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Kawasaki disease (KD) is a vasculitis of the middle and little caliber arteries that specifically affects the coronary arteries. It represents the main cause of acquired cardiopathy in the pediatric population of western countries and in 25% of cases it leads to coronary artery aneurism (1). Atypical KD is nowadays considered a challenge for pediatrics, due to all of its possible clinical manifestations: it is characterized by a long-lasting fever (5 or more days) associated with some of the typical signs and symptoms and other unusual clinical manifestations (as neurological, nephrological, respiratory, gastrointestinal, rheumatological manifestations), variably associated with coronary arteries aneurism. Early diagnosis and treatment are essential to reduce the risk of coronary artery aneurysms (2,3). We report the case of a 5-year-old boy who came to our attention for skin rash, abdominal pain, increased liver enzymes, and hydrops of the gallbladder. He presented a fever for less than 5 days. 2 weeks later, the appearance of peeling on the fingers and the toes confirmed the diagnosis of an atypical, incomplete KD.

Pediatric ear, nose, and throat (ENT) surgery is very common, and its outcomes may improve with family education. In this regard, mobile health (mHealth) applications (apps), which are on the rise due to digital transformation, can be beneficial in healthcare. This study outlines the user-centered design and development of a mHealth app (version 5.15.0) to support family caregivers during the perioperative process of pediatric ENT surgery. Conducted over two years in an Italian maternal and child health hospital (January 2020–May 2022), the study employed a participatory design method based on the Information System Research (ISR) framework and guided by the principles of Slow Medicine. Utilizing the Relevance, Rigor, and Design cycles of the ISR framework, the mHealth app’s content, functionalities, and technical features were defined and developed. A committee of fifteen experts guided the process with input from 25 family caregivers and 24 healthcare providers enrolled in the study. The mHealth app content was structured around five crucial educational moments characterizing the ENT perioperative period, providing evidence-based information on surgical procedures, strategies for preparing children for hospitalization and surgery, pain management, and post-discharge care. The mHealth app featured a function that sends customized notifications to guide caregivers at specific perioperative stages. The development of mHealth apps by implementing a rigorous, participatory, and Slow design process can foster accessible and family-centered information and care in the field of maternal and child health and beyond.

BackgroundSeveral attempts have been made to introduce mHealth solutions to support maternal, newborn, and child health (MNCH). However, most of the available apps do not meet the needs of end-users, underscoring the urgency of involving them in the co-design of telehealth interventions.ObjectiveWith this in mind, we investigated the needs and expectations of hospital-based health professionals (i.e., secondary users) providing care to pregnant women and new mothers with their babies for a hypothetical mHealth app to support the first 1000 days of life.MethodsFrom November 2021 to March 2022, we surveyed health professionals using a questionnaire that explored the perceived importance of specific content, functionalities, and technical features of the proposed app. We also collected sociodemographic information from secondary users. We performed descriptive analysis and then used Ward hierarchical clustering method to classify respondents according to their response patterns.ResultsWe recorded the needs and expectations of 145 hospital-based health professionals from obstetrics/gynecology, nursery/neonatology, and pediatrics. We found general agreement with the proposed content of the app, particularly general information about health during pregnancy (92%) and potential risky infections during pregnancy (91%). Three clusters emerged from the analysis, with the high and medium demanding clusters rating the importance of app content and technical features as very high and high, respectively, while low demanding cluster expressing more skepticism, especially about some of the proposed functionalities of the app.ConclusionsAssessing the needs and expectations of end-users is an essential process for developing tailored and effective mHealth solutions. This study has shown that hospital-based health professionals generally recognize the value of the proposed app, suggesting their propensity to integrate such a telehealth solution into mainstream clinical practice.

Objectives Evidence showed that, even in high-income countries, children and adolescents may not receive high quality of care (QOC). We describe the development and initial validation, in Italy, of two WHO standards-based questionnaires to conduct an assessment of QOC for children and young adolescents at inpatient level, based on the provider and user perspectives.Design Multiphase, mixed-methods study.Setting, participants and methods The two questionnaires were developed in four phases equally conducted for each tool. Phase 1 which included the prioritisation of the WHO Quality Measures according to predefined criteria and the development of the draft questionnaires. In phase 2 content face validation of the draft questionnaires was assessed among both experts and end-users. In phase 3 the optimised questionnaires were field tested to assess acceptability, perceived utility and comprehensiveness (N=163 end-users). In phase 4 intrarater reliability and internal consistency were evaluated (N=170 and N=301 end-users, respectively).Results The final questionnaires included 150 WHO Quality Measures. Observed face validity was excellent (kappa value of 1). The field test resulted in response rates of 98% and 76% for service users and health providers, respectively. Among respondents, 96.9% service users and 90.4% providers rated the questionnaires as useful, and 86.9% and 93.9%, respectively rated them as comprehensive. Intrarater reliability was good, with Cohen’s kappa values exceeding 0.70. Cronbach alpha values ranged from 0.83 to 0.95, indicating excellent internal consistency.Conclusions Study findings suggest these tools developed have good content and face validity, high acceptability and perceived utility, and good intrarater reliability and internal consistency, and therefore could be used in health facilities in Italy and similar contexts. Priority areas for future research include how tools measuring paediatric QOC can be more effectively used to help health professionals provide the best possible care.

About 5000 people in Italy suffer from hemophilia, the most common coagulation disorder. As for other chronic diseases, even in the case of hemophilia, the engagement of the patient is essential: the patients, in fact, must be empowered and helped to become strong partners of the care team and sensitized with respect to their rights and duties for the successful achievement of the goals set by their healthcare path. Hence the initiative to start a new research-intervention project in the field of hemophilia. The study had different phases of research: a first moment inspired by the principles of narrative medicine, aimed at collecting stories and narratives of patients with hemophilia related to the experience of the disease and therapy and expectations of active involvement in the relationship with the clinician. At the same time, among hematologists and patients has been surveyed the experience of therapeutical relationship and communication, to capture the aspects in which they feel effective and the areas of improvement and unmet needs. Subsequently, a workshop dedicated to patients and hematologists was organized to foster mutual awareness between these two targets and the formation of a better communication and relational skills of clinicians. The results of the project formed the basis for a policy brief document, aimed at disseminating recommendations to support better relationship and empathic communication between clinicians and patients.

Objective The aim of this retrospective observational study is to analyse clinical, serological and radiological predictors of outcome in patients with COVID-19 pneumonia treated with tocilizumab, providing clinical guidance to its use in real-life. Method This is a retrospective, monocentric observational cohort study. All consecutive patients hospitalized between February the 11th and April 14th 2020 for severe COVID-19 pneumonia at Reggio Emilia AUSL and treated with tocilizumab were enrolled. The patient’s clinical status was recorded every day using the WHO ordinal scale for clinical improvement. Response to treatment was defined as an improvement of one point (from the status at the beginning of tocilizumab treatment) during the follow-up on this scale. Bivariate association of main patients’ characteristics with outcomes was explored by descriptive statistics and Fisher or Kruskal Wallis tests (respectively for qualitative or quantitative variables). Each clinically significant predictor was checked by a loglikelihood ratio test (in univariate logistic models for each of the considered outcomes) against the null model. Results A total of 173 patients were included. Only hypertension, the use of angiotensin-converting enzyme inhibitors, PaO2/FiO2, respiratory rate and C-reactive protein were selected for the multivariate analysis. In the multivariable model, none of them was significantly associated with response. Conclusions Evaluating a large number of clinical variables, our study did not find new predictors of outcome in COVID19 patients treated with tocilizumab. Further studies are needed to investigate the use of tocilizumab in COVID-19 and to better identify clinical phenotypes which could benefit from this treatment.

ObjectivesWe aimed to identify clinical, anamnestic, and sociodemographic characteristics associated with a positive swab for SARS-CoV2, and to provide a predictive score to identify at risk population in children aged 2-14 years attending school and tested for clinical symptoms of COVID-19.DesignCross sectional study.SettingOutpatient clinic of the IRCCS Burlo Garofolo, a maternal and child health tertiary care hospital and research centre in Italy.Data collection and analysisData were collected through a predefined form, filled out by parents, and gathered information on sociodemographic characteristics, and specific symptoms, which were analysed to determine their association with a positive SARS-CoV-2 swab. The regression coefficients of the variables included in the multivariate analysis were further used in the calculation of a predictive score of the positive or negative test.ResultsBetween September 20th and December 23rd 2020, from 1484 children included in the study, 127 (8.6%) tested positive. In the multivariate analysis, the variables retained by the model were the presence of contact with a cohabiting, non-cohabiting or unspecified symptomatic case (respectively OR 37.2, 95% CI 20.1-68.7; 5.1, 95% CI 2.7-9.6; 15.6, 95% CI 7.3-33.2); female sex (OR 1.49, 95% CI 1.0-2.3); age (6-10 years old: OR 3.2, 95% CI 1.7-6.1 p10 years old: OR 4.8, 95% CI 2.7-8.8 pConclusionsThe timely identification of SARS-CoV2 cases among children is useful to reduce the dissemination of the disease and its related burden. The predictive score may be adopted in a public health perspective to rapidly identify at risk children.

In this study, incorporation of the cytotoxic agent paclitaxel and the P-glycoprotein inhibitor elacridar in hyaluronic acid (HA)-modified nanoemulsions was studied for intraductal delivery and breast cancer localized treatment. To improve cytotoxicity, we investigated the incorporation of perillyl alcohol or tributyrin as components of the nanoemulsion oil phase. The nanoemulsions presented size 50 by 1.6 to 3-fold in MCF-7 and MDA-MB-231 cells compared to the nanoemulsion containing only paclitaxel (P-NE). This nanoemulsion also produced a 3.3-fold reduction in the viability of MDA-MB-231 spheroids. Elacridar incorporated in the nanoemulsion was capable of inhibiting P-glycoprotein in membranes. In vivo intraductal administration of the NE containing HA resulted in a three-fold higher retention of a fluorescent marker compared to a solution or nanoemulsion without HA, demonstrating the importance of HA. The nanoemulsion produced no histological changes in the mammary tissue. These results support the potential applicability of the nanoemulsion for local breast cancer management.

An analysis of the structural effect of the mutations of the B.1.640.2 (IHU) Spike Receptor Binding Domain (RBD) and N-terminal Domain (NTD) is reported along with a comparison with the sister lineage B.1.640.1. and a selection of variants of concern. The effect of the mutations on the RBD–ACE2 interaction was also assessed. The structural analysis applied computational methods that are able to carry out in silico mutagenesis to calculate energy minimization and the folding energy variation consequent to residue mutations. Tools for electrostatic calculation were applied to quantify and display the protein surface electrostatic potential. Interactions at the RBD–ACE2 interface were scrutinized using computational tools that identify the interactions and predict the contribution of each interface residue to the stability of the complex. The comparison among the RBDs shows that the most evident differences between the variants is in the distribution of the surface electrostatic potential: that of B.1.640.1 is as that of the Alpha RBD, while B.1.640.2 appears to have an intermediate surface potential pattern with characteristics between those of the Alpha and Delta variants. Moreover, the B.1.640.2 Spike includes the mutation E484K that in other variants has been suggested to be involved in immune evasion. These properties may hint at the possibility that B.1.640.2 emerged with a potentially increased infectivity with respect to the sister B.1.640.1 variant, but significantly lower than that of the Delta and Omicron variants. However, the analysis of their NTD domains highlights deletions, destabilizing mutations and charge alterations that can limit the ability of the B.1.640.1 and B.1.640.2 variants to interact with cellular components, such as cell surface receptors.

We assessed the impact of chest CT body composition parameters on outcomes and disease severity at hospital presentation of COVID-19 patients, focusing also on the possible mediation of body composition in the relationship between age and death in these patients. Chest CT scans performed at hospital presentation by consecutive COVID-19 patients (02/27/2020-03/13/2020) were retrospectively reviewed to obtain pectoralis muscle density and total, visceral, and intermuscular adipose tissue areas (TAT, VAT, IMAT) at the level of T7-T8 vertebrae. Primary outcomes were: hospitalization, mechanical ventilation (MV) and/or death, death alone. Secondary outcomes were: C-reactive protein (CRP), oxygen saturation (SO2), CT disease extension at hospital presentation. The mediation of body composition in the effect of age on death was explored. Of the 318 patients included in the study (median age 65.7 years, females 37.7%), 205 (64.5%) were hospitalized, 68 (21.4%) needed MV, and 58 (18.2%) died. Increased muscle density was a protective factor while increased TAT, VAT, and IMAT were risk factors for hospitalization and MV/death. All these parameters except TAT had borderline effects on death alone. All parameters were associated with SO2 and extension of lung parenchymal involvement at CT; VAT was associated with CRP. Approximately 3% of the effect of age on death was mediated by decreased muscle density. In conclusion, low muscle quality and ectopic fat accumulation were associated with COVID-19 outcomes, VAT was associated with baseline inflammation. Low muscle quality partly mediated the effect of age on mortality.

Rheumatoid arthritis (RA) is a chronic systemic inflammatory disease characterized by chronic symmetrical erosive synovitis and extra-articular manifestations, including interstitial lung disease (ILD), whose treatment is nowadays challenging due to high infectious risk and possible pulmonary iatrogenic toxicity. Janus kinase inhibitors, namely, tofacitinib, baricitinib, and upadacitinib, are the latest drug class for the treatment of RA with a good safety profile. We present the case of a patient with RA-ILD successfully treated with tofacitinib. A 52-year-old man was referred to our multidisciplinary clinic for rheumatic and pulmonary diseases for an active erosive seropositive RA and progressive ILD. Previous treatments were GC, hydroxychloroquine, methotrexate, etanercept, withdrawn after ILD detection, and tocilizumab, discontinued due to relapsing infections. After our evaluation, we proposed rituximab in addition to low-dose GC and hydroxychloroquine, ineffective on joint involvement. Therefore, we proposed tofacitinib which allowed us to control joint involvement, stabilize ILD improving respiratory symptoms, and manage the frequent infectious episodes that occurred initially. The short half-life and rapid-acting of tofacitinib are two helpful characteristics regarding this aspect. Despite limited data from randomized trials and real-life, tofacitinib could represent a safe therapeutic option for RA-ILD patients. Longitudinal studies are required to confirm this encouraging report.

Rheumatoid arthritis (RA) is a chronic, systemic, inflammatory disease characterized by joint and extra-articular involvement. Among them, interstitial lung disease (ILD) is one of the most common and severe extra-articular manifestations, with a negative impact on both therapeutic approach and overall prognosis. ILD can occur at any point of the natural history of RA, sometimes before the appearance of joint involvement. Since no controlled studies are available, the therapeutic approach to RA-ILD is still debated and based on empirical approaches dependent on retrospective studies and case series. Here, we report the case of a 75-year-old patient affected by RA complicated by ILD successfully treated with a combination therapy of an antifibrotic agent, nintedanib, and an inhibitor of IL-6 receptor, sarilumab. We obtained a sustained remission of the joint involvement and, simultaneously, a stabilization of the respiratory symptoms and function, with a good safety profile. To date, this is the first report describing a combination therapy with nintedanib and a disease-modifying antirheumatic drug (DMARD) for the management of RA complicated by ILD. Future prospective studies are needed to better define efficacy and safety of this approach in the treatment of these subjects.

Abstract The expression of host inflammatory and Candida albicans putative virulence factors was studied in women with vulvovaginal candidiasis (VVC; twenty) or colonized by the fungus but asymptomatic (carriers; fifteen) or non-colonized asymptomatic (ten subjects). Overexpression of genes encoding NLRP3 and caspase-1 inflammasome components sharply differentiated VVC patients from asymptomatic colonized or non-colonized women. Inflammasome expression was coupled with neutrophils recruitment in the vagina of VVC women and IL-1β and IL-8 production. Both cytokines were present, though to a lower concentration, also in the vaginal fluid of colonized and non-colonized women. Secretory aspartyl proteinases (SAPs) and hyphae associated genes HWP1 and ECE1 were upregulated in VVC but with some differences among infected women. The most overexpressed SAP gene was SAP2, that correlated with neutrophils accumulation. Our data provide clinical evidence that the intracytoplasmic activation of NLRP3 inflammasome complex plays a critical, pathogenesis-relevant role in human VVC.

Abstract Methanol, the simplest alcohol, and dimethyl ether, the simplest ether, are central compounds in the search for alternative “green” combustion fuels. In fact, they are generally considered as the cornerstones of the envisaged “Methanol Economy” scenario, as they are able to efficiently produce energy in an environmentally friendly manner. However, despite a massive amount of research in this field, the synthesis of dimethyl ether from liquid methanol has never so far been reported. Here we present a computational study, based on ab initio Molecular Dynamics, which suggests a novel synthesis route to methanol dehydration – leading thus to the dimethyl ether synthesis – through the application of strong electric fields. Besides proving the impressive catalytic effects afforded by the field, our calculations indicate that the obtained dimethyl ether is stable and that it can be progressively accumulated thanks to the peculiar chemical pathways characterising the methanol reaction network under electric field. These results suggest that the experimental synthesis of dimethyl ether from liquid methanol could be achieved, possibly in the proximity of field emitter tips.

Abstract Sera from candidemic and non-candidemic subjects were examined for antibodies against the cell wall β1,3- and β1,6-glucans, as well as the β-glucan-associated protein MP65 of Candida species. Although antibodies against each of the above components were detected in all subjects, candidemic patients had lower antibody titers against β1,3-glucan, but higher antibody titers against β1,6-glucan and MP65, than non-candidemic subjects. The elevated levels of anti-β1,6-glucan and -MP65 antibodies found in candidemic patients were independent on the patient risk category, APACHE II score, presence of co-morbidities, β1,3-glucanemia level, Candida isolate, and antifungal treatment. Interestingly, however, the anti-MP65, but not the anti-β1,6-glucan antibodies, of candidemic patients had higher titers in survivors than in non-survivors, particularly in those subject categories with the highest mortality (>65-years old, diabetic, or septic shock patients). Thus, candidemic patients are capable of boosting anti-Candida immune responses upon infection, and some of these responses might be associated to the generation of protective immunity in patients with candidemia.

OBJECTIVES/GOALS: We investigated the association between gut microbiota features in newly admitted nursing facility (NF) patients and the acquisition of vancomycin-resistant Enterococcus (VRE) and/or resistant Gram-negative bacteria (rGNB) within 14 days. METHODS/STUDY POPULATION: Patients were recruited at 6 Michigan NFs from 09/16-08/18. VRE or rGNB colonization status was determined by culture swabs collected from multiple body sites at enrolment, day 7, and day 14. Our analysis focused on patients with no colonization at baseline, a perirectal swab collected at baseline, and at least one follow-up visit. The V4 hypervariable region of the 16S rRNA gene from bacterial DNA in each sample was PCR-amplified and sequenced on the MiSeq platform. Sequencing results were then processed with the mothur bioinformatics pipeline to classify bacterial taxa present in each sample. Taxa typically associated with the skin microbiota were removed. The primary outcome was acquisition of VRE and/or rGNB within 14 days. Exposures of interest included patient and microbiota characteristics. RESULTS/ANTICIPATED RESULTS: Among 61 patients, 18 (30%) acquired AROs within 14 days of enrolment (3 VRE, 13 rGNB, 2 both) (Table 1). The baseline microbiota features differed significantly in those who acquired a new ARO. Of the major 8 phyla found across samples, patients who acquired an ARO were depleted in the number of phyla present (5.74 ± 1.20 vs 5.06 ± 1.43; p = 0.037) (Fig. 1). The log10-transformed relative abundance of Enterococcus was enriched in patients who acquired an ARO (−0.32 ± 1.47) compared to those who did not (−1.68 ± 1.76; p = 0.021) (Fig. 2). Patients who did not acquire an ARO tended to harbour more butyrate-producing bacterial taxa and strict anaerobes, although the differences were not statistically significant (relative abundance of butyrate producer: 29.49 ± 22.09 vs 22.05 ± 17.76; anaerobes: 64.78 ± 23.54 vs 53.68 ± 27.61). DISCUSSION/SIGNIFICANCE OF IMPACT: Microbiota metrics calculated from perirectal samples are predictive of ARO acquisition. The clinical utility of perirectal samples thus warrants further assessment.

A 57-year-old woman with a diagnosis of antisynthetase syndrome (ASSD) underwent a nailfold videocapillaroscopy (NVC) showing a scleroderma pattern. Alterations in capillary morphology have been reported in adults with inflammatory myositis (IM) but only recently have the differences in NVC findings between these two diseases been established. ASSD is currently classified as a subset of IM, for which reason only a few studies in literature evaluate its specific hallmarks, showing nonspecific features of NVC in patients with polymyositis and dermatomyositis (DM) and antisynthetase antibodies. To our knowledge, this is the first description of ASSD capillaroscopy features, and the first report of NVC in ASSD with evidence of scleroderma pattern. Further studies are needed to define clearly frequency, typical features, and possible correlation with clinical and serological data of NVC changes in ASSD, differences between microangiopathy in ASSD and systemic sclerosis or DM.

For nearly 400 million years, insects and plants have been embattled in an evolutionary arms race. Insects have developed diverse feeding strategies and behaviors in an effort to circumvent and overcome an extensive collection of plant defense tactics. Sap-sucking insects often inject saliva into hosts plants, which contains a suite of effector proteins and even microbial communities that can alter the plant's defenses. Lacking salivary glands, leaf-feeding beetles represent an interesting group of phytophagous insects. Feeding beetles regurgitate onto leaf surfaces and it is thought that these oral secretions influence insect-plant interactions and even play a role in virus-vector specificity. Since the molecular and biological makeup of the regurgitant is virtually unknown, we carried out RNA sequencing and 16S rDNA analysis on a major soybean pest, Epilachna varivestis, to generate the first ever beetle "regurgitome" and characterize its microbiome. Interestingly, the regurgitant is comprised of a rich molecular assortment of genes encoding putative extracellular proteins involved in digestion, molting, immune defense, and detoxification. By carrying out plant inoculation assays, we reinforced the fundamental role of the regurgitant in beetle-borne virus specificity. Ultimately, these studies begin to characterize the importance of regurgitant in virus transmission and beetle-plant interactions.

In birds, seasonal reproduction is regulated by day length, with long days in the spring activating the hypothalamic-pituitary-gonadal axis and reproductive behaviors. The photoreceptors mediating this process remain unknown, but recently, the premammillary nucleus (PMM) of the hypothalamus has been implicated as the site of photoperiodic signaling in turkeys. We performed electrolytic lesions of the PMM to elucidate its role in the photoactivation and maintenance of egg production in female turkeys. Our results show that ablation of the PMM does not alter the normal lay cycle. No differences were found between lesioned birds and sham controls in the latency to lay following photostimulation, nor in subsequent egg production over a period of 29 weeks. No differences in the incidence of gonadal regression were found, indicating that the PMM is not essential for the termination of breeding. We conclude that any role of the PMM in photoperiodic regulation, if it exists, is redundant with other components of the system.

Background. In adult female mosquitoes, the renal (Malpighian) tubules play an important role in the post-prandial diuresis, which removes excess ions and water from the hemolymph of mosquitoes following a blood meal. After the post-prandial diuresis, the roles that Malpighian tubules play in the processing of blood meals are not well described. Methods. We used a combination of next-generation sequencing (paired-end RNA sequencing) and physiological/biochemical assays in adult female Asian tiger mosquitoes (Aedes albopictus) to generate molecular and functional insights into the Malpighian tubules and how they may contribute to blood meal processing (3–24 h after blood ingestion). Results/Discussion. Using RNA sequencing, we sequenced and assembled the first de novo transcriptome of Malpighian tubules from non-blood-fed (NBF) and blood-fed (BF) mosquitoes. We identified a total of 8,232 non-redundant transcripts. The Malpighian tubules of NBF mosquitoes were characterized by the expression of transcripts associated with active transepithelial fluid secretion/diuresis (e.g., ion transporters, water channels, V-type H+-ATPase subunits), xenobiotic detoxification (e.g., cytochrome P450 monoxygenases, glutathione S-transferases, ATP-binding cassette transporters), and purine metabolism (e.g., xanthine dehydrogenase). We also detected the expression of transcripts encoding sodium calcium exchangers, G protein coupled-receptors, and septate junctional proteins not previously described in mosquito Malpighian tubules. Within 24 h after a blood meal, transcripts associated with active transepithelial fluid secretion/diuresis exhibited a general downregulation, whereas those associated with xenobiotic detoxification and purine catabolism exhibited a general upregulation, suggesting a reinvestment of the Malpighian tubules’ molecular resources from diuresis to detoxification. Physiological and biochemical assays were conducted in mosquitoes and isolated Malpighian tubules, respectively, to confirm that the transcriptomic changes were associated with functional consequences. In particular, in vivo diuresis assays demonstrated that adult female mosquitoes have a reduced diuretic capacity within 24 h after a blood meal. Moreover, biochemical assays in isolated Malpighian tubules showed an increase in glutathione S-transferase activity and the accumulation of uric acid (an end product of purine catabolism) within 24 h after a blood meal. Our data provide new insights into the molecular physiology of Malpighian tubules in culicine mosquitoes and reveal potentially important molecular targets for the development of chemical and/or gene-silencing insecticides that would disrupt renal function in mosquitoes.

Circadian rhythms are fundamental properties of most eukaryotes, but evidence of biological clocks that drive these rhythms in prokaryotes has been restricted to Cyanobacteria. In vertebrates, the gastrointestinal system expresses circadian patterns of gene expression, motility and secretion in vivo and in vitro, and recent studies suggest that the enteric microbiome is regulated by the host's circadian clock. However, it is not clear how the host's clock regulates the microbiome. Here, we demonstrate at least one species of commensal bacterium from the human gastrointestinal system, Enterobacter aerogenes, is sensitive to the neurohormone melatonin, which is secreted into the gastrointestinal lumen, and expresses circadian patterns of swarming and motility. Melatonin specifically increases the magnitude of swarming in cultures of E. aerogenes, but not in Escherichia coli or Klebsiella pneumoniae. The swarming appears to occur daily, and transformation of E. aerogenes with a flagellar motor-protein driven lux plasmid confirms a temperature-compensated circadian rhythm of luciferase activity, which is synchronized in the presence of melatonin. Altogether, these data demonstrate a circadian clock in a non-cyanobacterial prokaryote and suggest the human circadian system may regulate its microbiome through the entrainment of bacterial clocks.

There is a real medical need of new diagnostic tools for the early recognition of invasive Candida infections. We exploited a rather simple and rapid redox methodology to construct a bispecific monoclonal antibody (bsmAb) that combines a monoclonal antibody (mAb) directed against 1,3-β-D-glucan, a well-known, pan-fungal diagnostic biomarker, with a mAb recognizing MP65, a major immunogenic mannoprotein secreted by C.albicans and other Candida species. The bsmAb (MP65/bglu mAb) was successfully produced and purified at high yields and proved to bind and reveal simultaneously, with high sensitivity, the β-glucan and MP65 antigens in both purified and native forms. The MP65/bglu mAb is the first bispecific antibody generated against a fungal microorganism and may prove useful for the concurrent detection of different and clinically significant Candida biomarkers in patient sera.

OBJETIVO: O Osteosarcoma e o Sarcoma de Ewing são as principais neoplasias malignas primárias ósseas, que acometem indivíduos menores de 15 anos. O objetivo deste estudo é comparar, retrospectivamente, os resultados funcionais dos pacientes submetidos à ressecção da extremidade distal do fêmur e à reconstrução com endoprótese não convencional, total ou parcial, do joelho. MÉTODOS: Foram analisados 26 pacientes portadores de sarcomas ósseos da extremidade distal do fêmur, acompanhados no Centro Infantil Boldrini, no período de 1990 a 2003. Vinte e quatro eram portadores de Osteossarcoma e 2 de Sarcoma de Ewing. O sistema de avaliação foi o proposto por Enneking (1987), preconizado pela Musculoskeletal Tumor Society. Para a comparação das médias entre cada critério e também entre os escores finais, utilizou-se o teste de Wilcoxon, com erro alfa de 5%. RESULTADOS: A idade variou de 5 a 17 anos, média=11,9 anos. A predominância foi no sexo feminino (61,5%). Na avaliação funcional, a comparação entre as médias de cada critério, foi encontrada diferença estatisticamente significativa somente relacionada ao item estabilidade (p=0,0037). Nos demais critérios, não foi observado diferença estatisticamente significativa: movimento (p=0,7546), dor (p=0,4848), deformidade (p=0,8695), força (p=1,0000), atividade funcional (p=0,9127) e resultado funcional (p=0,5866). CONCLUSÕES: O escore final global da avaliação funcional não apresentou diferença estatisticamente significativa (p=0,6027). O tipo de endoprótese utilizado para reconstrução do fêmur não interferiu nos resultados funcionais dos pacientes.OBJECTIVES: Osteosarcoma and Ewing's sarcoma are the most common malignant primary bone tumors in individuals under the age of 15 years. The purpose of the study is to retrospectively compare functional outcomes of patients submitted to resection of the distal femoral end and to reconstruction with total or partial non-conventional prosthesis of the knee. METHODS: We assessed 26 patients with bone sarcomas in the distal femoral end treated at the Boldrini's Children Center between 1990 and 2003. Twenty-four presented with Osteosarcoma and two had Ewing's sarcoma. The assessment system employed was the one proposed by Enneking (1987) as recognized by the Musculoskeletal Tumor Society. For the statistical analysis between the criteria and final scores, we have used the Wilcoxon test, with an alpha error of 5%. RESULTS: Ages ranged from 5 to 17 years, (mean: 11 years and 9 months); with prevalence being higher in females (61.5%). The only statistically significant difference found in this study was concerned to stability (p=0.0037). No statistical significance was found on any other criteria such as movement (p=0.7546), pain (p=0.4848), deformity (p=0.8695), strength (p=1.0000), functional activities (p=0.9127) and final functional outcome (p=0.5866). CONCLUSIONS: The overall functional end score did not show statistically significant differences (p=0.6027). The type of prosthesis for femoral reconstruction did not affect patients' functional outcomes.

Mitochondrial (Mt) dysfunction contributes to the pathophysiology of renal function and promotes cardiovascular disease such as hypertension. We hypothesize that renal Mt-genes derived from female spontaneously hypertensive rats (SHR) that exhibit hypertension have reduced expression specific to kidney cortex. After breeding a female Okamoto-Aoki SHR (SAP = 188mmHg) with Brown Norway (BN) males (SAP = 100 and 104 mmHg), hypertensive female progeny were backcrossed with founder BN for 5 consecutive generations in order to maintain the SHR mitochondrial genome in offspring that contain over increasing BN nuclear genome. Mt-protein coding genes (13 total) and nuclear transcription factors mediating Mt-gene transcription were evaluated in kidney, heart and liver of normotensive (NT: n = 20) vs. hypertensive (HT: n = 20) BN/SHR-mtSHR using quantitative real-time PCR. Kidney cortex, but not liver or heart Mt-gene expression was decreased ~2-5 fold in 12 of 13 protein encoding genes of HT BN/SHR-mtSHR. Kidney cortex but not liver mRNA expression of the nuclear transcription factors Tfam, NRF1, NRF2 and Pgc1α were also decreased in HT BN/SHR-mtSHR. Kidney cortical tissue of HT BN/SHR-mtSHR exhibited lower cytochrome oxidase histochemical staining, indicating a reduction in renal oxidative phosphorylation but not in liver or heart. These results support the hypothesis that renal cortex of rats with SHR mitochondrial genome has specifically altered renal expression of genes encoding mitochondrial proteins. This kidney-specific coordinated reduction of mitochondrial and nuclear oxidative metabolism genes may be associated with heritable hypertension in SHR.

For real-time reverse transcription-PCR (qRT-PCR) in soybean, reference genes in different tissues, developmental stages, various cultivars, and under stress conditions have been suggested but their usefulness for research on soybean under various biotic stresses occurring in North-Central U.S. is not known. Here, we investigated the expression stabilities of ten previously recommended reference genes (ABCT, CYP, EF1A, FBOX, GPDH, RPL30, TUA4, TUB4, TUA5, and UNK2) in soybean under biotic stress from Bean pod mottle virus (BPMV), powdery mildew (PMD), soybean aphid (SBA), and two-spotted spider mite (TSSM). BPMV, PMD, SBA, and TSSM are amongst the most common pest problems on soybean in North-Central U.S. and other regions. Reference gene stability was determined using three software algorithms (geNorm, NormFinder, BestKeeper) and a web-based tool (RefFinder). Reference genes showed variability in their expression as well as stability across various stressors and the best reference genes were stress-dependent. ABCT and FBOX were found to be the most stable in soybean under both BPMV and SBA stress but these genes had only minimal to moderate stability during PMD and TSSM stress. Expression of TUA4 and CYP was found to be most stable during PMD stress; TUB4 and TUA4 were stable under TSSM stress. Under various biotic stresses on soybean analyzed, GPDH expression was found to be consistently unstable. For all biotic stressors on soybean, we obtained pairwise variation (V2/3) values less than 0.15 which suggested that combined use of the two most stable reference genes would be sufficient for normalization. Further, we demonstrated the utility of normalizing the qRT-PCR data for target genes using the most stable reference genes validated in current study. Following of the recommendations from our current study will enable an accurate and reliable normalization of qRT-PCR data in soybean under biotic stress.