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Purpose: This study aimed to develop and psychometrically evaluate a patient-reported outcome measure (PROM), SAlivary, LAcrimal, NaSal (SALANS), to document patients' symptoms after radioactive iodine (RAI) treatment for differentiated thyroid cancer (DTC)., Methods: We generated and iteratively revised SALANS items based on expert input, focus group discussions and feedback from cognitive testing (n = 17). We administered an initial SALANS measure with 39 items to patients diagnosed with DTC in the past two years (n = 105). Exploratory factor analysis (EFA) examined the factor structure of the SALANS items. We assessed the consistency reliability and related the total and subscale scores of the final SALANS to existing PROMs to assess validity., Results: The final SALANS consisted of 33 items and six subscales (sialadenitis, taste, xerostomia, dry eyes, epiphora, and nasal) with six factors extracted by EFA. The six subscales demonstrated good internal reliability (α range = 0.87-0.92). The SALANS total score showed good convergent validity with the Xerostomia Inventory (r = 0.86) and good discriminant validity with a measure of spirituality (r =  - 0.05). The mean SALANS total score was significantly higher (d = 0.5, p < 0.04) among patients who had RAI compared to those who did not have RAI., Conclusion: Preliminary evidence suggests that SALANS is a novel and reliable PROM to assess the type and frequency all symptoms experienced after RAI treatment for DTC. Future work is needed to further validate and develop the scale., (© 2024. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Organoids can meet the needs between the use of cell culture and in vivo work, bringing together aspects of multicellular tissues, providing a more similar in vitro system for the study of various components, including host-interactions with pathogens and drug response. Organoids are structures that resemble organs in vivo, originating from pluripotent stem cells (PSCs) or adult stem cells (ASCs). There is great interest in deepening the understanding of the use of this technology to produce information about fungal infections and their treatments. This work aims the use 2D human lung organoid derived from human embryonic stem cells (hESCs), to investigate Cryptococcus neoformans-host interactions. C. neoformans is an opportunistic fungus acquired by inhalation that causes systemic mycosis mainly in immunocompromised individuals. Our work highlights the suitability of human minilungs for the study of C. neoformans infection (adhesion, invasion and replication), the interaction with the surfactant and induction of the host's alveolar pro-inflammatory response., Competing Interests: Declaration of competing interest The authors have declared that no competing interest exists., (Copyright © 2023 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Extracellular vesicles (EVs) are membrane-bound particles released by cells that play vital roles in intercellular communication by transporting diverse biologically active molecules, including RNA molecules, including mRNA, miRNA, lncRNA, and other regulatory RNAs. These RNA types are protected within the lipid bilayer of EVs, ensuring their stability and enabling long-distance cellular interactions. Notably, EVs play roles in infection, where pathogens and host cells use EV-mediated RNA transfer to influence immune responses and disease outcomes. For example, bacterial EVs play a crucial role in infection by modulating host immune responses and facilitating pathogen invasion. This review explores the complex interactions between EV-associated RNA and host-pathogen dynamics in bacteria, parasites, and fungi, aiming to uncover molecular mechanisms in infectious diseases and potential therapeutic targets.

Objectives: To assess long-term sexual outcome and quality of life after perineal reconstruction by pedicled anterolateral thigh (ALT) flaps after Fournier's gangrene. Postoperative surgical outcomes were assessed; quality of life and sexual function were assessed at long term follow-up (>12 months) with 2 scientifically validated questionnaires., Methods: We conducted a retrospective analysis of a prospectively maintained database. Long-term sexual function and quality of life were assessed by standardized questionnaires. Descriptive statistics were conducted., Results: 8 patients were included in our study, 5 patients responded to quality of life and sexual function analysis. Surgical outcomes were in line with literature: one minor complication (minor dehiscence requiring a skin graft), one major complication (flap loss, requiring a second, contralateral flap) occurred. No reconstructive failure occurred. Average time to complete wound healing was 17 days (SD ±5). Quality of life scores over 70/100 in four out of five categories; social function was rated lowest: patients reported very few residual complaints. Sexual outcome analysis emphasizes the positive impact of the reconstruction. As expected, Fournier's Gangrene heavily affected patient's sexual health., Conclusions: Perineal reconstruction with ALT shows excellent quality of life, and good sexual health outcomes. Currently the lack of reliable and relatable data leads to the impossibility to compare different reconstructive procedures. We emphasize that assessing sexual function and quality of life after perineal reconstruction is paramount to weigh reconstructive success., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2022 Rossi, de Schoulepnikoff, Guillier, Raffoul and Di Summa.)

The new generation of organoids derived from human pluripotent stem cells holds a promising strategy for modeling host-bacteria interaction studies. Organoids recapitulate the composition, diversity of cell types, and, to some extent, the functional features of the native organ. We generated lung bud organoids derived from human embryonic stem cells to study the interaction of Streptococcus pneumoniae (pneumococcus) with the alveolar epithelium. Invasive pneumococcal disease is an important health problem that may occur as a result of the spread of pneumococcus from the lower respiratory tract to sterile sites. We show here an efficient experimental approach to model the main events of the pneumococcal infection that occur in the human lung, exploring bacterial adherence to the epithelium and internalization and triggering an innate response that includes the interaction with surfactant and the expression of representative cytokines and chemokines. Thus, this model, based on human minilungs, can be used to study pneumococcal virulence factors and the pathogenesis of different serotypes, and it will allow therapeutic interventions in a reliable human context. IMPORTANCE Streptococcus pneumoniae is responsible for high morbidity and mortalities rates worldwide, affecting mainly children and adults older than 65 years. Pneumococcus is also the most common etiologic agent of bacterial pneumonia and nonepidemic meningitis, and it is a frequent cause of bacterial sepsis. Although the introduction of pneumococcal vaccines has decreased the burden of pneumococcal disease, the rise of antibiotic-resistant strains and nonvaccine types by serotype replacement is worrisome. To study the biology of pneumococcus and to establish a reliable human model for pneumococcal pathogenesis, we generated human minilungs from embryonic stem cells. The results show that these organoids can be used to model some events occurring during the interaction of pneumococcus with the lung, such as adherence, internalization, and the initial alveolar innate response. This model also represents a great alternative for studying virulence factors involved in pneumonia, drug screening, and other therapeutic interventions.

Buttock augmentation is a commonly performed aesthetic surgery. Several methods have been described, but only the use of implants or autologous fat is consensually deemed safe and effective. Synthetic fillers in gluteal augmentation have been described despite potential severe long-term complications, both medical and aesthetic. The aim of this study is to report a series of 2 consecutive cases who underwent buttock and hip augmentation with large volumes of permanent copolyamide filler requiring surgical removal due to significant complications. Based on these cases and a review of recent literature, a management algorithm is proposed. The authors conducted a retrospective chart review of 2 consecutive cases of failed copolyamide filler augmentations in the gluteal and inguinal regions. The authors conducted a literature overview using PubMed (National Institutes of Health, Bethesda, MD) and Google Scholar (Google, Mountain View, CA) to include all articles concerning the removal of large quantities of permanent copolyamide fillers. Based on the physical properties of copolyamide, resection of the filler was performed by percutaneous aspiration with liposuction cannulas, with varying infiltration protocols. Both cases showed successful removal of major parts of the filler; however, residual material tended toward migration, requiring a secondary intervention. Hydrated low-pressure aspiration can manage nonintegrated gluteal copolyamide filler but will achieve only partial resection. Literature shows that radical excision is possible, however, with major drawbacks in function and aesthetics. Moreover, in acute inflammation and infection, an open approach should be preferred., (© 2022 The Aesthetic Society.)

Background: The vertical rectus abdominis flap is considered the gold standard in perineal reconstruction after oncological abdominoperineal resection; however, it has a nonnegligible donor site morbidity. The anterolateral thigh flap offers reliable soft tissue coverage., Objective: The aim was to analyze long-term outcomes of composite anterolateral thigh-vastus lateralis flaps in oncological abdominoperineal resections., Design: We conducted a retrospective cohort analysis of a prospectively maintained database., Settings: This study was conducted in the Lausanne University Hospital. Annually, approximately 10 oncological abdomioperineal resections are performed. Literature reports 7% to 20% of patients undergoing abdominoperineal resection require flap reconstruction; in our institution, approximately 2 patients with large defects after abdominoperineal resections required reconstruction., Patients: Twenty-nine pedicled anterolateral thigh-vastus lateralis flaps in 27 consecutive patients (mean age 63 years +/-11.2, 23 with radiochemotherapy) after abdominoperineal resection to cover large defects (median 190 cm2, 48-600 cm2) were analyzed., Intervention: Pedicled composite anterolateral thigh-vastus lateralis flaps were performed after oncological abdominoperineal resection., Main Outcome Measures: Descriptive statistical analysis was conducted. Short- and long-term outcomes were analyzed, univariate and multivariate analyses were performed. Median follow-up was 16 months (12-48 months)., Results: Flap-related postoperative complications occurred in 16 flaps; flap-survival was 100%. Multivariate logistic analysis identified initial defect size as predictive for complications. Patients with larger defects (≥ 190 cm2) had higher complication rates (p = 0.006). Long-term analysis revealed 3 chronic fistulae, 2 tumor recurrences, 1 flap dysesthesia, and one perineal acne inversa., Limitations: Limitations include retrospective analysis, selection bias, and lacking a control group. Sample size limits statistical power., Conclusions: The pedicled anterolateral thigh-vastus lateralis flap offers reliable, stable tissue with low morbidity and good long-term outcomes. Complications compared favorably with current literature describing perineal reconstructions with rectus abdominis flaps. The composite anterolateral thigh flap is a valid alternative without the setback of abdominal donor site morbidity. See Video Abstract at http://links.lww.com/DCR/B757.RESULTADOS DEL COLGAJO COMPUESTO ANTEROLATERAL DE MUSLO PARA LA RECONSTRUCCIÓN PERINEAL DESPUÉS DE LA RESECCIÓN ABDOMINOPERINEAL POST ONCOLÓGICAANTECEDENTES:El colgajo vertical de recto abdominal se considera el estándar de oro en la reconstrucción perineal después de la resección abdominoperineal oncológica, sin embargo, tiene una morbilidad no despreciable en el sitio donante. El colgajo anterolateral del muslo ofrece una cobertura confiable de los tejidos blandos.OBJETIVO:El objetivo fue analizar los resultados a largo plazo de los colgajos compuestos anterolaterales del muslo - vasto lateral - en resecciones abdominoperineales oncológicas.DISEÑO:Realizamos un análisis, retrospectivo, de tipo cohorte, de una base de datos mantenida prospectivamente.AJUSTES:Este estudio fue realizado en el hospital universitario de Lausanne. Anualmente se realizan aproximadamente 10 resecciones abdominoperineales oncológicas. La literatura reporta que entre el 7 y el 20% de los pacientes que se someten a una resección abdominoperineal requieren de reconstrucción con colgajo; en nuestra institución, aproximadamente 2 pacientes con grandes defectos tras la resección abdominoperineal requirieron reconstrucción.PACIENTES:Fueron analizados veintinueve colgajos pediculados anterolaterales de muslo - vasto lateral - en 27 pacientes consecutivos (edad media 63 años +/- 11,2, 23 con radio quimioterapia) después de la resección abdominoperineal para cubrir defectos grandes (mediana 190 cm2, 48-600 cm2).INTERVENCIÓN:Tras la resección abdominoperineal oncológica se realizaron colgajos pediculados compuestos anterolaterales de muslo - vasto lateral.PRINCIPALES MEDIDAS DE RESULTADO:Fue realizado un análisis estadístico descriptivo. Fueron analizados los resultados a corto y largo plazo - fueron realizados así mismo análisis uni y multivariados. La mediana de seguimiento fue de 16 meses (12-48 meses).RESULTADOS:Complicaciones postoperatorias relacionadas con el colgajo ocurrieron en 16 colgajos, la supervivencia del colgajo fue del 100%. El análisis logístico multivariado identificó al tamaño del defecto inicial como predictor de complicaciones. Aquellos pacientes con defectos más grandes (≥190 cm2) tuvieron mayores tasas de complicaciones (p = 0,006). El análisis a largo plazo reveló tres fístulas crónicas, dos recidivas tumorales, una disestesia de colgajo y un acné perineal inverso.LIMITACIONES:Las limitaciones incluyen análisis retrospectivo, sesgo de selección y falta de grupo de control. El tamaño de la muestra limita el poder estadístico.CONCLUSIONES:El colgajo pediculado anterolateral de muslo - vasto lateral - ofrece tejido confiable y estable con baja morbilidad y buenos resultados a largo plazo. Los resultados de las complicaciones se mostraron favorables con respecto a la literatura actual que describe reconstrucciones perineales con colgajos de recto abdominal. El colgajo compuesto anterolateral de muslo es una alternativa válida sin el revés de la morbilidad del sitio donante abdominal. Consulte Video Resumen en http://links.lww.com/DCR/B757. (Traducción-Dr. Osvaldo Gauto)., (Copyright © The ASCRS 2021.)

As there are more than 6 million human deaths due to mycoses each year, there is an urgent need to develop fungal vaccines. Moreover, given the similarities among pathogenic fungi, it may be possible to create a multi-fungi vaccine. In this study, we combined immunoproteomic and immunopeptidomic methods, for which we have adapted a technique based on co-immunoprecipitation (Co-IP) that made it possible to map Histoplasma capsulatum epitopes for the first time in a natural context using murine dendritic cells (DCs) and macrophages (Mφ). Although polysaccharide epitopes exist, this research focused on mapping protein epitopes as these are more immunogenic. We used different algorithms to screen proteins and peptides identified by two-dimensional electrophoresis (2-D) and Co-IP. Seventeen proteins were revealed by 2-D gels, and 45 and 24 peptides from distinct proteins were presented by DCs and Mφ, respectively. We then determined which epitopes were restricted to MHC-I and II from humans and mice and showed high promiscuity, but lacked identity with human proteins. The 4 most promising peptides were synthesized, and the peptides with and without incorporation into glucan particles induced CD4+ and CD8+ T cell proliferation and produced a Th1 and Th17 response marked by the secretion of high levels of IFN-γ, IL-17 and IL-2. These epitopes were from heat shock protein 60, enolase, and the ATP-dependent molecular chaperone HSC82, and they each have a high degree of identity with proteins expressed by other medically important pathogenic fungi. Thus, the epitopes described in this study have the potential for use in the development of vaccines that could result in cross-protection among fungal species., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Kischkel, Boniche-Alfaro, Menezes, Rossi, Angeli, de Almeida, Palmisano, Lopes-Bezerra, Nosanchuk and Taborda.)

Due to its location, the fungal cell wall is the compartment that allows the interaction with the environment and/or the host, playing an important role during infection as well as in different biological functions such as cell morphology, cell permeability and protection against stress. All these processes involve the activation of signaling pathways within the cell. The cell wall integrity (CWI) pathway is the main route responsible for maintaining the functionality and proper structure of the cell wall. This pathway is highly conserved in the fungal kingdom and has been extensively characterized in Saccharomyces cerevisiae. However, there are still many unknown aspects of this pathway in the pathogenic fungi, such as Cryptococcus neoformans . This yeast is of particular interest because it is found in the environment, but can also behave as pathogen in multiple organisms, including vertebrates and invertebrates, so it has to adapt to multiple factors to survive in multiple niches. In this review, we summarize the components of the CWI pathway in C. neoformans as well as its involvement in different aspects such as virulence factors, morphological changes, and its role as target for antifungal therapies among others.

Purpose: Despite having a generally favorable prognosis, differentiated thyroid cancer is known to have a significant, long-term impact on the quality of life of survivors. We wished to investigate short- and long-term effects among thyroid cancer survivors following radioactive iodine therapy., Methods: We conducted eight focus groups (N = 47) to understand patients' experiences of short- and long-term effects after radioactive iodine treatment and the impact these treatment-related side effects had on patients' quality of life. We elicited responses regarding experiences with side effects following radioactive iodine treatment, particularly salivary, lacrimal, and nasal symptoms. We transcribed audiotapes and conducted qualitative analyses to identify codes and themes., Results: We identified eight broad themes from the qualitative analyses. Themes reflecting physical symptoms included dry mouth, salivary gland dysfunction, altered taste, eye symptoms such as tearing or dryness, and epistaxis. Psychosocial themes included lack of knowledge and preparation for treatment, regret of treatment, and distress that thyroid cancer is labeled as a "good cancer.", Conclusions: Thyroid cancer survivors reported a wide range of radioactive iodine treatment-related effects and psychosocial concerns that appear to reduce quality of life. The psychosocial concerns reported by participants underscore the significant unmet information and support needs prior to and following RAI treatment among individuals diagnosed with thyroid cancer., Implications for Cancer Survivors: Future research is needed to help both patients and physicians understand the effect of radioactive iodine on quality of life, and to better assess the benefits versus the risks of radioactive iodine therapy.

Treatment modalities for systemic mycoses are still limited. Currently, the main antifungal therapeutics include polyenes, azoles, and echinocandins. However, even in the setting of appropriate administration of antifungals, mortality rates remain unacceptably high. Moreover, antifungal therapy is expensive, treatment periods can range from weeks to years, and toxicity is also a serious concern. In recent years, the increased number of immunocompromised individuals has contributed to the high global incidence of systemic fungal infections. Given the high morbidity and mortality rates, the complexity of treatment strategies, drug toxicity, and the worldwide burden of disease, there is a need for new and efficient therapeutic means to combat invasive mycoses. One promising avenue that is actively being pursued is nanotechnology, to develop new antifungal therapies and efficient vaccines, since it allows for a targeted delivery of drugs and antigens, which can reduce toxicity and treatment costs. The goal of this review is to discuss studies using nanoparticles to develop new therapeutic options, including vaccination methods, to combat systemic mycoses caused by Candida sp., Cryptococcus sp., Paracoccidioides sp., Histoplasma sp., Coccidioides sp., and Aspergillus sp., in addition to providing important information on the use of different types of nanoparticles, nanocarriers and their corresponding mechanisms of action., (Copyright © 2020 Kischkel, Rossi, Santos Junior, Nosanchuk, Travassos and Taborda.)

Amphotericin B (AmB) is the antifungal with the strongest fungicidal activity, but its use has several limitations, mainly associated with its toxicity. Although some lipidic and liposomal formulations that present reduced toxicity are available, their price limits their application in developing countries. Flucytosine (5FC) has shown synergistic effect with AmB for treatment of some fungal infections, such as cryptococcosis, but again, its price is a limitation for its use in many regions. In the present work, we aimed to identify new drugs that have a minor effect on Cryptococcus neoformans , reducing its growth in the presence of subinhibitory concentrations of AmB. In the initial screening, we found fourteen drugs that had this pattern. Later, checkerboard assays of selected compounds, such as erythromycin, riluzole, nortriptyline, chenodiol, nisoldipine, promazine, chlorcyclizine, cloperastine, and glimepiride, were performed and all of them confirmed for their synergistic effect (fractional inhibitory concentration index [FICI] < 0.5). Additionally, toxicity of these drugs in combination with AmB was tested in mammalian cells and in zebrafish embryos. Harmless compounds, such as the antibiotic erythromycin, were found to have synergic activity with AmB, not only against C. neoformans but also against some Candida spp., in particular against Candida albicans In parallel, we identified drugs that had antifungal activity against C. neoformans and found 43 drugs that completely inhibited the growth of this fungus, such as ciclopirox and auranofin. Our results expand our knowledge about antifungal compounds and open new perspectives in the treatment of invasive mycosis based on repurposing off-patent drugs., (Copyright © 2020 American Society for Microbiology.)

: The increasing incidence in systemic fungal infections in humans has increased focus for the development of fungal vaccines and use of monoclonal antibodies. Invasive mycoses are generally difficult to treat, as most occur in vulnerable individuals, with compromised innate and adaptive immune responses. Mortality rates in the setting of our current antifungal drugs remain excessively high. Moreover, systemic mycoses require prolonged durations of antifungal treatment and side effects frequently occur, particularly drug-induced liver and/or kidney injury. The use of monoclonal antibodies with or without concomitant administration of antifungal drugs emerges as a potentially efficient treatment modality to improve outcomes and reduce chemotherapy toxicities. In this review, we focus on the use of monoclonal antibodies with experimental evidence on the reduction of fungal burden and prolongation of survival in in vivo disease models. Presently, there are no licensed monoclonal antibodies for use in the treatment of systemic mycoses, although the potential of such a vaccine is very high as indicated by the substantial promising results from several experimental models., Competing Interests: The authors declare no conflict of interest.

Candida auris is an emerging fungal pathogen of great concern among the scientific community because it is causing an increasing number of hospital outbreaks of difficult management worldwide. In addition, isolates from this species frequently present reduced susceptibility to azole and echinocandin drugs. For this reason, it is necessary to develop new antifungal strategies to better control the disease caused by this yeast. In this work, we screened drugs from the Prestwick chemical library, which contains 1,280 off-patent compounds that are already approved by the Food and Drug Administration, with the aim of identifying molecules with antifungal activity against C. auris . In an initial screening, we looked for drugs that inhibited the growth of three different C. auris strains and found 27 of them which it did so. Ten active compounds were selected to test the susceptibility profile by using the EUCAST protocol. Antifungal activity was confirmed for seven drugs with MICs ranging from 0.5 to 64 mg/L. Some of these drugs were also tested in combination with voriconazole and anidulafungin at sub-inhibitory concentrations. Our results suggest synergistic interactions between suloctidil and voriconazole with fractional inhibitory concentration index (FICI) values of 0.11 to 0.5 and between ebselen and anidulafungin (FICI, 0.12 to 0.44). Our findings indicate that drug repurposing could be a viable alternative to managing infections by C. auris .

Cryptococcus neoformans is an encapsulated pathogenic yeast that can change the size of the cells during infection. In particular, this process can occur by enlarging the size of the capsule without modifying the size of the cell body, or by increasing the diameter of the cell body, which is normally accompanied by an increase of the capsule too. This last process leads to the formation of cells of an abnormal enlarged size denominated titan cells. Previous works characterized titan cell formation during pulmonary infection but research on this topic has been hampered due to the difficulty to obtain them in vitro. In this work, we describe in vitro conditions (low nutrient, serum supplemented medium at neutral pH) that promote the transition from regular to titan-like cells. Moreover, addition of azide and static incubation of the cultures in a CO2 enriched atmosphere favored cellular enlargement. This transition occurred at low cell densities, suggesting that the process was regulated by quorum sensing molecules and it was independent of the cryptococcal serotype/species. Transition to titan-like cell was impaired by pharmacological inhibition of PKC signaling pathway. Analysis of the gene expression profile during the transition to titan-like cells showed overexpression of enzymes involved in carbohydrate metabolism, as well as proteins from the coatomer complex, and related to iron metabolism. Indeed, we observed that iron limitation also induced the formation of titan cells. Our gene expression analysis also revealed other elements involved in titan cell formation, such as calnexin, whose absence resulted in appearance of abnormal large cells even in regular rich media. In summary, our work provides a new alternative method to investigate titan cell formation devoid the bioethical problems that involve animal experimentation., Competing Interests: The authors have declared that no competing interests exist.

Cryptococcus neoformans is an environmental encapsulated yeast that behaves as an opportunistic pathogen in immunocompromised individuals. The capsule is the main virulence factor of this pathogen. This structure is highly dynamic, and it can change its size and structure according to the environmental conditions. During infection, C. neoformans significantly enlarges the size of the capsule by the addition of new polysaccharide. It is believed that capsule growth is an energy-cost process, but this aspect has never been addressed. In this work, we have evaluated the role of mitochondrial activity on capsule growth using specific inhibitors of the electron respiratory chain. We observed that capsule growth was impaired in the presence of inhibitors of the respiratory chain as salicylhydroxamic acid or antimycin A. Furthermore, capsule growth correlated with an increase of the mitochondrial membrane potential and higher production of reactive oxygen species. Our results confirm that capsule growth depends on mitochondrial activity, and open new insights to understand the regulation of this process.

Pathogenic fungi have developed many strategies to evade the host immune system. Multiple escape mechanisms appear to function together to inhibit attack by the various stages of both the adaptive and the innate immune response. Thus, after entering the host, such pathogens fight to overcome the immune system to allow their survival, colonization and spread to different sites of infection. Consequently, the establishment of a successful infectious process is closely related to the ability of the pathogen to modulate attack by the immune system. Most strategies employed to subvert or exploit the immune system are shared among different species of fungi. In this review, we summarize the main strategies employed for immune evasion by some of the major pathogenic fungi.

Cryptococcus neoformans and C. gattii are fungal pathogens that are most commonly found in infections of the central nervous system, which cause life-threatening meningoencephalitis and can grow as a biofilm. Biofilms are structures conferring protection and resistance of microorganism to the antifungal drugs. This study compared the virulence of planktonic and biofilm cells of C. neoformans and C. gattii in Galleria mellonella model, as well as, the quantification of gene transcripts LAC1, URE1, and CAP59 by real time PCR. All three of the genes showed significantly increased expressions in the biofilm conditions for two species of Cryptococcus, when compared to planktonic cells. C. neoformans and C. gattii cells in the biofilm forms were more virulent than the planktonic cells in G. mellonella. This suggests that the biofilm conditions may contribute to the virulence profile. Our results contribute to a better understanding of the agents of cryptococcosis in the host-yeast aspects of the interaction.

Cryptococcus sp. are responsible for around 1 million cases of meningitis every year. Fluconazole (FLU) is commonly used in the treatment of cryptococcosis, mainly in immunocompromised patients and the resistance is usually reported after long periods of treatment. In this study, the morphological characterization and virulence profile of FLU-susceptible and FLU-resistant clinical and environmental isolates of C. neoformans and C. gattii were performed both in vitro and in vivo using the Galleria mellonella model. FLU-susceptible isolates from C. neoformans were significantly more virulent than the FLU-resistant isolates. FLU-susceptible C. gattii isolates showed a different virulence profile from C. neoformans isolates where only the environmental isolate, CL, was more virulent compared with the resistant isolates. Cell morphology and capsule size were analyzed and the FLU-resistant isolates did not change significantly compared with the most sensitive isolates. Growth at 37°C was also evaluated and in both species, the resistant isolates showed a reduced growth at this temperature, indicating that FLU resistance can affect their growth. Based on the results obtained is possible suggest that FLU resistance can influence the morphology of the isolates and consequently changed the virulence profiles. The most evident results were observed for C. neoformans showing that the adaptation of isolates to antifungal selective pressure influenced the loss of virulence.

Members of the Paracoccidioides genus are the etiologic agents of paracoccidioidomycosis (PCM). This genus is composed of two species: Paracoccidioides brasiliensis and Paracoccidioides lutzii. The correct molecular taxonomic classification of these fungi has created new opportunities for studying and understanding their relationships with their hosts. Paracoccidioides spp. have features that permit their growth under adverse conditions, enable them to adhere to and invade host tissues and may contribute to disease development. Cell wall proteins called adhesins facilitate adhesion and are capable of mediating fungi-host interactions during infection. This study aimed to evaluate the adhesion profile of two species of the genus Paracoccidioides, to analyze the expression of adhesin-encoding genes by real-time PCR and to relate these results to the virulence of the species, as assessed using a survival curve in mice and in Galleria mellonella after blocking the adhesins. A high level of heterogeneity was observed in adhesion and adhesin expression, showing that the 14-3-3 and enolase molecules are the most highly expressed adhesins during pathogen-host interaction. Additionally, a survival curve revealed a correlation between the adhesion rate and survival, with P. brasiliensis showing higher adhesion and adhesin expression levels and greater virulence when compared with P. lutzii. After blocking 14-3-3 and enolase adhesins, we observed modifications in the virulence of these two species, revealing the importance of these molecules during the pathogenesis of members of the Paracoccidioides genus. These results revealed new insights into the host-pathogen interaction of this genus and may enhance our understanding of different isolates that could be useful for the treatment of this mycosis.

Cryptococcus neoformans is an encapsulated opportunistic fungal pathogen that is found in multiple niches in the environment and that can cause fatal meningoencephalitis in susceptible patients, mainly HIV+ individuals. Cryptococcus also infects environmental hosts such as nematodes, insects and plants. In particular, C. neoformans can kill the lepidopteran Galleria mellonella, which offers a useful tool to study microbial virulence and drug efficacy. Galleria mellonella immunity relies on innate responses based on melanization, accumulation of antimicrobial peptides, and cellular responses as phagocytosis or multicellular encapsulation. In this work we have investigated the immune response of G. mellonella during cryptococcal infection. We found that G. mellonella infected with C. neoformans had a high lytic activity in their hemolymph. This response was temperature- and capsule-dependent. During interaction with phagocytic cells, C. neoformans behaved as an intracellular pathogen since it could replicate within hemocytes. Non-lytic events were also observed. In contrast to Candida species, C. neoformans did not induce melanization of G. mellonella after infection. Finally, passage of C. neoformans through G. mellonella resulted in changes in capsule structure as it has been also reported during infection in mammals. Our results highlight that G. mellonella is an optimal model to investigate innate immune responses against C. neoformans.

This work aims to demonstrate that the gallic acid structure modification to the decyl gallate (G14) compound contributed to increase the antifungal activity against several species of pathogenic fungi, mainly, Candida spp., Cryptococcus spp., Paracoccidioides spp., and Histoplasma capsulatum, according to standardized microdilution method described by Clinical Laboratory Standard Institute (CLSI) documents. Moreover this compound has a particularly good selectivity index value, which makes it an excellent candidate for broad-spectrum antifungal prototype and encourages the continuation of subsequent studies for the discovery of its mechanism of action.

Cryptococcosis is an important systemic mycosis and the third most prevalent disease in human immunodeficiency virus (HIV)-positive individuals. The incidence of cryptococcosis is high among the 25 million people with HIV/acquired immunodeficiency syndrome (AIDS), with recent estimates indicating that there are one million cases of cryptococcal meningitis globally per year in AIDS patients. In Cryptococcus neoformans, resistance to azoles may be associated with alterations in the target enzyme encoded by the gene ERG11, lanosterol 14α-demethylase. These alterations are obtained through mutations, or by overexpressing the gene encoding. In addition, C. gattii and C. neoformans present a heteroresistance phenotype, which may be related to increased virulence. Other species beyond C. neoformans and C. gattii, such as C. laurentii, have been diagnosed mainly in patients with immunosuppression. Infections of C. albidus have been isolated in cats and marine mammals. Recent evidence suggests that the majority of infections produced by this pathogen are associated with biofilm growth, which is also related with increased resistance to antifungal agents. Therefore, there is a great need to search for alternative antifungal agents for these fungi. The search for new molecules is currently occurring from nanoparticle drugs of plant peptide origin. This article presents a brief review of the literature regarding the epidemiology of cryptococcosis, as well as fungal resistance and new alternatives for treatment.

For the first time, a general catalytic procedure for the cross-coupling of primary amides and alkylboronic acids is demonstrated. The key to the success of this reaction was the identification of a mild base (NaOSiMe3) and oxidant (di-tert-butyl peroxide) to promote the copper-catalyzed reaction in high yield. This transformation provides a facile, high-yielding method for the monoalkylation of amides.

Fungal infections in humans have increased alarmingly in recent years, particularly in immunocompromised individuals. Among the infections systemic candidiasis, aspergillosis, cryptococcosis, paracoccidioidomycosis, and histoplasmosis mortality are more prevalent and more severe in humans. The current high incidence of dermatophytosis is in humans, especially as the main etiologic agents Trichophyton rubrum and Trichophyton mentagrophytes. Molecules pristimerin and maytenin obtained from the plant Maytenus ilicifolia (Celastraceae) are known to show various pharmacological activities. This study aimed to evaluate the spectrum of antifungal activity of maytenin and pristimerin and their cytotoxicity in human keratinocytes (NOK cells of the oral mucosa). It was concluded that the best spectrum of antifungal activity has been shown to maytenin with MIC varying from 0.12 to 125 mg/L, although it is also active with pristimerin MIC ranging between 0.12 and 250 mg/L. Regarding the toxicity, both showed to have high IC(50). The SI showed high pristimerin against some species of fungi, but SI maytenin was above 1.0 for all fungi tested, showing a selective action of fungi. However, when comparing the two substances, maytenin also showed better results. The two molecules can be a possible prototype with a broad spectrum of action for the development of new antifungal agents.

To evaluate a potential tool for assessing the risk of a pathologic fracture of the femoral shaft, we examined whether fracture loads computed by our computed tomography scan-based finite element models are predictive of measured fracture loads. We also evaluated whether the precision of the computed fracture loads for shafts with metastases is altered if models are generated using mechanical property-density relationships for bone without metastases. We investigated whether femoral shafts with a hemispheric defect and shafts with metastases have qualitatively similar structural behavior. Using identical four-point bending loading conditions, we computed and measured fracture loads of femoral shafts with and without metastases and with a burred hemispheric defect to simulate a tumor. Finite element model fracture loads were strongly predictive of the measured fracture loads (range, 0.92-0.98) even when the models of bones with metastases used mechanical property relationships for bone without metastases. Specimens with hemispheric defects behaved structurally differently than specimens with metastases, indicating that these defects do not accurately simulate the effects of metastases. Results of our study show that these computed tomography scan-based finite element models can be used to estimate the strength of femoral shafts with and without metastases. These models may be useful for assessing the risk of pathologic fractures of femoral shafts.

Finite element (FE) models of the proximal femur are often used to study hip fracture. To interpret the results of these models, it is important to know whether the models accurately predict fracture location and/or type. This study evaluated the ability of automatically generated, CT scan-based linear FE models of the proximal femur to predict fracture location and fracture type. Fracture location was defined as the specific location of the fracture. Fracture type was a categorical variable defined as either a cervical or a trochanteric fracture. FE modeling and mechanical testing of 18 pairs of human femora were performed under two loading conditions, one similar to joint loading during single-limb stance and one simulating impact from a fall. For the stance condition, the predicted and actual fracture locations agreed in 13 of the 18 cases (72% agreement). For the fall condition, the predicted and actual fracture locations agreed in 10 of the 15 cases where the actual fractures could be identified (67% agreement). The FE models correctly predicted that only cervical fractures occurred in the stance configuration. For the fall configuration, FE-predicted and actual fracture types agreed in 11 of the 14 cases that could be compared (9 trochanteric, 2 cervical; 79% agreement). These results provide evidence that CT scan-based FE models of the proximal femur can predict fracture location and fracture type with moderate accuracy.

There is a need for a small, simple, and versatile intracorporeal ventricular assist device (IVAD) as an alternative to the large implantable electromechanical LVAD systems in current use. Because the basic design of the Thoratec paracorporeal VAD has been demonstrated in over 1,000 patients, weighing from 17 to 144 kg, and for durations up to 515 days including patient discharge (by using the portable driver), we are developing a new intracorporeal version of our VAD. This IVAD has a smooth contoured, polished titanium housing, and maintains the same blood flow path and Thoralon polyurethane blood pumping sac as the paracorporeal VAD. The IVAD is controlled with the Thoratec TLC-II Portable VAD Driver, which is a small briefcase sized, battery powered, pneumatic control unit. Intracorporeal LVADs and/or RVADs are implanted in a preperitoneal position, with a single small (9 mm OD) percutaneous pneumatic driveline for each VAD. The major advantages of the new IVAD design are size and simplicity. The IVAD weight (339 g) and implanted volume (252 ml) are substantially smaller than current implantable electromechanical LVAD systems. Only the small blood pump is implanted, leaving the more complex control unit external, where it can be serviced and replaced. The versatile design is intended for left and/or right heart support in large or small patients. The IVAD in combination with the TLC-II portable driver will be a viable and attractive alternative to large, implanted electromechanical systems.

Finite element (FE) models are often used to model bone failure. However, no failure theory for bone has been validated at this time. In this study, we examined the performance of nine stress- and strain-based failure theories, six of which could account for differences in tensile and compressive material strengths. The distortion energy, Hoffman and a strain-based Hoffman analog, maximum normal stress, maximum normal strain, maximum shear strain, maximum shear stress (tau(max)), Coulomb-Mohr, and modified Mohr failure theories were evaluated using automatically generated, computed tomographic scan-based FE models of the femur. Eighteen matched pairs of proximal femora were examined in two load configurations, one approximating joint loading during single-limb stance and one simulating impact from a fall. Mechanical testing was performed to assess model and failure theory performance in the context of predicting femoral fracture load. Measured and FE-computed fracture load were significantly correlated for both loading conditions and all failure criteria (p < or = 0.001). The distortion energy and tau(max) failure theories were the most robust of those examined, providing the most consistently strong FE model performance for two very different loading conditions. The more complex failure theories and the strain-based theories examined did not improve performance over the simpler distortion energy and tau(max) theories, and often degraded performance, even when differences between tensile and compressive failure properties were represented. The relatively strong performance of the distortion energy and tau(max) theories supports the hypothesis that shear/distortion is an important failure mode during femoral fracture.

Hip fracture is an important cause of morbidity and mortality among the elderly. Current methods of assessing a patient's risk of hip fracture involve local estimates of bone density (densitometry), and are limited by their inability to account for the complex structural features of the femur. In an effort to improve clinical and research tools for assessing hip fracture risk, this study investigated whether automatically generated, computed tomographic (CT) scan-based finite element (FE) models can be used to estimate femoral fracture load in vitro. Eighteen pairs of femora were examined under two loading conditions one similar to loading during the stance phase of gait, and one simulating impact from a fall. The femora were then mechanically tested to failure and regression analyses between measured fracture load and FE-predicted fracture load were performed. For comparison, densitometry measures were also examined. Significant relationships were found between measured fracture load and FE-predicted fracture load (r = 0.87, stance; r = 0.95, fall; r = 0.97, stance and fall data pooled) and between measured fracture load and densitometry data (r = 0.78, stance; r = 0.91, fall). These results indicate that this sophisticated technique, which is still early in its development, can achieve precision comparable to that of densitometry and can predict femoral fracture load to within -40% to +60% with 95% confidence. Therefore, clinical use of this approach, which would require additional X-ray exposure and expenditure for a CT scan, is not justified at this point. Even so, the potential advantages of this CT/FE technique support further research in this area.

The purpose of this study was to characterize gait initiation of persons with leg amputation and determine whether prosthetic alignment was a critical parameter in the initiation process. Gait initiation was chosen for study because of the difficult neuromuscular demands placed on the body in negotiating the transition from stance to ambulation. In this investigation, ground reaction force data were collected on seven persons with below-knee amputation. These subjects underwent a series of gait initiation trials while varying prosthetic alignment. An analysis of the data demonstrated key elements in the gait initiation process, including the motion of the center of gravity in preparation for steady-state walking. Significant asymmetries in the force profiles of the residual and nonamputated limbs were also found; gait initiation forces were consistently higher for the prosthetic limb and the timings of maxima and minima were indicative of an intact limb preference. Relatively small changes in prosthesis alignment proved not to have statistically significant effects on generalized force parameters. This result is consistent with the findings of other studies that gait initiation is a motor program with certain invariant characteristics.

Short-column (3.5 m) gas chromatography (GC)/tandem mass spectrometry (MS/MS) has been investigated for the detection of structurally related, underivatized anabolic steroids in urine. The approach described here demonstrates the ability to rapidly and qualitatively detect underivatized anabolic steroids in spiked urine matrices. In this approach, underivatized steroids are determined using a short-column GC separation, ionized by positive ion chemical ionization, and detected by selected reaction monitoring MS/MS. This approach permits positive identification of underivatized anabolic steroids based on retention time and the production of characteristic product ions. Preliminary detection limits studies in spiked urine samples showed quantitative results between 2 and 40 ng steroid per milliliter of uterine. The potential advantages of this approach compared to present screening methods based on conventional (30 m) GC/MS are its rapidity and selectivity. Reliable qualitative identification can be performed with a short-column GC/MS/MS analysis of less than 6 min with a reduction in sample preparation time due to the elimination of the derivatization step.

A gas chromatographic/mass spectrometric method based on the use of short capillary gas chromatograph columns (3-5 m) and electron ionization mass spectrometry has been optimized and evaluated for the determination of underivatized anabolic steroids. The short-column gas chromatographic/mass spectrometric method was shown to result in short analysis times and to require minimal sample preparation, but suffered from some loss in sensitivity and chromatographic resolution compared with conventional gas chromatographic/mass spectrometric techniques for derivatized steroids. Therefore, short-column gas chromatographic conditions were optimized to maximize the sample transfer efficiency (sensitivity) from the gas chromatograph into the ion source of the mass spectrometer, while maintaining chromatographic integrity and minimizing thermal decomposition. Mass spectrometric conditions were optimized to maximize ionization efficiency with respect to the intensity of the molecular ion and degree of fragmentation such that positive identification of each steroid could be made based on the resulting mass spectra. Under optimized conditions, we have shown that underivatized anabolic steroids spiked into urine samples can be determined at low-nanogram levels using short-column chromatography/full-scan electron ionization mass spectrometry.