Parasitic findings on threatened pudu deer from Central Chile accounts first genetic characterization of lice parasitizing P. puda in Chile and the first molecular report of Taenia hydatigena metacestodes in this species.

Southern pudu (Pudu puda) is a threatened endemic deer of the temperate forests of Chile. In recent years pudu populations rates have decreased mainly due to anthropogenic causes including forest loss and landscape fragmentation. In this context, the parasitic fauna of Chilean pudu has been scarcely investigated. The aim of this study was to determine the parasitic status of rescued pudu n = 13 from its natural habitat in Central Chile (Maule region) during March 2022 and June 2023 by applying morphological, histopathological, and molecular analyses. As result, we report the presence of transmission of parasites from dogs to pudus as showed by the presence of metacestodes of the parasite Taenia hydatigena on omentum, liver, and pleura of pudus during postmortem examinations, being the first molecular report on the presence of this parasite on Chilean pudu. Meanwhile, ectoparasite examinations determined the presence of chewing and sucking lice on pudu exemplars here analysed. Molecular and phylogenetic analysis of lice revealed new insights on Bovicola and Anoplura lice parasitizing P. puda in Chile, equally being the first genetic characterization of lice parasitizing pudu exemplars in Chile. In addition, parasite loads of lice and metacestodes were analysed. However, no statistically significance was observed when comparing environmental and individual traits influence on parasite load variation. Overall, the study area is the northern limit of habitat distribution of this specie in Chile and we here provide novel information on pudu deer parasites, thus making a useful and valuable contribution to the parasitological knowledge on this threatened species.

Human pluripotent stem cell-derived macrophages host Mycobacterium abscessus infection.

Human macrophages are a natural host of many mycobacterium species, including Mycobacterium abscessus (M. abscessus), an emerging pathogen affecting immunocompromised and cystic fibrosis patients with few available treatments. The search for an effective treatment is hindered by the lack of a tractable in vitro intracellular infection model. Here, we established a reliable model for M. abscessus infection using human pluripotent stem cell-derived macrophages (hPSC-macrophages). hPSC differentiation permitted reproducible generation of functional macrophages that were highly susceptible to M. abscessus infection. Electron microscopy demonstrated that M. abscessus was present in the hPSC-macrophage vacuoles. RNA sequencing analysis revealed a time-dependent host cell response, with differing gene and protein expression patterns post-infection. Engineered tdTOMATO-expressing hPSC-macrophages with GFP-expressing mycobacteria enabled rapid image-based high-throughput analysis of intracellular infection and quantitative assessment of antibiotic efficacy. Our study describes the first to our knowledge hPSC-based model for M. abscessus infection, representing a novel and accessible system for studying pathogen-host interaction and drug discovery.

Blocking phospholamban with VHH intrabodies enhances contractility and relaxation in heart failure.

The dysregulated physical interaction between two intracellular membrane proteins, the sarco/endoplasmic reticulum Ca2+ ATPase and its reversible inhibitor phospholamban, induces heart failure by inhibiting calcium cycling. While phospholamban is a bona-fide therapeutic target, approaches to selectively inhibit this protein remain elusive. Here, we report the in vivo application of intracellular acting antibodies (intrabodies), derived from the variable domain of camelid heavy-chain antibodies, to modulate the function of phospholamban. Using a synthetic VHH phage-display library, we identify intrabodies with high affinity and specificity for different conformational states of phospholamban. Rapid phenotypic screening, via modified mRNA transfection of primary cells and tissue, efficiently identifies the intrabody with most desirable features. Adeno-associated virus mediated delivery of this intrabody results in improvement of cardiac performance in a murine heart failure model. Our strategy for generating intrabodies to investigate cardiac disease combined with modified mRNA and adeno-associated virus screening could reveal unique future therapeutic opportunities.

Impact of dental cone-beam computed tomography on diagnostic evaluation and treatment plan for the maxillary sinus in dental implant planning / Impacto de la tomografía computarizada de haz cónico en el diagnóstico y plan detratamiento preimplantologico de senos maxilares

Objective: To determine the impact of CBCT on diagnostic evaluation and treatment plan for the maxillary sinus in dental implant planning. Material and Methods: Diagnostic evaluation and treatment plan for the maxillary sinus were evaluated by eight specialists experienced in dental implant placement. Eight panoramic radiographs (PAN) and CBCT examinations were obtained from five adult patients with a specific clinical need for dental implants. Evaluation was performed first on PAN then, at least 2 weeks later, on CBCT. Residual alveolar ridge height, mucosal thickening, radiographic findings and treatment plan were recorded. The confidence level was evaluated for both diagnostic evaluation and treatment plan. The kappa statistic for intra-observer reproducibility and McNemar test were performed. Results: In the diagnostic evaluation, CBCT showed significant impact on the diagnosis of radiographic findings. Availability of CBCT significantly changed the treatment plan, for less invasive treatment, or no treatment need. Observers had significantly greater confidence when using CBCT than PAN, when indicating presence of mucosal thickening and radiographic findings in the maxillary sinus. In addition, CBCT increased confidence in the treatment plan. Conclusion: The present study suggests that CBCT has an impact on the diagnostic evaluation of radiographic findings in the maxillary sinus and on the decision to place implants, owing to misdiagnosis of pathology and planning of more invasive treatments when using PAN. Availability of CBCT also improves clinician confidence. Further studies at higher levels of diagnostic efficacy should be performed, to justify the use of CBCT, by evaluating the actual treatment performed and its outcome.

Objetivo: Determinar el impacto de la tomografía computarizada de haz cónico (CBCT) en la evaluación diagnóstica y el plan de tratamiento del seno maxilar en la planificación de implantes dentales. Material y Métodos: La evaluación diagnóstica y el plan de tratamiento del seno maxilar fueron evaluados por ocho especialistas con experiencia en la colocación de implantes dentales. Se obtuvieron ocho radiografías panorámicas (PAN) y exámenes CBCT de cinco pacientes adultos con una necesidad clínica específica de implantes dentales. La evaluación se realizó primero en PAN y luego, al menos dos semanas después, en CBCT. Se registraron la altura del reborde alveolar residual, el engrosamiento de la mucosa, los hallazgos radiográficos y el plan de tratamiento. Se evaluó el nivel de confianza tanto para la evaluación diagnóstica como para el plan de tratamiento. Se realizó el estadístico kappa para la reproducibilidad intraobservador y la prueba de McNemar. Resultados: En la evaluación diagnóstica, CBCT mostró un impacto significativo en el diagnóstico de los hallazgos radiográficos. La disponibilidad de CBCT cambió significativamente el plan de tratamiento, para un tratamiento menos invasivo o sin necesidad de tratamiento. Los observadores tuvieron una confianza significativamente mayor al usar CBCT que PAN, al indicar la presencia de engrosamiento de la mucosa y hallazgos radiográficos en el seno maxilar. Además, CBCT aumentó la confianza en el plan de tratamiento. Conclusión: El presente estudio sugiere que la CBCT tiene un impacto en la evaluación diagnóstica de los hallazgos radiográficos en el seno maxilar y en la decisión de colocar implantes, debido al diagnóstico erróneo de la patología y la planificación de tratamientos más invasivos al usar PAN. La disponibilidad de CBCT también mejora la confianza del clínico. Se deben realizar más estudios a niveles más altos de eficacia diagnóstica para justificar el uso de CBCT, evaluando el tratamiento real realizado y su resultado.

Tratamiento con HeberFERON® del carcinoma basocelular en la Atención Primaria de Salud en Cuba / HeberFERON® Treatment of Basal Cell Carcinoma in Primary Healthcare in Cuba

Introducción: El cáncer de piel es el tipo de cáncer más frecuente en el ser humano, el carcinoma basocelular es el más común de todos los cánceres de piel (80-90 por ciento). Excepcionalmente producen metástasis, pero pueden causar significativa morbilidad e involucran a edades más jóvenes, se tratan con éxito mediante cirugía, radioterapia, quimioterapia y crioterapia, generalmente en el nivel secundario de salud, sin embargo, estos tratamientos no siempre son posibles o deseables. El HeberFERON® es una combinación de interferones alfa y gamma humanos recombinantes, que ha mostrado producir efectos sinérgicos en la reducción de la proliferación de varias líneas de células cancerosas, esta formulación ha sido aprobada en Cuba para el tratamiento del carcinoma basocelular. Presentación de casos: Se presentaron tres casos con diagnóstico de carcinoma basocelular, localizados en la cara, tratados con HeberFERON®, en dos casos fue observada la desaparición de la lesión al finalizar la tercera semana de tratamiento. En el tercer caso, una mujer de 84 años de edad, al finalizar el primer ciclo de tratamiento, fue reducido el tamaño de la lesión tratada y desapareció otra lesión adyacente que no recibió directamente tratamiento, la lesión residual, en esta paciente, fue valorada por ultrasonido para determinar su extensión y profundidad, en los tres casos las reacciones adversas fueron leves y transitorias. Conclusiones: El HeberFERON® es una opción efectiva y segura para el tratamiento del carcinoma basocelular en la atención primaria de salud en Cuba(AU)

Introduction: Skin cancer is the commonest type of cancer in humans. Basal cell carcinoma is the commonest of all skin cancers, accounting for 80 percent to 90 percent of all cases. Exceptionally, they metastasize, but can cause significant morbidity and involve younger ages. They are successfully treated by surgery, radiotherapy, chemotherapy and cryotherapy, mostly at the secondary level of health. However, these treatments are not always possible or desirable. HeberFERON® is a combination of recombinant human alpha and gamma interferons, which has been shown to produce synergistic effects in reducing the proliferation of several lines of cancer cells. This formulation has been approved in Cuba for treating basal cell carcinoma. Case presentation: Three cases are presented with a diagnosis of basal cell carcinoma, located on the face, treated with HeberFERON®. In two cases, the lesion was observed to disappear at the end of the third week of treatment. In the third case, an 84-year-old woman, at the end of the first cycle of treatment, the size of the treated lesion was reduced and another adjacent lesion disappeared, which did not receive direct treatment. In this patient, the residual lesion was assessed by ultrasound to determine its extension and depth. In the three cases, the adverse reactions were mild and transitory. Conclusions: HeberFERON® is an effective and safe option for treating basal cell carcinoma in primary health care in Cuba(AU)

Tomografía computarizada de haz cónico como examen concluyente de hiperplasia de procesos coronoides bilateral. Reporte de tres casos / Cone beam computed tomography as a conclusive examination of bilateral coronoid process hyperplasia. Report of three cases

RESUMEN La hiperplasia del proceso coronoides (HPC) es un crecimiento exagerado del proceso coronoides. La HPC es poco frecuente y genera limitación asintomática de la apertura bucal debido a la impactación del proceso coronoides con el segmento posterior del hueso cigomático. La HPC es un posible diagnóstico en pacientes con limitación progresiva tanto de la apertura bucal como del movimiento protrusivo mandibular. En el presente artículo, se reportan tres casos de pacientes con limitación de apertura bucal. Mediante examen de tomografía computarizada de haz cónico (TCHC), se observó un aumento de tamaño uniforme de los procesos coronoides, confirmando el diagnóstico de HPC bilateral. La TCHC con campo de visión grande determinó el diagnóstico final, pues mostró la impactación del proceso coronoides con el segmento posterior del hueso cigomático en apertura bucal. Además, la TCHC permitió distinguir entre una HPC y una neoplasia del proceso coronoides y/o estructuras anatómicas vecinas.

SUMMARY Coronoid process hyperplasia (CPH) is an excessive coronoid process growing. CPH is infrequent and produces an asymptomatic mouth opening limitation due to the impaction of the coronoid process in the posterior portion of the zygomatic bone. CPH is a possible diagnosis in patients with both progressive mouth opening and protrusive movement limitations. In the present article, cases of three patients with mouth opening limitation are reported. In cone-beam computed tomography (CBCT) examination, a uniform growing of coronoid processes was observed, which confirmed the diagnosis of bilateral CPH. CBCT with large field of view allowed the determination of the final diagnosis, because the impaction of the coronoid process in the posterior portion of the zygomatic bone during mouth opening could be observed. Furthermore, CBCT allowed to distinguish between CPH and neoplasia of coronoid process and/or neighboring anatomical structures.

Cysteine auxotrophy drives reduced susceptibility to quinolones and paraquat by inducing the expression of efflux-pump systems and detoxifying enzymes in S. Typhimurium.

Currently, Salmonella enterica serovar Typhimurium (S. Typhimurium), is a major global public health problem, which has caused food-borne illnesses in many countries. Today, with the extensive use of antimicrobials, antimicrobial resistance is increasing at a serious rate in S. Typhimurium isolates. The present study sought the role of cysteine (Cys) auxotrophy on the resistance to quinolones and paraquat in S. Typhimurium. Cys auxotrophy was achieved by deleting either the cysDNC, cysJIH or cysQ loci. Deletion of these loci resulted in loss of susceptibility against nalidixic acid, levofloxacin, ciprofloxacin (CIP) and paraquat. Further studies with cysJIH mutant indicated increased expression of multi-antibiotic resistance genes marA and ramA, and consequently increased expression of efflux-pump systems. The cysJIH mutant presented a smaller increase of reactive oxygen species (ROS) in presence of paraquat or CIP. Expression of katG and sodA (expressing for a catalase and a superoxide dismutase, respectively) genes was increased in presence of paraquat in the cysJIH mutant; while expression of the superoxide dismutase gene sodB was decreased. These results indicate that deletion of cysDNC, cysJIH or cysQ genes of S. Typhimurium renders Cys auxotrophy along with decreased susceptibility in response to quinolone and paraquat. Overexpression of efflux-pump systems AcrB-TolC and SmvA-OmpD and antioxidant enzymes KatG and SodA could explain the mechanisms of antimicrobial resistance in the Cys auxotrophic mutants.

A PCR-RFLP assay for discrimination of Echinococcus granulosus sensu stricto and Taenia spp. in dogs stool.

In order to develop a method of identification and discrimination of Echinococcus granulosus sensu stricto from faecal samples of dogs infected with taeniid eggs (Echinococcus spp., Taenia spp.), a combined strategy of Polymerase Chain Reaction (PCR) and Restriction Fragments of Lenght Polymorphisms (RFLP) was proposed. Initially, a pair of primers was designed to amplify a fragment of the 12 Subunit of ribosomal RNA gene (12SrRNA) from mitochondrial DNA. The amplified product was digested by SspI restriction enzyme, which in E. granulosus kept the intact fragment of 160 basis pairs (bp), while in Taenia spp. produced two fragments (62 bp and another of 98 bp). The method was tested using positive controls of DNA, in faecal samples experimentally contaminated with eggs of E. granulosus and Taenia spp. and in dogs naturally infected. In all of them, reproducible results were obtained and the primers were specific to amplify only Taeniidae DNA. The sensitivity of the technique was tested, achieving amplification of DNA extractions with a single egg. In conclusion, the technique developed was optimal and easy to identify patent infections by E. granulosus s.s., constituting a possible alternative for epidemiological studies in dogs, especially in endemic areas where this infection occurs.

DNA extraction in Echinococcus granulosus and Taenia spp. eggs in dogs stool samples applying thermal shock.

The extraction of DNA in taeniid eggs shows complications attached to the composition of stool samples and the high resistance of eggs to degradation. The objective of this study was to test a method of DNA extraction in taeniid eggs by applying a thermal shock to facilitate the chemical-enzymatic degradation of these elements. A group of six tubes containing 1 ml of dog stool sample was spiked with eggs of Echinococcus granulosus and another group of six with Taenia pisiformis. Samples were floated with supersaturated sugar solution and centrifuged. The upper portion of each tube (500 µl) was aspirated and deposited in 1.5 ml tubes. Three tubes from each group were incubated at -20 °C and then at 90 °C, the remaining three from each group, incubated at room temperature. Proteinase K and lysis buffer were added to each tube and incubated for 12 h at 58 °C. The lysis effect was evaluated by microscopy at 3, 6 and 12 h and integrity by electrophoresis in 1% agarose gels. With the same experimental scheme, the thermal shock effect was evaluated in extractions of 1, 2, 3 and 4 eggs of each species and the DNA was quantified. Additionally, the protocol was applied in samples of 4 dogs diagnosed with natural infection by Taeniidae worms. Finally, all the extractions were tested by PCR amplification. Both E. granulosus and T. pisiformis eggs showed a similar response in the tests. In samples without treatment, the lysis effect was poor and showed no differences over time, but in those subjected to thermal shock, eggs degradation increased with time. In both treatments, there was no DNA loss integrity. The protocol applied to limited amounts of eggs yielded PCR products in 100% of the samples exposed to thermal shock, allowing PCR amplifications up to 1 egg. In non-exposed samples, the results were not replicable. However, DNA quantification showed low values in both treatments. In turn, DNA extractions with thermal shock in infected dog samples finally yielded PCR amplifications in 100%. It was concluded that thermal shock facilitates the DNA extraction for molecular analysis in taeniid eggs. The technique is effective extracting DNA even from a single egg and also to analyze natural infections samples with a relatively simple implementation.

Diagnostic accuracy of a charge-coupled device and a photostimulable phosphor plate in detection of non-cavitated proximal caries

Objective: the aim of this study was to compare the diagnostic accuracy of two direct digital radiography systems: the charge-coupled device (CCD) XIOS XG Sirona and the photostimulable storage phosphor (PSP) VistaScan DürrDental, in the detection of non-cavitated proximal caries lesions. materials and methods: in this experimental and cross-sectional study 112 proximal surfaces from 27 molars and 31 premolars with or without proximal caries lesions were evaluated and randomly allocated in a study unit. bitewing radiographs were acquired with a CCD XIOS XG and with the PSP VistaScan. a single x-ray unit was used for both systems. radiographic images were assessed independently by two calibrated radiologists. histological evaluation on a stereomicroscope was used as gold standard. results: sensitivity values were found to be 0.35 for CCD and 0.31 for PSP. specificity values were found to be similar for both systems (0.867). az values showed a low diagnostic accuracy for both sensors: 0.61 for CCD and 0.59 for PSP, no statistical difference was found between these two values (p=0.78). conclusion: both digital radiology systems have a high diagnostic accuracy to detect sound surfaces but low diagnostic accuracy to detect proximal carious lesions.

SPI-9 of Salmonella enterica serovar Typhi is constituted by an operon positively regulated by RpoS and contributes to adherence to epithelial cells in culture.

The genomic island 9 (SPI-9) from Salmonella enterica serovar Typhi (S. Typhi) carries three ORFs (STY2876, STY2877, STY2878) presenting 98 % identity with a type 1 secretory apparatus (T1SS), and a single ORF (STY2875) similar to a large RTX-like protein exhibiting repeated Ig domains. BapA, the Salmonella enterica serovar Enteritidis orthologous to S. Typhi STY2875, has been associated with biofilm formation, and is described as a virulence factor in mice. Preliminary in silico analyses revealed that S. Typhi STY2875 ORF has a 600 bp deletion compared with S. Enteritidis bapA, suggesting that S. Typhi STY2875 might be non-functional. At present, SPI-9 has not been studied in S. Typhi. We found that the genes constituting SPI-9 are arranged in an operon whose promoter was up-regulated in high osmolarity and low pH in a RpoS-dependent manner. All the proteins encoded by S. Typhi SPI-9 were located at the membrane fraction, consistent with their putative role as T1SS. Furthermore, SPI-9 contributed to adherence of S. Typhi to epithelial cells when bacteria were grown under high osmolarity or low pH. Under the test conditions, S. Typhi SPI-9 did not participate in biofilm formation. SPI-9 is functional in S. Typhi and encodes an adhesin induced under conditions normally found in the intestine, such as high osmolarity. Hence, this is an example of a locus that might be designated a pseudogene by computational approaches but not by direct biological assays.

Oestrus ovis infection of grazing sheep during summer in southern Chile

Oestrus ovis is a botfly whose larvae cause nasal myiasis, an environmental-dependent disease in small ruminants, generating acute and chronic injuries in the cranial cavities of sheep. Chile is a country of the southernmost worldwide distribution of this parasite, and there is few information about. Whence, the objective of this study was to approximate the epidemiological situation of O. ovis infection in Chilean sheep. From December 2009 to March 2010, a total of 87 samples were obtained by necropsy for skull inspection. The larvae were collected, and microscopically identified. The prevalence in the sampled sheep was 60.9%. From those that were infected, 85.7% (18/21) of sheep were 1 to 3 years old, constituting the stratum with the highest prevalence. The difference of infection in females and males was not significant. The high risk of infection seems to be dependent upon the environmental conditions of this southern region, especially during summer when the first larval stage (L1) could be found as the evidence. Therefore, the disease should be considered as a significant problem for this kind of livestock production.

Infusion of endomorphin-1 (EM-1) in the MPOA and the Me modulate sexual and socio-sexual behavior in the male rat.

Several neurotransmitters, among them neuropeptides, have been implicated in the control of male sexual behavior. Opioids are involved in mediating the positive affective states generated by the execution of sexual behavior in both males and females. We have previously shown that intracerebroventricular administration of endomorphin-1 (EM-1), the specific ligand for the µ opioid receptor, increased the ejaculation latency and interintromission interval and reduced the number of ejaculations during the test. In the present study we evaluated the effect of EM-1 upon male sexual behavior and socio-sexual interactions when infused in the medial preoptic area (MPOA) or the medial amygdala (Me). The results indicate that the administration of EM-1 in the MPOA increased mount and intromission latencies while infusion in the Me increased the number of mounts before ejaculation as well as the ejaculation latency. With respect to socio-sexual interactions, the duration of pursuit was significantly increased after administration of EM-1 in the MPOA. The effects upon sexual behavior and socio-sexual interactions were blocked by administration of the opioid antagonist naloxone. These results indicate that EM-1 modulates the appetitive aspect of sexual behavior in the MPOA and the consummatory phase in the Me.

Inecalcitol, an analog of 1,25D3, displays enhanced antitumor activity through the induction of apoptosis in a squamous cell carcinoma model system.

Epidemiological data suggest an important role of vitamin D signaling in cancer development and progression, and experimental studies demonstrate that the active vitamin D metabolite 1α, 25-dihydroxyvitamin D3 (1,25D3) has broad spectrum antitumor activity. Hypercalcemia has often been suggested to limit the clinical application of these data. The 14-epi-analog of 1,25D3, inecalcitol [19-nor-14-epi-23-yne-1,25-(OH)2D3; TX522], was developed to have superagonistic antitumor activities but low hypercalcemia potential. We examined the antitumor activity of inecalcitol and the underlying mechanisms in a murine squamous cell carcinoma (SCC) model system. In vitro, compared with 1,25D3, inecalcitol showed enhanced vitamin D receptor (VDR)-mediated transcriptional activity. Inecalcitol suppressed SCC cell proliferation in a dose-dependent manner with an IC50 value 30 times lower than that of 1,25D3. Both inecalcitol and 1,25D3 induced a comparable level of G0/G1 cell cycle arrest in SCC cells. The level of apoptosis induced by inecalcitol was markedly higher than that of 1,25D3. Apoptosis was mediated through the activation of the caspase 8/10- caspase 3 pathway. Further, inecalcitol markedly inhibited the mRNA and protein expression of c-IAP1 and XIAP compared with 1,25D3. In vivo, inecalcitol inhibits SCC tumor growth in a dose-dependent fashion. Notably, inecalcitol induced a significantly higher level of apoptosis in the SCC xenograft model. While in vitro inecalcitol demonstrates apparent enhanced VDR binding and antiproliferative effects compared to 1,25D3, in vivo these advantages disappear; at doses of inecalcitol that have equivalent antitumor effects, similar hypercalcemia is seen. This may be explained by the pharmacokinetics of 1,25D3 vs. inecalcitol and attributed to the much shorter serum half-life of inecalcitol.We show that inecalcitol has potent antitumor activity in the SCC model system, and this is associated with a strong induction of apoptosis. These findings support the further development of inecalcitol in cancer treatment.

Primitive cardiac cells from human embryonic stem cells.

Pluripotent stem cell-derived cardiomyocytes are currently being investigated for in vitro human heart models and as potential therapeutics for heart failure. In this study, we have developed a differentiation protocol that minimizes the need for specific human embryonic stem cell (hESC) line optimization. We first reduced the heterogeneity that exists within the starting population of bulk cultured hESCs by using cells adapted to single-cell passaging in a 2-dimensional (2D) culture format. Compared with bulk cultures, single-cell cultures comprised larger fractions of TG30(hi)/OCT4(hi) cells, corresponding to an increased expression of pluripotency markers OCT4 and NANOG, and reduced expression of early lineage-specific markers. A 2D temporal differentiation protocol was then developed, aimed at reducing the inherent heterogeneity and variability of embryoid body-based protocols, with induction of primitive streak cells using bone morphogenetic protein 4 and activin A, followed by cardiogenesis via inhibition of Wnt signaling using the small molecules IWP-4 or IWR-1. IWP-4 treatment resulted in a large percentage of cells expressing low amounts of cardiac myosin heavy chain and expression of early cardiac progenitor markers ISL1 and NKX2-5, thus indicating the production of large numbers of immature cardiomyocytes (~65,000/cm(2) or ~1.5 per input hESC). This protocol was shown to be effective in HES3, H9, and, to a lesser, extent, MEL1 hESC lines. In addition, we observed that IWR-1 induced predominantly atrial myosin light chain (MLC2a) expression, whereas IWP-4 induced expression of both atrial (MLC2a) and ventricular (MLC2v) forms. The intrinsic flexibility and scalability of this 2D protocol mean that the output population of primitive cardiomyocytes will be particularly accessible and useful for the investigation of molecular mechanisms driving terminal cardiomyocyte differentiation, and potentially for the future treatment of heart failure.

Dexamethasone enhances 1alpha,25-dihydroxyvitamin D3 effects by increasing vitamin D receptor transcription.

Calcitriol, the active form of vitamin D, in combination with the glucocorticoid dexamethasone (Dex) has been shown to increase the antitumor effects of calcitriol in squamous cell carcinoma. In this study we found that pretreatment with Dex potentiates calcitriol effects by inhibiting cell growth and increasing vitamin D receptor (VDR) and VDR-mediated transcription. Treatment with actinomycin D inhibits Vdr mRNA synthesis, indicating that Dex regulates VDR expression at transcriptional level. Real time PCR shows that treatment with Dex increases Vdr transcripts in a time- and a dose-dependent manner, indicating that Dex directly regulates expression of Vdr. RU486, an inhibitor of glucocorticoids, inhibits Dex-induced Vdr expression. In addition, the silencing of glucocorticoid receptor (GR) abolishes the induction of Vdr by Dex, indicating that Dex increases Vdr transcripts in a GR-dependent manner. A fragment located 5.2 kb upstream of Vdr transcription start site containing two putative glucocorticoid response elements (GREs) was evaluated using a luciferase-based reporter assay. Treatment with 100 nm Dex induces transcription of luciferase driven by the fragment. Deletion of the GRE distal to transcription start site was sufficient to abolish Dex induction of luciferase. Also, chromatin immunoprecipitation reveals recruitment of GR to distal GRE with Dex treatment. We conclude that Dex increases VDR and vitamin D effects by increasing Vdr de novo transcription in a GR-dependent manner.

Biochemical characterization of nuclear receptors for vitamin D3 and glucocorticoids in prostate stroma cell microenvironment.

The disruption of stromal cell signals in prostate tissue microenvironment influences the development of prostate cancer to androgen independence. 1α,25-Dihydroxyvitamin D(3) (1,25D(3)) and glucocorticoids, either alone or in combination, have been investigated as alternatives for the treatment of advanced prostate cancers that fails androgen therapies. The effects of glucocorticoids are mediated by the intracellular glucocorticoid receptor (GR). Similarly, the effect of 1,25D(3) is mediated by the 1,25D(3) nuclear receptor (VDR). In this study, fibroblasts from benign- (BAS) and carcinoma-associated stroma (CAS) were isolated from human prostates to characterize VDR and GR function as transcription factors in prostate stroma. The VDR-mediated transcriptional activity assessed using the CYP24-luciferase reporter was limited to 3-fold induction by 1,25D(3) in 9 out of 13 CAS (70%), as compared to >10-fold induction in the BAS clinical sample pair. Expression of His-tagged VDR (Ad-his-VDR) failed to recover the low transcriptional activity of the luciferase reporter in 7 out of 9 CAS. Interestingly, expression of Ad-his-VDR successfully recovered receptor-mediated induction in 2 out of the 9 CAS analyzed, suggesting that changes in the receptor protein itself was responsible for decreased response and resistance to 1,25D(3) action. Conversely, VDR-mediated transcriptional activity was more efficient in 4 out of 13 CAS (30%), as compared to the BAS sample pair. Consistent with the reduced response to 1,25D(3) observed in CAS, chromatin immunoprecipitation (ChIP) assays indicated decreased recruitment of coactivators SRC-1/CBP, without major changes in the recruitment of VDR to the CYP24 promoter. In addition, we observed that GR-mediated transcriptional activity was also altered in CAS, as compared to BAS. Disruption of coactivators SRC-1/CBP recruitment may promote hormone resistance in CaP, and highlights the relevance of molecular diagnosis and drug design in tumor cell microenvironment.

Glucocorticoid regulation of the vitamin D receptor.

Many studies indicate calcitriol has potent anti-tumor activity in different types of cancers. However, high levels of vitamin D can produce hypercalcemia in some patients. Glucocorticoids are used to ameliorate hypercalcemia and to enhance calcitriol anti-tumor activity. Calcitriol in combination with the glucocorticoid dexamethasone (Dex) increased vitamin D receptor (VDR) protein levels and ligand binding in squamous cell carcinoma VII (SCC). In this study we found that both calcitriol and Dex induce VDR- and glucocorticoid receptor (GR)-mediated transcription respectively, indicating both hormone receptors are active in SCC. Pre-treatment with Dex increases VDR-mediated transcription at the human CYP24A1 promoter. Whereas, pre-treatment with other steroid hormones, including dihydrotestosterone and R1881, has no effect on VDR-mediated transcription. Real-time PCR indicates treatment with Dex increases Vdr transcripts in a time-dependent manner, suggesting Dex may directly regulate expression of Vdr. Numerous putative glucocorticoid response elements (GREs) were found in the Vdr gene. Chromatin immuno-precipitation (ChIP) assay demonstrated GR binding at several putative GREs located within the mouse Vdr gene. However, none of the putative GREs studied increase GR-mediated transcription in luciferase reporter assays. In an attempt to identify the response element responsible for Vdr transcript regulation, future studies will continue to analyze newly identified GREs more distal from the Vdr gene promoter.

Altered VDR-mediated transcriptional activity in prostate cancer stroma.

The 1alpha,25-dihydroxy-vitamin D(3) (1alpha,25(OH)(2)D(3)) mediated gene transcription in primary cultures of human prostate cells was analyzed using an adenoviral luciferase expression reporter under the control of the 25-hydroxy-vitamin D(3)-24-hydroxylase (CYP24) gene promoter. Stromal cells isolated from benign and malignant associated stroma (BAS and CAS) of a human clinical sample have been determined to contain similar levels of functional 1alpha,25(OH)(2)D(3) receptor (VDR). However, VDR-mediated reporter activity of the luciferase reporter has been found to be limited 7-9-fold in CAS compared to 14-16-fold in BAS. Chromatin immunoprecipitation (ChIP) assays indicate that in the absence of added ligand VDR interact with the silencing mediator for retinoid and thyroid hormone (SMRT) corepressor in both cell types, with higher recruitment in CAS as compared to BAS cells. In the presence of added ligand, VDR in CAS cells exhibited decreased ligand-inducible DNA binding activity, altered recruitment of coregulators SRC-1 and CBP, and increased recruitment of SMRT corepressor, as compared to BAS. Additionally, overexpression of wild-type VDR recovered VDR-mediated transaction of CYP24 luciferase reporter. These results indicate that VDR structure/function and coregulator recruitment to 1alpha,25(OH)(2)D(3) regulated genes is altered in the CaP stroma microenvironment.