Unveiling Novel ERCC1-XPF Complex Inhibitors: Bridging the Gap from In Silico Exploration to Experimental Design.

Modifications in DNA repair pathways are recognized as prognostic markers and potential therapeutic targets in various cancers, including non-small cell lung cancer (NSCLC). Overexpression of ERCC1 correlates with poorer prognosis and response to platinum-based chemotherapy. As a result, there is a pressing need to discover new inhibitors of the ERCC1-XPF complex that can potentiate the efficacy of cisplatin in NSCLC. In this study, we developed a structure-based virtual screening strategy targeting the inhibition of ERCC1 and XPF interaction. Analysis of crystal structures and a library of small molecules known to act against the complex highlighted the pivotal role of Phe293 (ERCC1) in maintaining complex stability. This residue was chosen as the primary binding site for virtual screening. Using an optimized docking protocol, we screened compounds from various databases, ultimately identifying more than one hundred potential inhibitors. Their capability to amplify cisplatin-induced cytotoxicity was assessed in NSCLC H1299 cells, which exhibited the highest ERCC1 expression of all the cell lines tested. Of these, 22 compounds emerged as promising enhancers of cisplatin efficacy. Our results underscore the value of pinpointing crucial molecular characteristics in the pursuit of novel modulators of the ERCC1-XPF interaction, which could be combined with cisplatin to treat NSCLC more effectively.

Immunotherapy as a Turning Point in the Treatment of Melanoma Brain Metastases.

The incidence of tumor metastases in the brain is many times more frequent than primary brain tumors, affecting a very large share of patients suffering from systemic cancer. Advanced malignant melanoma is well known for its ability to invade the brain space and current treatment options, such as surgery and radiation therapy, are not very efficient and cause notable complications and morbidity. The aim of this review is to explore the recent advances and future potential of using immunotherapy in the treatment of melanoma brain metastases. Several FDA approved immunotherapeutic drugs have shown to be able to at least double the overall survival rates in such patients. Clinical trials of varying phases are underway and available results are promising, significantly prolonging survival rates in patients with previously untreated melanoma brain metastases. Nevertheless, not all patients respond to these immunotherapies, facing a high percentage of resistant cases, without yet knowing the mechanisms and causes of resistance behind. Also, at the time of immunotherapy, a small percentage of patients is affected by pseudoprogression, which can be difficult to distinguish from true progression given the similarity of symptoms. Therefore, there is a pressing need for future research about treatment effectiveness in patients with brain metastases from melanoma, including outcomes from the perspective of patients.

The Redox-Active Manganese(III) Porphyrin, MnTnBuOE-2-PyP5+, Impairs the Migration and Invasion of Non-Small Cell Lung Cancer Cells, Either Alone or Combined with Cisplatin.

Manganese(III) porphyrin MnTnBuOE-2-PyP5+ (MnBuOE, BMX-001) is a third-generation redox-active cationic substituted pyridylporphyrin-based drug with a good safety/toxicity profile that has been studied in several types of cancer. It is currently in four phase I/II clinical trials on patients suffering from glioma, head and neck cancer, anal squamous cell carcinoma and multiple brain metastases. There is yet an insufficient understanding of the impact of MnBuOE on lung cancer. Therefore, this study aims to fill this gap by demonstrating the effects of MnBuOE on non-small cell lung cancer (NSCLC) A549 and H1975 cell lines. The cytotoxicity of MnBuOE alone or combined with cisplatin was evaluated by crystal violet (CV) and/or 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-Tetrazolium (MTS) reduction assays. Intracellular ROS levels were assessed using two fluorescent probes. Furthermore, the impact of MnBuOE alone or in combination with cisplatin on collective cell migration, individual chemotactic migration and chemoinvasion was assessed using the wound-healing and transwell assays. The expression of genes related to migration and invasion was assessed through RT-qPCR. While MnBuOE alone decreased H1975 cell viability at high concentrations, when combined with cisplatin it markedly reduced the viability of the more invasive H1975 cell line but not of A549 cell line. However, MnBuOE alone significantly decreased the migration of both cell lines. The anti-migratory effect was more pronounced when MnBuOE was combined with cisplatin. Finally, MnBuOE alone or combined with cisplatin significantly reduced cell invasion. MnBuOE alone or combined with cisplatin downregulated MMP2, MMP9, VIM, EGFR and VEGFA and upregulated CDH1 in both cell lines. Overall, our data demonstrate the anti-metastatic potential of MnBuOE for the treatment of NSCLC.

Correction: The Meaning of Lymphadenopathies During Adjuvant Durvalumab After Chemoradiotherapy for Lung Cancer: Thinking Beyond Disease Progression.

[This corrects the article DOI: 10.7759/cureus.26729.].

MnTnHex-2-PyP5+ Displays Anticancer Properties and Enhances Cisplatin Effects in Non-Small Cell Lung Cancer Cells.

The manganese(III) porphyrin MnTnHex-2-PyP5+ (MnTnHex) is a potent superoxide dismutase mimic and modulator of redox-based transcriptional activity that has been studied in the context of different human disease models, including cancer. Nevertheless, for lung cancer, hardly any information is available. Thus, the present work aims to fill this gap and reports the effects of MnTnHex in non-small cell lung cancer (NSCLC) cells, more specifically, A549 and H1975 cells, in vitro. Both cell lines were initially characterized in terms of innate levels of catalase, glutathione peroxidase 1, and peroxiredoxins 1 and 2. To assess the effect of MnTnHex in NSCLC, alone or in combination with cisplatin, endpoints related to the cell viability, cell cycle distribution, cell motility, and characterization of the volatile carbonyl compounds (VCCs) generated in the extracellular medium (i.e., exometabolome) were addressed. The results show that MnTnHex as a single drug markedly reduced the viability of both NSCLC cell lines, with some IC50 values reaching sub-micromolar levels. This redox-active drug also altered the cell cycle distribution, induced cell death, and increased the cytotoxicity pattern of cisplatin. MnTnHex also reduced collective cell migration. Finally, the metabolomics study revealed an increase in the levels of a few VCCs associated with oxidative stress in MnTnHex-treated cells. Altogether these results suggest the therapeutic potential of MnTnHex to be further explored, either alone or in combination therapy with cisplatin, in NSCLC.

Case report: Using DNA short tandem repeats to confirm nongestational origin of pulmonary choriocarcinoma.

Gestational trophoblastic neoplasias (GTN) are malignant neoplasms that occur in pregnant or recently pregnant women. Choriocarcinoma (CCA) is a highly aggressive and rare GTN, and cases outside the female genital tract are commonly seen as secondary manifestations of gynecologic disease. In this paper, we describe the case of a 40 years-old female patient with a primary pulmonary CCA who was surgically treated and for whom the confirmation of the primary origin of the tumor was possible using a DNA short tandem repeat genotyping. Distinction between gestational and non-gestational trophoblastic neoplasia is crucial as they require different therapeutic approach and have different prognoses.

The Meaning of Lymphadenopathies During Adjuvant Durvalumab After Chemoradiotherapy for Lung Cancer: Thinking Beyond Disease Progression.

Immune-checkpoint inhibitors (ICIs) have become the mainstay of treatment for many malignancies. With this new strategy, relevant immune-related adverse events (irAEs) have been reported, some of which can be mistaken for disease progression. To better illustrate the current challenges in diagnosing and managing a patient under adjuvant ICI treatment, we present the case of a 67-year-old female patient with stage IIIB unresectable, epidermal growth factor receptor (EGFR)-mutated, non-small-cell lung cancer who was initially treated with chemoradiotherapy, followed by immunotherapy with durvalumab. During the course of immunotherapy, the patient presented with madarosis and erythematous and endured skin lesions, in addition to lymphadenopathies and pulmonary infiltrates. She was started on first-line palliative treatment with an EGFR tyrosine kinase inhibitor. After reviewing the case, a multidisciplinary team meeting suggested diagnostic procedures, including a transbronchial needle aspiration from mediastinal lymph nodes. The histologic examination showed chronic systemic inflammation and non-caseating granulomas of the sarcoid type. In this case, palliative treatment was suspended and systemic therapy with prednisolone was initiated. The patient became asymptomatic and the previously observed radiologic abnormalities resolved. This case highlights the importance of early recognition and appropriate treatment of irAEs, mainly because these conditions remain poorly understood and are probably underdiagnosed. Considering differential diagnosis is paramount to guide clinical management, despite curative or palliative treatment intent.

A narrative review of the migration and invasion features of non-small cell lung cancer cells upon xenobiotic exposure: insights from in vitro studies.

Lung cancer (LC) is the leading cause of cancer deaths worldwide, being non-small lung cancer (NSCLC) sub-types the most prevalent. Since most LC cases are only detected during the last stage of the disease the high mortality rate is strongly associated with metastases. For this reason, the migratory and invasive capacity of these cancer cells as well as the mechanisms involved have long been studied to uncover novel strategies to prevent metastases and improve the patients' prognosis. This narrative review provides an overview of the main in vitro migration and invasion assays employed in NSCLC research. While several methods have been developed, experiments using conventional cell culture models prevailed, specifically the wound-healing and the transwell migration and invasion assays. Moreover, it is provided herewith a summary of the available information concerning chemical contaminants that may promote the migratory/invasive properties of NSCLC cells in vitro, shedding some light on possible LC risk factors. Most of the reported agents with pro-migration/invasion effects derive from cigarette smoking [e.g., Benzo(a)pyrene and cadmium] and air pollution. This review further presents several studies in which different dietary/plant-derived compounds demonstrated to impair migration/invasion processes in NSCLC cells in vitro. These chemicals that have been proposed as anti-migratory consisted mainly of natural bioactive substances, including polyphenols non-flavonoids, flavonoids, bibenzyls, terpenes, alkaloids, and steroids. Some of these compounds may eventually represent novel therapeutic strategies to be considered in the future to prevent metastasis formation in LC, which highlights the need for additional in vitro methodologies that more closely resemble the in vivo tumor microenvironment and cancer cell interactions. These studies along with adequate in vivo models should be further explored as proof of concept for the most promising compounds.

Nivolumab-induced seronegative encephalitis.

Immune checkpoint inhibitors (ICIs) have emerged as a new therapeutic tool for numerous types of cancer. Neurological complications have been reported in 1% of patients who have undergone checkpoint inhibition therapy. ICIs-induced encephalitides occur in 0.1-0.2% of patients within weeks after ICIs initiation; are usually seronegative and have nonspecific changes on imaging, CSF and electroencephalogram (EEG) studies. Early recognition and prompt treatment are important to prevent significant morbidity and mortality. We present a case of nivolumab-induced encephalitis with very subtle clinical symptoms and full recovery following ICIs suspension and steroids.

Impact of the APE1 Redox Function Inhibitor E3330 in Non-small Cell Lung Cancer Cells Exposed to Cisplatin: Increased Cytotoxicity and Impairment of Cell Migration and Invasion.

Elevated expression levels of the apurinic/apyrimidinic endonuclease 1 (APE1) have been correlated with the more aggressive phenotypes and poor prognosis of non-small cell lung cancer (NSCLC). This study aimed to assess the impact of the inhibition of the redox function of APE1 with E3330 either alone or in combination with cisplatin in NSCLC cells. For this purpose, complementary endpoints focusing on cell viability, apoptosis, cell cycle distribution, and migration/invasion were studied. Cisplatin decreased the viability of H1975 cells in a time- and concentration-dependent manner, with IC50 values of 9.6 µM for crystal violet assay and 15.9 µM for 3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay. E3330 was clearly cytotoxic for concentrations above 30 µM. The co-incubation of E3330 and cisplatin significantly decreased cell viability compared to cisplatin alone. Regarding cell cycle distribution, cisplatin led to an increase in sub-G1, whereas the co-treatment with E3330 did not change this profile, which was then confirmed in terms of % apoptotic cells. In addition, the combination of E3330 and cisplatin at low concentrations decreased collective and chemotactic migration, and also chemoinvasion, by reducing these capabilities up to 20%. Overall, these results point to E3330 as a promising compound to boost cisplatin therapy that warrants further investigation in NSCLC.

Pulmonary Administration: Strengthening the Value of Therapeutic Proximity.

In recent years inhaled systems have shown momentum as patient-personalized therapies emerge. A significant improvement in terms of therapeutic efficacy and/or reduction adverse systemic effects is anticipated from their use owing these systems regional accumulation. Nevertheless, whatever safety and efficacy evidence required for inhaled formulations regulatory approval, it still poses an additional hurdle to gaining market access. In contrast with the formal intravenous medicines approval, the narrower adoption of pulmonary administration might rely on discrepancies in pre-clinical and clinical data provided by the marketing authorization holder to the regulatory authorities. Evidences of a diverse and inconsistent regulatory framework led to concerns over toxicity issues and respiratory safety. However, an overall trend to support general concepts of good practices exists. Current regulatory guidelines that supports PK/PD (pharmacokinetics/pharmacodynamic) assessment seeks attention threatening those inhaled formulations set to be approved in the coming years. A more complex scenario arises from the attempt of implementing nanomedicines for pulmonary administration. Cutting-edge image techniques could play a key role in supporting diverse stages of clinical development facilitating this pharmaceutics take off and speed to patients. The ongoing challenge in adapting conventional regulatory frameworks has proven to be tremendously difficult in an environment where market entry relies on multiple collections of evidence. This paper intention is to remind us that an acceptable pre-clinical toxicological program could emerge from, but not only, an accurate and robust data imaging collection. It is our conviction that if implemented, inhaled nanomedicines might have impact in multiple severe conditions, such as lung cancer, by fulfilling the opportunity for developing tailored treatments while solving dose-related toxicity issues; the most limiting threat in conventional lung cancer clinical management.

ProMenteSã: Formação de professores para promoção da saúde mental na escola / ProMenteSã: formación de profesores para promover la salud mental en la escuela / ProMenteSã: a teacher training intervention program for mental health promotion at schools

Resumo Objetivo: Promover a saúde mental nas escolas através da (in)formação e capacitação de professores do 2º e 3º ciclo do ensino básico. Métodos: Realizado um estudo não-experimental, analítico, inserido no Projeto MaiSaúdeMental (Referência: Centro - 01-0145-FEDER-023293). Após identificarmos necessidades de formação na área da saúde mental em professores, foi construído um programa de formação designado ' ProMenteSã' com vista à capacitação dos professores do 2º e 3º ciclo do ensino básico na área da promoção da saúde mental em crianças e adolescentes em meio escolar. O programa completo foi aplicado a 13 professores, maioritariamente do género feminino 11(84,6%), com uma média de idade 51,00±6,58 anos, casados e a residir em meio urbano. A eficácia do programa e a aquisição de conhecimentos em áreas da saúde mental e literacia em saúde mental foi avaliada com recurso a um questionário aplicado 'antes' e 'após' a intervenção. Resultados: Ao compararmos as duas amostras ('antes' e 'após') não foram encontradas diferenças significativas quanto à eficácia do programa de formação. Contudo, avaliada a aquisição de conhecimentos no domínio da saúde mental, verificou-se que após a aplicação do Programa ProMenteSã os professores apresentaram um aumento significativo de conhecimentos nas temáticas 'consumos e dependências-novos comportamentos aditivos' (p=0,03) e 'importância do sono na promoção da saúde mental' (p=0,04). Conclusão: O programa de formação teve impacto no aumento dos conhecimentos dos professores na área da saúde mental, na vertente da promoção da saúde.

Resumen Objetivo: Promover la salud mental en las escuelas a través de la (in)formación y capacitación de profesores de 5º a 9º año de primaria. Métodos: Estudio no experimental, analítico, dentro del Proyecto "MaiSaúdeMental" (Más Salud Mental) (Referencia: Centro - 01-0145-FEDER-023293). Luego de identificar las necesidades de formación de profesores en el área de la salud mental, se elaboró un programa de formación denominado " ProMenteSã" (Programa Mente Sana), con el fin de capacitar a profesores de 5º a 9º año de primaria en el área de promoción de la salud mental en niños y adolescentes en medio escolar. El programa completo fue aplicado con 13 profesores, mayormente de género femenino 11 (84,6 %), con un promedio de edad de 51,00±6,58 años, casados y residentes en medio urbano. La eficacia del programa y la adquisición de conocimientos en áreas de salud mental e instrucción en salud mental fue evaluada mediante un cuestionario aplicado antes y después de la intervención. Resultados: Al comparar las dos muestras (antes y después), no se encontraron diferencias significativas respecto a la eficacia del programa de formación. No obstante, al analizar la adquisición de conocimientos en el dominio de la salud mental después de aplicar el programa ProMenteSã , se verificó que los profesores demostraron un aumento significativo de conocimientos en las temáticas "consumos y dependencias: nuevos comportamientos adictivos" (p=0,0) e "importancia del sueño en la promoción de la salud mental" (p=0,04). Conclusión: El programa de formación tuvo impacto en el aumento de los conocimientos de los profesores en el área de la salud mental, en la vertiente de la promoción de la salud.

Abstract Objective: To promote mental health at schoolsthrough a training intervention program for teachers of the 2nd (5th and 6th years) and 3rd (7th, 8th and 9th years) Cycles of Basic Education. Method: Non-experimental analytical study conducted as part of the MAISaúdeMental (More Mental Health) Project (ref. number: 01-0145-FEDER-023293). After identifying needs in teachers' mental health training, the ProMenteSã training intervention program was designed to train teachers of the 2nd and 3rd Cycles of Basic Education to promote mental health in children and adolescents within the school setting. The full program was applied to 13 teachers who were mainly women (11, 84.6%) with a mean age of 51.00 ±6,58 years, married, and living in urban settings. Efficacy and acquisition of mental health knowledge and literacy were assessed with a questionnaire applied before and after the training intervention program. Results: When comparing the two samples (before and after the training intervention program), no significant differences in efficacy were found. However, when assessing the acquisition of mental health knowledge, after the application of the ProMenteSã Program teachers showed a significant increase in knowledge areas "Abuse and dependence — New behaviors of addiction" (p=0.03) and "Significance of sleep in promoting mental health" (p=0.04). Conclusion: The training intervention program increased teachers' mental health knowledge for promotion of health.

Immunomodulatory Drugs in Melanoma Brain Metastases.

Brain metastases are about ten times more frequent than a brain primary tumor, being present in 20-40% of adults with systemic cancer. Together with lung cancer and breast cancer, skin cancers such as melanoma are top primary tumors which metastasizes to the brain. Advanced melanoma is well known for its propensity to metastasize to the brain, with 80% of patients presenting brain metastasis at the autopsy. However, current therapies are not very efficient and brain metastases are in most of the cases lethal. Treatment of melanoma brain metastases with surgery and/or radiation therapy results in a median overall survival of only about four months after diagnosis. New immunotherapies such as targeted or immunomodulatory drugs, many in clinical trials, have shown promise, with some immunomodulatory drugs being able to at least double the overall survival rates for patients with melanoma brain metastases. This review focuses on the recent advances and future potential of using immunotherapy, such as the newly developed immunomodulatory drugs, for melanoma brain metastases therapy. Immunomodulatory drugs bring a great promise as new tools for melanoma treatment in particular and for the treatment of other types of malignancies in general.

Pulmonary side effects of nivolumab in a patient treated for gynecological malignancy.

We report a case of a 26-year-old female patient treated with nivolumab for peritoneal implants from small-cell ovarian cancer. Pulmonary changes were depicted on follow-up examinations, namely, consolidations and scattered ground glass opacities, without any remarkable pulmonary complaints or laboratory changes. Both infectious and progressive diseases were ruled out. The condition was attributed to nivolumab side effect, and total remission was documented, followed by corticosteroids administration. We present clinical details and imaging findings, followed by a discussion where pulmonary toxicity can often be mistaken with infection or secondary involvement.

Genotype diversity in the honey bee parasite Nosema ceranae: multi-strain isolates, cryptic sex or both?

BACKGROUND: There is great controversy as to whether Microsporidia undergo a sexual cycle. In the paradigmatic case of Nosema ceranae, although there is no morphological evidence of sex, some meiosis-specific genes are present in its reduced genome and there is also high intraspecific variability, with incongruent phylogenies having been systematically obtained. The possibility of sexual recombination is important from an epidemiological standpoint, particularly as N. ceranae is considered to be a major factor in the current disquieting epidemic of widespread bee colony losses. This parasite apparently originated in oriental honey bees, spreading out of Asia and Australia to infect honey bees worldwide. This study had three main objectives: i) to obtain genetic markers that are not part of known multi-copy arrays for strain determination; ii) to shed light on the intraspecific variability and recombination of N. ceranae; and iii) to assess the variability in N. ceranae populations. The answers to these questions are critical to understand the capacity of adaptation of microsporidia. RESULTS: Biallelic polymorphisms were detected at a number of specific points in the five coding loci analyzed from European and Australian isolates of N. ceranae. Heterozygous genotypes were abundant and cloning experiments demonstrate that they reflect the existence of multiple alternative sequences in each isolate. The comparisons of different clones and genotypes clearly indicate that new haplotypes are generated by homologous recombination. CONCLUSIONS: The N. ceranae isolates from honey bees correspond to genotypically distinct populations, revealing that individual honey bees may not be infected by a particular clone but rather, a pool of different strains. Homologous recombination implies the existence of a cryptic sex cycle yet to be described in N. ceranae. There are no diagnostic alleles associated with Australian or European origins, nor are there differences between the two hosts, A. cerana and A. mellifera, supporting the absence of biological barriers for N. ceranae transmission. Diversity is high among microsporidia of both these origins, and the maintenance of a high heterozygosis in the recently invaded European populations, could hypothetically underlie the stronger virulence of N. ceranae observed in A. mellifera.

Strategies to overcome resistance to tyrosine kinase inhibitors in non-small-cell lung cancer.

The use of molecularly targeted agents has dramatically improved the prognosis of defined subsets of patients with non-small-cell lung cancer harboring somatically activated oncogenes, such as mutant EGFR or rearranged ALK. However, after initial marked responses to EGFR or ALK tyrosine kinase inhibitors (TKIs), almost all patients inevitably progress due to development of acquired resistance. Multiple molecular mechanisms of resistance have been identified; the best characterized are secondary mutations in the tyrosine kinase domain of the oncogene, such as T790M in EGFR and L1196M in ALK, which prevent target inhibition by the corresponding TKI. Other mechanisms include copy number gain of the ALK fusion gene and the activation of bypass signaling pathways that can maintain downstream proliferation and survival signals despite inhibition of the original drug target. Here, the authors provide an overview of the known mechanisms of resistance to TKIs and outline the therapeutic strategies, including new investigational agents and targeted therapies combinations, that have been developed to overcome resistance.

C1-C2 pigmented villonodular synovitis and clear cell carcinoma: unexpected presentation of a rare disease and a review of the literature.

INTRODUCTION: Pigmented Villonodular synovitis (PVNS) is a rare vertebral pathology--around 50 reports, only 3 concerning C1-C2 location. CASE REPORT: A 64-year-old man, submitted to a right nephrectomy for a clear cell carcinoma, presented with an asymptomatic osteolytic C1-C2 lesion. Even though the diagnosis of metastatic disease was the most probable, the presence of a solitary lesion without other osseous or systemic localization and the predicted low risk of recurrence imposed a surgical biopsy. A pigmented villonodular synovitis diagnosis was made, a rare vertebral pathology--around 50 reports, only 3 concerning C1-C2 location. No further treatment was assigned precluding the iatrogenic consequences of empirical treatments based on clinical diagnosis with no histopathological support. The patient remains stable at 18 months of follow-up. CONCLUSION: A large differential diagnosis should be made when the typical findings for metastatic disease are absent precluding the iatrogenic consequences of empirical treatments based on clinical diagnosis with no histopathological support.

Microsporidia detection and genotyping study of human pathogenic E. bieneusi in animals from Spain.

Microsporidia are ubiquitous parasites infecting all animal phyla and we present evidence that supports their zoonotic potential. Fecal samples taken from domestic (cats and dogs), farm (pigs, rabbits and ostriches) and wild animals (foxes) from different provinces of Spain were evaluated for microsporidia infection by light microscopy and PCR. After Microsporidia species identification, E. bieneusi genotypes were additionally studied by sequence analysis of the ITS region. Eighty-five samples out of 159 exhibited structures that were compatible with microsporidia spores by Webers stain with 37 of them being confirmed by PCR. Microsporidia species identified included E. bieneusi, E. intestinalis and A. algerae. We report the first diagnosis of E. intestinalis and E. bieneusi in ostriches and A. algerae in pigs. We also provide new information on the molecular characterization of E. bieneusi isolates both in rabbits and ostriches. All of the E. bieneusi genotypes identified belonged to the zoonotic group of genotypes (Group I) including genotypes A (dogs), I (pigs), D (rabbits and foxes) and type IV (ostriches). Our results demonstrate that microsporidia are present in domestic, farm and wild animals in Spain, corroborating their potential role as a source of human infection and environmental contamination.

Ribosomal gene polymorphism in small genomes: analysis of different 16S rRNA sequences expressed in the honeybee parasite Nosema ceranae (Microsporidia).

To date, few organisms have been shown to possess variable ribosomal RNA, otherwise considered a classic example of uniformity by concerted evolution. The polymorphism for the 16S rRNA in Nosema ceranae analysed here is striking as Microsporidia are intracellular parasites which have suffered a strong reduction in their genomes and cellular organization. Moreover, N. ceranae infects the honeybee Apis mellifera, and has been associated with the colony-loss phenomenon during the last decade. The variants of 16S rRNA include single nucleotide substitutions, one base insertion-deletion, plus a tetranucleotide indel. We show that different gene variants are expressed. The polymorphic sites tend to be located in particular regions of the rRNA molecule, and the comparison to the Escherichia coli 16S rRNA secondary structure indicates that most variations probably do not preclude ribosomal activity. The fact that the polymorphisms in such a minimal organism as N. ceranae are maintained in samples collected worldwide suggest that the existence of differently expressed rRNA may play an adaptive role in the microsporidian.

Variability in minimal genomes: analysis of tandem repeats in the microsporidia Encephalitozoon intestinalis.

Microsporidia are ubiquitous fungi with genomes that have undergone a strong reduction to the extreme cases of Encephalitozoon cuniculi and Encephalitozoon intestinalis. Genetic variability within species of the Encephalitozoon genus has been reported, with most of the studies based on the internal transcribed spacer (ITS) of the rDNA. However, in contrast to the picture of E. cuniculi and Encephalitozoon hellem, where different strains have been identified, no genetic variability has yet been observed in E. intestinalis. We have analysed tandem repeats included in putative coding sequences which could be used as polymorphic markers in E. intestinalis. Eight candidate loci (M2, M2A, M3, M5, M7, M7A, M8 and PTP1) were established and 9 E. intestinalis cultured strains from North America, South America and Europe were analysed. M2, M7 and PTP1 nucleotide sequences were identical among the different strains and the GenBank sequence. In contrast, we observed variants in 4 markers (M2A, M3, M7A and M8) which did not correspond to their respective reference sequences. The most noticeable finding was that with the M5 marker two genotypes were defined among the different strains studied, demonstrating genotypic variability of E. intestinalis. Although the diversity described is certainly not high, which can be explained by a lower chance of genetic variability in its minimal genome, we have demonstrated that polymorphisms actually exist in E. intestinalis. Epidemiological studies using this genetic marker should now be conducted to elucidate the genetic variability in E. intestinalis and improve our knowledge of the epidemiology of this microsporidia.