental masses during the Early Cretaceous. Further specimens and more detailed analysis are needed to elucidate if this Aptian fauna is caused by the presence of previously unnoticed Aptian land bridges, or it represents a relict fauna from an earlier dispersal event.

Colonización rectal por Enterobacterales productores de múltiples carbapenemasas: Reporte de un caso de coproducción / Rectal colonization by Enterobacterales producing multiple carbapenemases: a co-production case report

Resumen El constante aumento de Enterobacterales productores de carbapenemasas (CPE) se constituye en un problema de salud pública a nivel mundial, por el impacto generado en la mortalidad de los pacientes. El tracto gastrointestinal es el principal reservorio de este tipo de microorganismos, por lo cual, la colonización rectal se convierte en un importante factor de riesgo para el desarrollo de posteriores infecciones. Una de las estrategias de vigilancia epidemiológica activa, es la búsqueda de pacientes colonizados, a través de cultivos de tamización para detectar estos microrganismos multirresistentes. Reportamos el caso de un paciente, con historia de sepsis de origen pulmonar, colonizado por Klebsiella pneumoniae con coproducción de carbapenemasas NDM + KPC y Escherichia coli con carbapenemasa NDM. Este hallazgo es cada vez más frecuente, lo cual implica un reto en su detección y diagnóstico. Se describen características del paciente, procedimientos realizados y hallazgos microbiológicos.

Abstract The constant increase in carbapenemase-producing Enterobacterales (CPE) constitutes a public health problem worldwide, due to the impact generated on the mortality of patients. The gastrointestinal tract is the main reservoir for this microorganism, which is why, rectal colonization becomes an important risk factor for the development of subsequent infections. One of the active epidemiological surveillance strategies is the search for colonized patients through screening cultures, to detect these multi-resistant microorganisms. We report the case of a patient, with a history of sepsis of pulmonary origin, colonized by Klebsiella pneumoniae with co-production of NDM + KPC carbapenemases and NDM carbapenemase-producing Escherichia coli. This finding is more and more frequent, which implies a challenge in its detection and diagnosis. Patient characteristics, procedures performed and microbiological findings are described.

Bacteremia por Campylobacter fetus subespecie fetus en Colombia: Reporte de un caso / Bacteremia by Campylobacter fetus subsp. fetus in Colombia: Case Report

Las bacteremias por microorganismos del genero Campylobacter son poco comunes en nuestro medio, debido a su baja incidencia y son escasos los reportes en la literatura. El siguiente reporte describe un caso de bacteremia por la especie Campylobacter fetus subespecie fetus, en un paciente de edad avanzada, quien presentó manifestaciones neurológicas que inicialmente desviaron el diagnóstico oportuno. Finalmente el paciente resuelve la infección tras el tratamiento antibiótico, sin complicaciones. Es de interés mencionar la presencia de este microorganismo como causante de diversas patologías en pacientes con inmunosupresión, considerándose un patógeno oportunista, del cual no está bien definida su patogénesis ni la fuente de exposición o los factores de riesgo en estos pacientes. Igualmente, es importante la vigilancia activa de este microorganismo ya que por su complicada recuperación, altas exigencias nutricionales y su crecimiento lento, dificulta su aislamiento y diagnóstico en el laboratorio de microbiología.

The bacteremias by microorganism of genera Campylobacter are uncommon in our medium, because of its low incidence and few reports in the literature. The report describe a bacteremia by Campylobacter fetus, a gram negative rod, spiral shape, in an older patient who presented initial neurogycal manifestations that shifted the timely diagnosis. Finally, the patient solves the infection after the antibiotic treatment, uncomplicated. It`s important to say that, this microorganism is the cause of different pathologies in patients with inmunosuppression considering as opportunistic pathogen. It`s pathogenesis source of exposure and risk factor in these patients has not yet been defined. Also, it is important the active surveillance in these microorganism, due to its complicated recovery, high nutritional requirements and slow growth. As described above is the reason because of its difficult isolation and diagnosis in the laboratory of microbiologic.

Enfermedad pélvica inflamatoria por Actinomyces sp. en paciente con dispositivo intrauterino: reporte de un caso / Pelvic inflammatory disease by Actinomyces sp. in a patient with an intrauterine device: Case report

Dentro de las infecciones pélvicas que se presentan en mujeres, se encuentran las causadas por microorganismos anaerobios del género Actinomyces. Esta etiología es poco sospechada, debido a los esporádicos casos que se presentan, por lo que la infección permanece por largos periodos de tiempo y conlleva diferentes complicaciones que solo pueden resolverse con procedimientos quirúrgicos y tratamiento antibiótico prolongado. Por esto, se hace el reporte de caso de una paciente de 48 años de edad portadora de dispositivo intrauterino desde hace 13 años, el cual se consideró como el principal factor de riesgo. Se describen las características de la infección, procedimientos realizados, hallazgos microbiológicos y tratamiento, con lo cual se resuelve de manera satisfactoria.

Among the pelvic infections in women there exist those caused by anaerobic microorganisms of the genera Actinomyces . This etiology is rarely suspected, due to the few number of cases presented, thus the infection continues for long periods of time. This persistence can lead to various complications that can only be resolved with surgical procedures and prolonged antibiotic treatment. We present a case report on a 48-year old patient with a 13-year history of intrauterine device use, which we considered to be the principal risk factor. We describe the characteristics of the infection, the procedures performed, microbiological findings and the treatment, with which it was satisfactorily resolved.

Factores relacionados con el control exitoso de un brote por Klebsiella pneumoniae productora de KPC-2 en una unidad de cuidado intensivo en Bogotá, Colombia / Factors related to successful control of an outbreak by Klebsiella pneumonia -producing KPC-2 in an intensive care unit in Bogotá, Colombia

La resistencia a carbapenémicos en Klebsiella pneumoniae ha aumentado de manera considerable, incrementando las tasas de morbimortalidad. El objetivo de este trabajo fue describir las características epidemiológicas, microbiológicas y las medidas de intervención que permitieron el control exitoso de un brote de Klebsiella pneumoniae productora de KPC-2. Métodos: El estudio se realizó en 2 periodos: el primero durante el brote, con instauración de un protocolo de medidas de intervención; y el segundo, de seguimiento posbrote. Se realizaron pruebas de identificación y susceptibilidad por sistema automatizado, tamización de carbapenemasas por test de Hodge modificado, PCR para detección de los genes bla KPC, bla KPC-2, NDM-1 y estudio de clonalidad por electroforesis de campos pulsados. Resultados: Durante el brote, se identificaron 18 aislamientos de Klebsiella pneumoniae productora de KPC en 11 pacientes. Tres casos fueron confirmados como infección intrahospitalaria. La técnica de PCR reveló la presencia del gen bla KPC en 21 de 22 aislamientos (pacientes y medio ambiente) y se identificó la presencia de un clon con una similitud superior al 75%. En el periodo posbrote los cultivos ambientales y de búsqueda de colonizados fueron negativos. Discusión: Se evidenció un control exitoso del brote producido por un clon. La implementación de un protocolo de intervención y la monitorización de su cumplimiento, la comunicación efectiva y el trabajo en equipo fueron indispensables para evitar su propagación y evitar un comportamiento endémico posbrote.

The considerable increase in carbapenem-resistant Klebsiella pneumoniae has caused an increase in mortality and morbidity rates. The aim of this study was to describe its epidemiological and microbiological characteristics and the intervention measures that controlled an outbreak caused by K. pneumoniae- producing KPC-2 B-lactamase. Methods: The study was divided into 2 periods: the first during the outbreak with the implementation of a bundle and the second a post-outbreak surveillance. We performed tests for identification and susceptibility by using an automated system, screening carbapenemases by the Modified-Hodge test, bla KPC, bla KPC-2 and NDM-1 identification by PCR and clonal relationship characterisation by PFGE. Results: During the outbreak, there were 18 isolates of Klebsiella pneumoniae -producing KPC- 2 in 11 patients. Three cases were confirmed as hospital-acquired infection. Of 22 isolates, 21 were positive to bla KPC by PCR (samples from patients and environment) and a clone was identified with a similarity of greater than 75%. During the post-outbreak surveillance, we did not find any new positive cultures from surfaces and there were no new colonisations. Discussion: This was a successful control of an outbreak produced by a clone. The implementation of a bundle and a subsequent surveillance to monitor its fulfilment, effective communication and teamwork were crucial to inhibit propagation of the infection and to prevent an endemic behaviour post-outbreak.

Reversión de la diabetes en el ratón por el xenotrasplante de islotes porcinos en un páncreas bioartificial / Reversion of diabetes type I in the mice with diffusion chambers of pig islets an bioartificial pancreas model

Antecedentes: El páncreas bioartificial permitiría la utilización de islotes porcinos para la reversión de la diabetes tipo I en humanos. Distintos modelos se han creado hasta el presente, presentando diversos problemas, que limitan su aplicación clínica. Objetivo: Evaluar el modelo de páncreas bioartificial, con cámaras de difusión en un xenotrasplante discordante (cerdo-ratón). Material y Métodos: Ratones hembras diabéticos por Streptozotocin, fueron utilizados como receptores de islotes porcinos. Se introdujeron en agarosa, 2000 islotes en cada cámara, formadas con arandelas de acrílico adheridas a 2 membranas semipermeables de policarbonato. El diseño experimental: Grupo 1 (n = 4): 4000 islotes porcinos no encapsulados en cápsula renal. Grupo 2 (n = 4): 1 cámara c/u en el abdomen. Grupo 3 (n = 6): 2 cámaras c/u. Grupo 4 (n = 2): 3 cámaras c/u. Se evaluó en cada grupo las variaciones de glucemia y la reacción histológica pericámara. Resultados: En el grupo 1, la reversión de la diabetes no superó los 3 días. De los 12 ratones con cámaras de difusión, el 50 por ciento alcanzó la normoglucemia, manteniéndose en el 33,3 por ciento hasta la 5ta. semana fecha en la que se sacrificaron. Ninguno de los ratones con una cámara normaliza la glucemia, como en el 100 por ciento de los que poseían tres. Importante reacción inflamatoria pericámara, se halla en la 1era. y 5ta. semana. Conclusiones: Resultados auspiciosos; necesidad de ampliar la experiencia utilizando 6000 o más islotes por ratón, y un mayor número de estos para encontrar nuevas estrategias que disminuyan la inflamación pericámara, mejorando a largo plazo la supervivencia del injerto

Reversión de la diabetes en el ratón por el xenotrasplante de islotes porcinos en un páncreas bioartificial / Reversion of diabetes type I in the mice with diffusion chambers of pig islets an bioartificial pancreas model

Antecedentes: El páncreas bioartificial permitiría la utilización de islotes porcinos para la reversión de la diabetes tipo I en humanos. Distintos modelos se han creado hasta el presente, presentando diversos problemas, que limitan su aplicación clínica. Objetivo: Evaluar el modelo de páncreas bioartificial, con cámaras de difusión en un xenotrasplante discordante (cerdo-ratón). Material y Métodos: Ratones hembras diabéticos por Streptozotocin, fueron utilizados como receptores de islotes porcinos. Se introdujeron en agarosa, 2000 islotes en cada cámara, formadas con arandelas de acrílico adheridas a 2 membranas semipermeables de policarbonato. El diseño experimental: Grupo 1 (n = 4): 4000 islotes porcinos no encapsulados en cápsula renal. Grupo 2 (n = 4): 1 cámara c/u en el abdomen. Grupo 3 (n = 6): 2 cámaras c/u. Grupo 4 (n = 2): 3 cámaras c/u. Se evaluó en cada grupo las variaciones de glucemia y la reacción histológica pericámara. Resultados: En el grupo 1, la reversión de la diabetes no superó los 3 días. De los 12 ratones con cámaras de difusión, el 50 por ciento alcanzó la normoglucemia, manteniéndose en el 33,3 por ciento hasta la 5ta. semana fecha en la que se sacrificaron. Ninguno de los ratones con una cámara normaliza la glucemia, como en el 100 por ciento de los que poseían tres. Importante reacción inflamatoria pericámara, se halla en la 1era. y 5ta. semana. Conclusiones: Resultados auspiciosos; necesidad de ampliar la experiencia utilizando 6000 o más islotes por ratón, y un mayor número de estos para encontrar nuevas estrategias que disminuyan la inflamación pericámara, mejorando a largo plazo la supervivencia del injerto (AU)