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Sarcoidosis is a multisystemic syndrome characterized by non-caseous granulomatous inflammation, although necrotizing sarcoid granulomatosis is considered part of the spectrum of the disease. Drug induced sarcoidosis-like reaction (DISR) is a systemic granulomatous reaction, which is histopathologically identical to primary sarcoidosis - mostly described after the use of biologics like tumour necrosis factor alpha antagonists but also anti-CD20 (rituximab). The authors present the very rare case of a woman with a primary Sjögren's syndrome (pSS) started on rituximab for disease control, which evolved with a 3-year indolent progressive systemic sarcoid reaction. There has been much speculation about the potential role of B cells in sarcoidosis. Findings show a decrease of B memory cells and an increase in naïve and active subsets of regulatory B cells in sarcoidosis patients, which resembles the repopulation with naïve B cells after treatment with rituximab. Moreover, granulomatous lymphocytic interstitial lung disease associated with common variable immunodeficiency and immune reconstitution syndrome in patients wirh human immunodeficiency virus show clinical similarities to DISR and can help unveil new cytogenic and physiologic pathways. To the authors' knowledge this is the first report of a systemic sarcoidosis-like reaction with necrotizing granulomas following an anti-CD20 therapy and also the first described in a pSS patient - underlining the importance of recognizing necrotizing sarcoid granulomatous processes in the diferential diagnosis of patients with caseous inflammation. Although this is a very rare adverse effect, the case enhances the importance of actively searching for DISR after biologics, even in patients undergoing rescue on-label therapies, such as rituximab., Learning Points: First report of a systemic sarcoidosis-like reaction with necrotizing granulomas following an anti-CD20 therapy, in a patient with primary Sjögren's syndrome.Recognizing immunotherapy and biological therapies as the possible causative agents of rare and underrecognized adverse effects in patients with rare diseases in the era of biologics.Recognizing necrotizing sarcoid granulomas in the diferential diagnosis of patients with caseous inflammation., Competing Interests: Conflicts of Interests: The Authors declare that there are no competing interests., (© EFIM 2024.)

Insect pest control can be achieved by the application of RNA interference (RNAi), a key molecular tool in functional genomics. Whereas most RNAi research has focused on insect pests, few studies have been performed on natural enemies. Validating the efficacy of RNAi in natural enemies is crucial for assessing its safety and enabling molecular research on these organisms. Here, we assessed the efficacy of RNAi in the ladybird beetle Eriopis connexa Germar (Coleoptera: Coccinellidae), focusing on genes related to reproduction, such as vitellogenin (Vg) and its receptor (VgR). In the transcriptome of E. connexa, we found one VgR (EcVgR) and two Vg genes (EcVg1 and EcVg2). These genes have been validated by in silico analyses of functional domains and evolutionary relationships. Five-day-old females were injected with 500 ng/µL of a specific double-stranded RNA (dsRNA) (dsEcVg1, dsEcVg2, or dsEcVgR) for RNAi tests, while nonspecific dsRNA (dsGFP or dsAgCE8.1) was used as a control. Interestingly, dsEcVg2 was able to knockdown both Vg genes, while dsEcVg1 could silence only EcVg1. Additionally, the viability of the eggs was significantly reduced when both Vg genes were knocked down at the same time (after treatment with dsEcVg2 or "dsEcVg1+dsEcVg2"). Ultimately, malformed, nonviable eggs were produced when EcVgR was silenced. Interestingly, no dsRNA treatment had an impact on the quantity of eggs laid. Therefore, the feasibility of RNAi in E. connexa has been confirmed, suggesting that this coccinellid is an excellent Neotropical model for molecular research on natural enemies and for studying RNAi nontarget effects., (© 2024 Wiley Periodicals LLC.)

Deforestation and high human mobility due to mining activities have been key to the increase in malaria cases in the Americas. Here, we review the epidemiological and control aspects of malaria in the Amazon mining areas. Epidemiological evidence shows: 1) a positive correlation between illegal mining activity and malaria incidence, mostly in the Amazon region; 2) most Brazilian miners are males aged 15-29 years who move between states and even countries; 3) miners do not fear the disease and rely on medical care, diagnosis, and medication when they become ill; 4) illegal mining has emerged as the most reported anthropogenic activity within indigenous lands and is identified as a major cause of malaria outbreaks among indigenous people in the Amazon; and 5) because mining is largely illegal, most areas are not covered by any healthcare facilities or activities, leading to little assistance in the diagnosis and treatment of malaria. Our review identified five strategies for reducing the malaria incidence in areas with mining activities: 1) reviewing legislation to control deforestation and mining expansion, particularly in indigenous lands; 2) strengthening malaria surveillance by expanding the network of community health agents to support rapid diagnosis and treatment; 3) reinforcing vector control strategies, such as the use of insecticide-treated nets; 4) integrating deforestation alerts into the national malaria control program; and 5) implementing multi-sectoral activities and providing prompt assistance to indigenous populations. With this roadmap, we can expect a decrease in malaria incidence in the Amazonian mining areas in the future.

Background: Atrioventricular block (AVB) describes an impairment of conduction from the atria to the ventricles. Although the clinical course of AVB has been evaluated, the findings are from high-income countries and, therefore, cannot be extrapolated to the Latinx population., Objective: Evaluate the association between AVB and mortality., Methods: Patients from the CODE (Clinical Outcomes in Digital Electrocardiology) study, older than 16 years who underwent digital electrocardiogram (ECG) from 2010 to 2017 were included. ECGs were reported by cardiologists and by automated software. To assess the relationship between AVB and mortality, the log-normal model and the Kaplan-Meier curves were used with two-tailed p-values < 0.05 considered statistically significant., Results: The study included 1,557,901 patients; 40.2% were men, and mean age was 51.7 (standard deviation ± 17.6) years. In a mean follow-up of 3.7 years, the mortality rate was 3.35%. The AVB prevalence was 1.38% (21,538). Patients with first-, second-, and third-degree AVB were associated with 24% (relative survival rate [RS] = 0.76; 95% confidence interval [CI]: 0.71-0.81; p < 0.001), 55% (RS = 0.45; 95% CI: 0.27-0.77; p = 0.01), and 64% (RS = 0.36; 95% CI: 0.26-0.49; p < 0.001) lower survival rate when compared to the control group, respectively. Patients with 2:1 AVB had 79% (RS = 0.21; 95% CI: 0.08-0.52; p = 0.005) lower survival rate than the control group. Only Mobitz type I was not associated with higher mortality (p = 0.27)., Conclusion: AVB was an independent risk factor for overall mortality, with the exception of Mobitz type I.

Intraspecific competition between mosquito larvae can affect several adult traits, particularly size. This study tested the hypothesis that intraspecific competition during the larval stage affects wing length in Ae. aegypti and Ae. albopictus adults, in turn influencing locomotor activity. L1 larvae of both species were reared in trays under conditions of low and high competition. After adults had emerged, the locomotor activity of virgin females of Ae. aegypti and Ae. albopictus was evaluated under light-dark cycles of 12:12 h at 25 °C and 70% relative humidity. At the end of the locomotor activity experiment, the left wings of the mosquitoes were removed to be measured, and wing length was used as an indicator of adult female size. Although the results showed that the wing lengths of Ae. aegypti and Ae. albopictus females reared under low larval competition were significantly greater than those of females reared under high larval competition, this difference did not affect locomotor activity in females of either species, demonstrating that locomotor activity in small Ae. aegypti and Ae. albopictus females is not lower than in larger females. Our findings reinforce the idea that intraspecific competition alters the wing length of Ae. aegypti and Ae. albopictus females and provide new evidence about this effect on the locomotor activity of these species., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Background: Insecticide resistance in arthropods is an inherited trait that has become a major cause of insect pest control failure. Monitoring the level of susceptibility and characterization of the type of resistance of key pest species aims to determine the risk of resistance selection in time to take action to mitigate control failures. Seven populations of the boll weevil, Anthonomus grandis grandis, collected from cotton fields in the Semiarid and Cerrado areas of Brazil, were screened for their resistance to malathion and beta-cyfluthrin, insecticides widely recommended for control of boll weevil and other pests., Results: The levels of adult mortality were variable for beta-cyfluthrin (0-82%) but invariant (100%) for malathion. Bioassays of concentration-mortality were used to determine lethal concentrations (LCs) for each insecticide. The LC-values corroborate the lack of resistance to field rates of malathion but high levels of resistance to beta-cyfluthrin from 62.7- to 439.7-fold. Weevils resistant to beta-cyfluthrin were found through genome sequencing to possess a kdr mutation through the L1014F substitution in the voltage gated-sodium channel gene., Conclusions: This study found boll weevil resistance to beta-cyfluthrin to be not mediated by carboxylesterases, but with cross-resistance to DDT and carbaryl, and kdr mutation as the major mechanism of the resistance in our samples. Caution is recommended in further use of beta-cyfluthrin against boll weevil due to potential resistance. Monitoring studies using other boll weevil populations are recommended to determine the geographic pattern and extent of pyrethroid resistance. © 2021 Society of Chemical Industry., (© 2021 Society of Chemical Industry.)

Background: The hemolymph and insect gut together have an essential role in the immune defense against microorganisms, including the production of antimicrobial peptides (AMP). AMPs are mainly induced by two specific signaling pathways, Toll and immune deficiency (IMD). Here, we characterize the expression profile of four genes from both pathways and describe the importance of AgraRelish in the immune defense of Anthonomus grandis against the entomopathogenic fungus Metarhizium anisopliae by RNA interference (RNAi)., Results: To characterize the pathway that is activated early during the A. grandis-M. anisopliae interaction, we assessed the expression profiles of AgraMyD88 and AgraDorsal (Toll pathway), AgraIMD and AgraRelish (IMD pathway), and several AMP genes. Interestingly, we found that IMD pathway genes are upregulated early, and Toll pathway genes are upregulated just 3 days after inoculation (DAI). Furthermore, nine AMPs were upregulated 24 h after fungus inoculation, including attacins, cecropins, coleoptericins, and defensins. AgraRelish knockdown resulted in a reduction in median lethal time (LT 50 ) for M. anisopliae-treated insects of around 2 days compared to control treatments. In addition, AgraRelish remained knocked down at 3 DAI. Finally, we identified that AgraRelish knockdown increased fungal loads at 2 DAI compared to control treatments, possibly indicating a faster infection., Conclusions: Our data indicate the influence of the IMD pathway on the antifungal response in A. grandis. Combining biocontrol and RNAi could significantly improve cotton boll weevil management. Hence, AgraRelish is a potential target for the development of biotechnological tools aimed at improving the efficacy of M. anisopliae against A. grandis., (© 2021 Society of Chemical Industry.)

Natural products are a valuable source of biologically active compounds and continue to play an important role in modern drug discovery due to their great structural diversity and unique biological properties. Brazilian biodiversity is one of the most extensive in the world and could be an effective source of new chemical entities for drug discovery. Mosquitoes are vectors for the transmission of dengue, Zika, chikungunya, yellow fever, and many other diseases of public health importance. These diseases have a major impact on tropical and subtropical countries, and their incidence has increased dramatically in recent decades, reaching billions of people at risk worldwide. The prevention of these diseases is mainly through vector control, which is becoming more difficult because of the emergence of resistant mosquito populations to the chemical insecticides. Strategies to provide efficient and safe vector control are needed, and secondary metabolites from plant species from the Brazilian biodiversity, especially Cerrado, that are biologically active for mosquito control are herein highlighted. Also, this is a literature revision of targets as insights to promote advances in the task of developing active compounds for vector control. In view of the expansion and occurrence of arboviruses diseases worldwide, scientific reviews on bioactive natural products are important to provide molecular models for vector control and contribute with effective measures to reduce their incidence., Competing Interests: The authors declare that they have no conflict of interest., (Thieme. All rights reserved.)

The cotton boll weevil, Anthonomus grandis , is the most economically important pest of cotton in Brazil. Pest management programs focused on A. grandis are based mostly on the use of chemical insecticides, which may cause serious ecological impacts. Furthermore, A. grandis has developed resistance to some insecticides after their long-term use. Therefore, alternative control approaches that are more sustainable and have reduced environmental impacts are highly desirable to protect cotton crops from this destructive pest. RNA interference (RNAi) is a valuable reverse genetics tool for the investigation of gene function and has been explored for the development of strategies to control agricultural insect pests. This study aimed to evaluate the biological role of the Laccase2 ( AgraLac2 ) gene in A. grandis and its potential as an RNAi target for the control of this insect pest. We found that AgraLac2 is expressed throughout the development of A. grandis with significantly higher expression in pupal and adult developmental stages. In addition, the immunolocalization of the AgraLac2 protein in third-instar larvae using specific antibodies revealed that AgraLac2 is distributed throughout the epithelial tissue, the cuticle and the tracheal system. We also verified that the knockdown of AgraLac2 in A. grandis resulted in an altered cuticle tanning process, molting defects and arrested development. Remarkably, insects injected with ds AgraLac2 exhibited defects in cuticle hardening and pigmentation. As a consequence, the development of ds AgraLac2 -treated insects was compromised, and in cases of severe phenotypic defects, the insects subsequently died. On the contrary, insects subjected to control treatments did not show any visible phenotypic defects in cuticle formation and successfully molted to the pupal and adult stages. Taken together, our data indicate that AgraLac2 is involved in the cuticle tanning process in A. grandis and may be a promising target for the development of RNAi-based technologies., (Copyright © 2020 Firmino, Pinheiro, Moreira-Pinto, Antonino, Macedo, Martins-de-Sa, Arraes, Coelho, Fonseca, Silva, Engler, Silva, Lourenço-Tessutti, Terra and Grossi-de-Sa.)

Objective: This study aimed to assess the antibacterial and antibiofilm effects of essential oils and herbal toothpastes against bacteria associated with oral diseases., Methods: The minimum inhibitory concentration (MIC) and antibiofilm activity of 13 essential oils against Staphylococcus aureus, Streptococcus mutans, Lactobacillus lactis, and Enterococcus faecalis. were determined. Toothpastes were formulated with different concentrations of the most active essential oils, alone and in combination, and evaluated for antibacterial and antibiofilm activities., Results: Clove, oregano, thyme, and cinnamon essential oils were effective in inhibiting all bacterial strains. The antibacterial activity of cinnamon essential oil was similar to that of the control (0.12 % chlorhexidine gluconate mouthwash). Cinnamon essential oil was a strong inhibitor of S. mutans growth. The antibiofilm activity of clove, oregano, thyme, and cinnamon essential oils at 1, 2, and 4 × MIC against S. mutans did not differ from that of the control. In the hole-plate diffusion assay, 17 out of the 18 tested toothpastes produced an inhibition halo at least half as large as that of the control. Toothpastes containing clove, clove and oregano, or clove, oregano, thyme, and cinnamon essential oils were able to completely disrupt S. mutans biofilms, not differing from the control. Thyme essential oil was found to act synergistically with chlorhexidine against S. mutans., Conclusions: The results indicate that clove, oregano, thyme, and cinnamon essential oils may be added to fluoride-free toothpastes to enhance inhibitory effects against bacteria associated with cavities and periodontal disease. Thyme essential oil may increase the efficiency of chlorhexidine-containing dentifrices., Competing Interests: Declaration of Competing Interest The authors declare that there is no conflict of interests regarding the publication of this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Galls induced by plant-parasitic nematodes involve a hyperactivation of the plant mitotic and endocycle machinery for their profit. Dedifferentiation of host root cells includes drastic cellular and molecular readjustments. In such a background, potential DNA damage in the genome of gall cells is evident. We investigated whether DNA damage checkpoint activation followed by DNA repair occurred, or was eventually circumvented, in nematode-induced galls. Galls display transcriptional activation of the DNA damage checkpoint kinase WEE1, correlated with its protein localization in the nuclei. The promoter of the stress marker gene SMR7 was evaluated under the WEE1-knockout background. Drugs inducing DNA damage and a marker for DNA repair, PARP1, were used to understand the mechanisms for coping with DNA damage in galls. Our functional study revealed that gall cells lacking WEE1 conceivably entered mitosis prematurely, disturbing the cell cycle despite the loss of genome integrity. The disrupted nuclei phenotype in giant cells hinted at the accumulation of mitotic defects. In addition, WEE1-knockout in Arabidopsis and downregulation in tomato repressed infection and reproduction of root-knot nematodes. Together with data on DNA-damaging drugs, we suggest a conserved function for WEE1 in controlling G1/S cell cycle arrest in response to a replication defect in galls., (© 2019 The Authors. New Phytologist © 2019 New Phytologist Trust.)

Coffee production is a global industry valued at approximately 173 billion US dollars. One of the main challenges facing coffee production is the management of the coffee berry borer (CBB), Hypothenemus hampei, which is considered the primary arthropod pest of coffee worldwide. Current control strategies are inefficient for CBB management. Although biotechnological alternatives, including RNA interference (RNAi), have been proposed in recent years to control insect pests, characterizing the genetics of the target pest is essential for the successful application of these emerging technologies. In this study, we employed RNA-seq to obtain the transcriptome of three developmental stages of the CBB (larva, female and male) to increase our understanding of the CBB life cycle in relation to molecular features. The CBB transcriptome was sequenced using Illumina Hiseq and assembled de novo. Differential gene expression analysis was performed across the developmental stages. The final assembly produced 29,434 unigenes, of which 4,664 transcripts were differentially expressed. Genes linked to crucial physiological functions, such as digestion and detoxification, were determined to be tightly regulated between the reproductive and nonreproductive stages of CBB. The data obtained in this study help to elucidate the critical roles that several genes play as regulatory elements in CBB development.

Cell cycle control in galls provoked by root-knot nematodes involves the activity of inhibitor genes like the Arabidopsis ICK/KRP members. Ectopic KRP1, KRP2 and KRP4 expression resulted in decreased gall size by inhibiting mitotic activity, whereas KRP6 induces mitosis in galls. Herein, we investigate the role of KRP3, KRP5 and KRP7 during gall development and compared their role with previously studied members of this class of cell cycle inhibitors. Overexpression of KRP3 and KRP7 culminated in undersized giant cells, with KRP3 OE galls presenting peculiar elongated giant cells. Nuclei in KRP3 OE and KRP5 OE lines presented a convoluted and apparently connected phenotype. This appearance may be associated with the punctuated protein nuclear localization driven by specific common motifs. As well, ectopic expression of KRP3 OE and KRP5 OE affected nematode development and offspring. Decreased mitotic activity in galls of KRP3 OE and KRP7 OE lines led to a reduced gall size which presented distinct shapes - from more elongated like in the KRP3 OE line to small rounded like in the KRP7 OE line. Results presented strongly support the idea that induced expression of cell cycle inhibitors such as KRP3 and KRP7 in galls can be envisaged as a conceivable strategy for nematode feeding site control in crop species attacked by phytopathogenic nematodes., (© 2017 John Wiley & Sons Ltd.)

Root-knot nematodes induce galls that contain giant-feeding cells harboring multiple enlarged nuclei within the roots of host plants. It is recognized that the cell cycle plays an essential role in the set-up of a peculiar nuclear organization that seemingly steers nematode feeding site induction and development. Functional studies of a large set of cell cycle genes in transgenic lines of the model host Arabidopsis thaliana have contributed to better understand the role of the cell cycle components and their implication in the establishment of functional galls. Mitotic activity mainly occurs during the initial stages of gall development and is followed by an intense endoreduplication phase imperative to produce giant-feeding cells, essential to form vigorous galls. Transgenic lines overexpressing particular cell cycle genes can provoke severe nuclei phenotype changes mainly at later stages of feeding site development. This can result in chaotic nuclear phenotypes affecting their volume. These aberrant nuclear organizations are hampering gall development and nematode maturation. Herein we report on two nuclear volume assessment methods which provide information on the complex changes occurring in nuclei during giant cell development. Although we observed that the data obtained with AMIRA tend to be more detailed than Volumest (Image J), both approaches proved to be highly versatile, allowing to access 3D morphological changes in nuclei of complex tissues and organs. The protocol presented here is based on standard confocal optical sectioning and 3-D image analysis and can be applied to study any volume and shape of cellular organelles in various complex biological specimens. Our results suggest that an increase in giant cell nuclear volume is not solely linked to increasing ploidy levels, but might result from the accumulation of mitotic defects.

Streptomyces lunalinharesii strain 235 produces an antimicrobial substance that is active against sulfate reducing bacteria, the major bacterial group responsible for biofilm formation and biocorrosion in petroleum reservoirs. The use of this antimicrobial substance for sulfate reducing bacteria control is therefore a promising alternative to chemical biocides. In this study the antimicrobial substance did not interfere with the biofilm stability, but the sulfate reducing bacteria biofilm formation was six-fold smaller in carbon steel coupons treated with the antimicrobial substance when compared to the untreated control. A reduction in the most probable number counts of planktonic cells of sulfate reducing bacteria was observed after treatments with the sub-minimal inhibitory concentration, minimal inhibitory concentration, and supra-minimal inhibitory concentration of the antimicrobial substance. Additionally, when the treated coupons were analyzed by scanning electron microscopy, the biofilm formation was found to be substantially reduced when the supra-minimal inhibitory concentration of the antimicrobial substance was used. The coupons used for the biofilm formation had a small weight loss after antimicrobial substance treatment, but corrosion damage was not observed by scanning electron microscopy. The absence of the dsrA gene fragment in the scraped cell suspension after treatment with the supra-minimal inhibitory concentration of the antimicrobial substance suggests that Desulfovibrio alaskensis was not able to adhere to the coupons. This is the first report on an antimicrobial substance produced by Streptomyces active against sulfate reducing bacteria biofilm formation. The application of antimicrobial substance as a potential biocide for sulfate reducing bacteria growth control could be of great interest to the petroleum industry., (Copyright © 2016 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.)

Vitellogenin (Vg), a yolk protein precursor, is the primary egg nutrient source involved in insect reproduction and embryo development. The Cotton Boll weevil (CBW) Anthonomus grandis Boheman, the most important cotton pest in Americas, accumulates large amounts of Vg during reproduction. However, the precise role of this protein during embryo development in this insect remains unknown. Herein, we investigated the effects of vitellogenin (AgraVg) knockdown on the egg-laying and egg viability in A. grandis females, and also characterized morphologically the unviable eggs. AgraVg transcripts were found during all developmental stages of A. grandis, with highest abundance in females. Silencing of AgraVg culminated in a significant reduction in transcript amount, around 90%. Despite this transcriptional reduction, egg-laying was not affected in dsRNA-treated females but almost 100% of the eggs lost their viability. Eggs from dsRNA-treated females showed aberrant embryos phenotype suggesting interference at different stages of embryonic development. Unlike for other insects, the AgraVg knockdown did not affect the egg-laying ability of A. grandis, but hampered A. grandis reproduction by perturbing embryo development. We concluded that the Vg protein is essential for A. grandis reproduction and a good candidate to bio-engineer the resistance against this devastating cotton pest.

Plant defensins are antifungal peptides produced by the innate immune system plants developed to circumvent fungal infection. The defensin Drr230a, originally isolated from pea, has been previously shown to be active against various entomopathogenic and phytopathogenic fungi. In the present study, the activity of a yeast-expressed recombinant Drr230a protein (rDrr230a) was tested against impacting soybean and cotton fungi. First, the gene was subcloned into the yeast expression vector pPICZαA and expressed in Pichia pastoris. Resulting rDrr230a exhibited in vitro activity against fungal growth and spore germination of Fusarium tucumaniae, which causes soybean sudden death syndrome, and against Colletotrichum gossypii var. cephalosporioides, which causes cotton ramulosis. The rDrr230a IC 50 corresponding to inhibition of fungal growth of F. tucumaniae and C. gossypii var. cephalosporioides was 7.67 and 0.84 µM, respectively, demonstrating moderate activity against F. tucumaniae and high potency against C. gossypii var. cephalosporioides. Additionally, rDrr230a at 25 ng/µl (3.83 µM) resulted in 100 % inhibition of spore germination of both fungi, demonstrating that rDrr230a affects fungal development since spore germination. Moreover, rDrr230a at 3 µg/µl (460.12 µM) inhibited 100 % of in vitro spore germination of the obligatory biotrophic fungus Phakopsora pachyrhizi, which causes Asian soybean rust. Interestingly, rDrr230a substantially decreased the severity of Asian rust, as demonstrated by in planta assay. To our knowledge, this is the first report of a plant defensin active against an obligatory biotrophic phytopathogenic fungus. Results revealed the potential of rDrr230a as a candidate to be used in plant genetic engineering to control relevant cotton and soybean fungal diseases.

Background: The etiology of lymphedema is multifactorial, and definition criteria of lymphedema, its limitation, and follow-up must be considered in studies related to risk factors. The aim of this study is to evaluate risk factors related to arm lymphedema in a cohort study with a long follow-up., Patients and Methods: The study was performed in 622 breast cancer patients. The main endpoint reported was the presence of clinical lymphedema reported in medical records. Univariate and multivariate regression analyses were performed to identify factors related to lymphedema., Results: 66.4% of the patients were submitted to mastectomy, 88.4% to level III axillary lymphadenectomy, 34.9% to radiotherapy in the supraclavicular fossa, and 4.3% to axillary radiotherapy. The mean follow-up was 96.7 months. 45 patients (7.2%) developed lymphedema, of which 82.2% had developed lymphedema at 60 months. Univariate regression analysis showed that supraclavicular radiotherapy, adjuvant/palliative chemotherapy, ≥ 15 lymph nodes dissected, and axillary surgery increase the lymphedema rate by 1.87, 2.28, 2.03, and 6.17, respectively. Adjusted multivariate regression analysis showed that the combination of axillary dissection and number of lymph nodes dissected was the main factor related to lymphedema (p = 0.017)., Conclusion: In the pre-sentinel era, axillary dissection and the number of lymph nodes resected are related to 10-year lymphedema.

Sugarcane is a widely cultivated plant that serves primarily as a source of sugar and ethanol. Its annual yield can be significantly reduced by the action of several insect pests including the sugarcane giant borer (Telchin licus licus), a lepidopteran that presents a long life cycle and which efforts to control it using pesticides have been inefficient. Although its economical relevance, only a few DNA sequences are available for this species in the GenBank. Pyrosequencing technology was used to investigate the transcriptome of several developmental stages of the insect. To maximize transcript diversity, a pool of total RNA was extracted from whole body insects and used to construct a normalized cDNA database. Sequencing produced over 650,000 reads, which were de novo assembled to generate a reference library of 23,824 contigs. After quality score and annotation, 43% of the contigs had at least one BLAST hit against the NCBI non-redundant database, and 40% showed similarities with the lepidopteran Bombyx mori. In a further analysis, we conducted a comparison with Manduca sexta midgut sequences to identify transcripts of genes involved in digestion. Of these transcripts, many presented an expansion or depletion in gene number, compared to B. mori genome. From the sugarcane giant borer (SGB) transcriptome, a number of aminopeptidase N (APN) cDNAs were characterized based on homology to those reported as Cry toxin receptors. This is the first report that provides a large-scale EST database for the species. Transcriptome analysis will certainly be useful to identify novel developmental genes, to better understand the insect's biology and to guide the development of new strategies for insect-pest control.

Cotton plants are subjected to the attack of several insect pests. In Brazil, the cotton boll weevil, Anthonomus grandis, is the most important cotton pest. The use of insecticidal proteins and gene silencing by interference RNA (RNAi) as techniques for insect control are promising strategies, which has been applied in the last few years. For this insect, there are not much available molecular information on databases. Using 454-pyrosequencing methodology, the transcriptome of all developmental stages of the insect pest, A. grandis, was analyzed. The A. grandis transcriptome analysis resulted in more than 500.000 reads and a data set of high quality 20,841 contigs. After sequence assembly and annotation, around 10,600 contigs had at least one BLAST hit against NCBI non-redundant protein database and 65.7% was similar to Tribolium castaneum sequences. A comparison of A. grandis, Drosophila melanogaster and Bombyx mori protein families' data showed higher similarity to dipteran than to lepidopteran sequences. Several contigs of genes encoding proteins involved in RNAi mechanism were found. PAZ Domains sequences extracted from the transcriptome showed high similarity and conservation for the most important functional and structural motifs when compared to PAZ Domains from 5 species. Two SID-like contigs were phylogenetically analyzed and grouped with T. castaneum SID-like proteins. No RdRP gene was found. A contig matching chitin synthase 1 was mined from the transcriptome. dsRNA microinjection of a chitin synthase gene to A. grandis female adults resulted in normal oviposition of unviable eggs and malformed alive larvae that were unable to develop in artificial diet. This is the first study that characterizes the transcriptome of the coleopteran, A. grandis. A new and representative transcriptome database for this insect pest is now available. All data support the state of the art of RNAi mechanism in insects.

Background: Soybean pathogens and pests reduce grain production worldwide. Biotic interaction cause extensive changes in plant gene expression profile and the data produced by functional genomics studies need validation, usually done by quantitative PCR. Nevertheless, this technique relies on accurate normalization which, in turn, depends upon the proper selection of stable reference genes for each experimental condition. To date, only a few studies were performed to validate reference genes in soybean subjected to biotic stress. Here, we report reference genes validation in soybean during root-knot nematode (Meloidogyne incognita) parasitism and velvetbean caterpillar (Anticarsia gemmatalis) attack., Findings: The expression stability of nine classical reference genes (GmCYP2, GmELF1A, GmELF1B, GmACT11, GmTUB, GmTUA5, GmG6PD, GmUBC2 and GmUBC4) was evaluated using twenty-four experimental samples including different organs, developmental stages, roots infected with M. incognita and leaves attacked by A. gemmatalis. Two different algorithms (geNorm and NormFinder) were used to determine expression stability. GmCYP2 and GmUBC4 are the most stable in different organs. Considering the developmental stages, GmELF1A and GmELF1B genes are the most stable. For spatial and temporal gene expression studies, normalization may be performed using GmUBC4, GmUBC2, GmCYP2 and GmACT11 as reference genes. Our data indicate that both GmELF1A and GmTUA5 are the most stable reference genes for data normalization obtained from soybean roots infected with M. incognita, and GmCYP2 and GmELF1A are the most stable in soybean leaves infested with A. gemmatalis., Conclusions: Future expression studies using nematode infection and caterpilar infestation in soybean plant may utilize the reference gene sets reported here.

Trichoderma atroviride 676 was studied to evaluate its efficiency in the production of some lignocellulolytic enzymes, using lignocellulosic residual biomass. Best results were obtained when 3.0 % (w/v) untreated sugarcane bagasse was used (61.3 U mL(-1) for xylanase, 1.9 U mL(-1) for endoglucanase, 0.25 U mL(-1) for FPase, and 0.17 U mL(-1) for β-glucosidase) after 3-4 days fermentation. The maximal enzymatic activity for endoglucanase, FPase, and xylanase were observed at 50-60 °C and pH 4.0-5.0, whereas thermal stability at 50 °C (CMCase and FPase) or 40 °C (xylanase) was obtained after 8 h. Zymograms have shown two bands of 104 and 200 kDa for endoglucanases and three bands for xylanase (23, 36, and 55.7 kDa). The results obtained with T. atroviride strain 676 were comparable to those obtained with the cellulolytic strain Trichoderma reesei RUT-C30, indicating, in the studied conditions, its great potential for biotechnological application, especially lignocellulose biomass hydrolysis.

Four actinomycete strains previously isolated from Brazilian soils were tested for their antimicrobial activity against Bacillus pumilus LF-4 and Desulfovibrio alaskensis NCIMB 13491, bacteria that are well known to be involved in biofilm formation and biocorrosion. Strain 235, belonging to the species Streptomyces lunalinharesii, inhibited the growth of both bacteria. The antimicrobial activity was seen over a wide range of pH, and after treatment with several chemicals and heat but not with proteinase K and trypsin. The antimicrobial substances present in the concentrated supernatant from growth media were partially characterized by SDS-PAGE and extracellular polypeptides were seen. Bands in the size range of 12 to 14.4 kDa caused antimicrobial activity. Transmission electron microscopy of D. alaskensis cells treated with the concentrated supernatant containing the antimicrobial substances revealed the formation of prominent bubbles, the spherical double-layered structures on the cell membrane, and the periplasmic space completely filled with electron-dense material. This is the first report on the production of antimicrobial substances by actinomycetes against bacteria involved in biocorrosion processes, and these findings may be of great relevance as an alternative source of biocides to those currently employed in the petroleum industry.

Streptomyces misionensis strain PESB-25 was screened and selected for its ability to secrete cellulases. Cells were grown in a liquid medium containing sugarcane bagasse (SCB) as carbon source and corn steep liquor (CSL) as nitrogen source, whose concentrations were optimized using response surface methodology (RSM). A peak of endoglucanase accumulation (1.01 U · mL(-1)) was observed in a medium with SCB 1.0% (w/v) and CSL 1.2% (w/v) within three days of cultivation. S. misionensis PESB-25 endoglucanase activity was thermoacidophilic with optimum pH and temperature range of 3.0 to 3.6 and 62° to 70 °C, respectively. In these conditions, values of 1.54 U mL(-1) of endoglucanase activity were observed. Moreover, Mn(2+) was demonstrated to have a hyperactivating effect on the enzyme. In the presence of MnSO4 (8 mM), the enzyme activity increased threefold, up to 4.34 U · mL(-1). Mn(2+) also improved endoglucanase stability as the catalyst retained almost full activity upon incubation at 50 °C for 4 h, while in the absence of Mn(2+), enzyme activity decreased by 50% in this same period. Three protein bands with endoglucanase activity and apparent molecular masses of 12, 48.5 and 119.5 kDa were detected by zymogram.

In the present paper, endo-β-1,4-xylanase production by Aspergillus fumigatus was evaluated in solid-state fermentation using low-cost substrates such as sugarcane bagasse (SCB), brewer's spent grain (BSG), and wheat bran (WB). The partial characterization of the crude enzyme was also performed. In the experimental conditions, the highest levels of endo-β-1,4-xylanase production by A. fumigatus FBSPE-05 occurred within 8 days incubation when using SCB/liquid medium at 1:2 ratio (219.5 U g(-1)) and 4 days incubation when using WB/liquid medium at 1:1 ratio (215.6 U g(-1)). Crude enzyme from this last condition was used to enzyme characterization, showing best enzyme activity at 60 °C and pH 6.0, which suggests a thermophilic endoxylanase. The crude enzyme retained 73% of its activity after 1 h at 60 °C, and zymogram has shown three bands of endo-β-1,4-xylanase activity, with different molecular masses. A. fumigatus FBSPE-05 was able to grow and produce good levels of endo-β-1,4-xylanase using agro-industrial by-products, making this strain worthy for further investigation. To our knowledge, this is the first study reporting the use of SCB and/or BSG as sole substrates for endoxylanase production by solid-state fermentation using A. fumigatus.

Brewer's spent grain and corn steep liquor or yeast extract were used as the sole organic forms for proteinase production by Streptomyces malaysiensis in submerged fermentation. The influence of the C and N concentrations, as well as the incubation periods, were assessed. Eight proteolytic bands were detected through gelatin-gel-electrophoresis in the various extracts obtained from the different media and after different incubation periods, with apparent molecular masses of 20, 35, 43, 50, 70, 100, 116 and 212 kDa. The results obtained suggest an opportunity for exploring this alternative strategy for proteinases production by actinomycetes, using BSG and CSL as economically feasible substrates.

A cellulolytic bacterial strain, designated P118, isolated from the gut of the tropical fish Parotocinclus maculicauda was identified as belonging to the genus Paenibacillus based on phenotypic and chemotaxonomic characteristics and the 16S rRNA gene sequence. The novel strain was Gram-positive, spore-forming and rod-shaped. Catalase but not oxidase was produced. Carboxymethylcellulose was hydrolyzed but starch or gelatin was not. Acetoin production was negative whereas nitrate reduction and urease production were positive. Many carbohydrates served as carbon sources for growth. MK-7 was the predominant isoprenoid quinone. Anteiso-C15:0 (38.73%) and C16:0 (20.85%) were the dominant cellular fatty acids. Strain P118 was closely related to Paenibacillus amylolyticus NRRL NRS-290, P. pabuli HSCC 492, P. tundrae Ab10b, P. xylanexedens B22a, and P. tylopili MK2 with 98.3-98.8% 16S rRNA gene sequence similarity. The results presented here suggest that strain P118 represents a novel species of the genus Paenibacillus and it is a potential strain for further studies concerning its role in the production of industrially important products from cellulosic biomass.

An actinomycete strain, isolated from a soil sample under a sugar cane plantation in Brazil and identified as Streptomyces viridobrunneus SCPE-09, was selected as a promising cellulolytic strain, and tested for its ability to produce cellulases from agro-industrial residues. Sugar cane bagasse or wheat bran was tested as carbon source, and corn steep liquor tested as nitrogen source. Different concentrations of carbon and nitrogen were tested using factorial design to identify optimal cellulose production. The results showed that media containing wheat bran 2.0% (w/v) and corn steep liquid 0.19% (w/v) lead to the highest production, 2.0 U mL(-1) of CMCase, obtained on the fifth day of fermentation. The pH and temperature profile showed optimal activity at pH 4.9 and 50°C. As for thermostability, endoglucanases were most tolerant at 50°C, retaining more than 80% of maximal activity even after 2 h of incubation. Zymogram analyses using supernatant from growth under optimized conditions revealed the presence of two CMCase bands with apparent molecular masses of 37 and 119 kDa. The combination of pH tolerance and CMCase production from agro-industrial residues by S. viridobrunneus SCPE-09 offers promise for future bioethanol biotechnologies.

Litopenaeus vannamei, which is the most common shrimp species cultivated in the northeast of Brazil, is very susceptible to microbial diseases, and this consequently affects productivity. There are reports of bacteria, viruses and protozoa in these shrimp, but not fungi. This study aims to isolate and identify fungi present in shrimp Litopenaeus vannamei, and in their nursery waters, at two breeding farms in Brazil. The pathogenic potential of the isolates was assessed through the qualitative detection of proteases and aflatoxin B production. The 146 isolated fungi comprised 46 species. Aspergillus, Penicillium and Furarium were the three most relevant genera and Aspergillus flavus was the predominant species with a total of 33 isolates. Most of the isolated species are known as potentially pathogenic to humans and other animals. Eighteen isolates of A. flavus and two of A. parasiticus were able to produce aflatoxin B and 33 out of the 46 species produced protease, indicating that these fungi may also become pathogenic to shrimp and their consumers.

Three Bacillus species (B. subtilis LFB-FIOCRUZ 1270, B. subtilis LFB-FIOCRUZ 1273, and B. licheniformis LFB-FIOCRUZ 1274), isolated from the poultry industry, were evaluated for keratinase production using feathers or feather meal as the sole carbon and nitrogen sources in a submerged fermentation. The three Bacillus spp. produced extracellular keratinases and peptidases after 7 days. Feather meal was the best substrate for keratinase and peptidase production in B. subtilis 1273, with 412 U/mL and 463 U/ml. The three strains were able to degrade feather meal (62-75%) and feather (40-95%) producing 3.9-4.4 mg/ml of soluble protein in feather meal medium and 1.9-3.3 mg/ml when feather medium was used. The three strains produced serine peptidases with keratinase and gelatinase activity. B. subtilis 1273 was the strain which exhibited the highest enzymatic activity.

Aims: To evaluate cellulase production by Streptomyces malaysiensis in submerged fermentation using brewer's spent grain (BSG) and wheat bran (WB) as carbon source, and corn steep liquor (CSL) as nitrogen source, as compared to yeast extract (YE), and partial characterization of the crude enzyme., Methods and Results: Maximum cellulase production by Streptomyces malaysiensis (720 U l(-1)) occurred within 4 days incubation when using a growth medium containing BSG 0.5% (w/v) and CSL1.2% (w/v). CMCases activity showed to be stable over an acidic pH range (2.0-7.0) and in temperatures of 40-60 degrees C. Zymogram indicated three bands of CMCase activity, with different molecular masses., Conclusion: S. malaysiensis was able to grow and produce good levels of CMCases using solely brewer's spent grain and corn steep liquor as low-cost substrates, making this strain and these low cost by-product worthy for further investigation, and potentially feasible for biotechnological applications in different areas., Significance and Impact of the Study: To our knowledge, this is the first study reporting the use of the low-cost by-products brewer's spent grain and corn steep liquor, as sole substrates for microbial enzyme production.

This study evaluated the production of cellulolytic enzymes by an Aspergillus fumigatus strain, isolated from sugar cane bagasse, according to its ability to grow on microcrystalline cellulose as the sole carbon source. The effect of the carbon source (brewer's spent grain, sugarcane bagasse, and wheat bran) and of the nitrogen source (corn steep liquor and sodium nitrate) on cellulase production was studied using submerged and solid state cultivations at 30 degrees C. The highest levels of endoglucanase (CMCase) corresponded to 365 U L(-1) and was obtained using sugarcane bagasse (1%) and corn steep liquor (1.2%) in submerged fermentation within 6 days of cultivation. This supernatant was used to run a sodium dodecyl sulfate polyacrylamide gel electrophoresis that showed six bands with endoglucanase activity. CMCase activity was higher at 65 degrees C and pH 2.0, indicating that this microorganism produces a thermophilic and acid endoglucanase. Solid state cultivation favored FPase production, that reached 47 U g(-1) of dry substrate (wheat bran and sugarcane bagasse) within 3 days.

A novel chitinolytic actinomycete isolated from a Brazilian cerrado soil, designated strain RCQ1071(T), was assigned to the genus Streptomyces on the basis of chemical and morphological characteristics. The almost-complete nucleotide sequence of the 16S rRNA gene of strain RCQ1071(T) was determined and also placed this strain in the genus Streptomyces. Phylogenetic analyses of 16S rRNA gene sequences showed that strain RCQ1071(T) formed a long branch in a group related to Streptomyces albulus, sharing approximately 98 % sequence similarity. Levels of DNA-DNA relatedness between strain RCQ1071(T) and members of this group, namely S. albulus DSM 40492(T), Streptomyces noursei DSM 40635(T) and Streptomyces yunnanensis DSM 41793(T), were 38.3, 27.8 and 46 %, respectively, strongly indicating that strain RCQ1071(T) was not a member of any of these species. The relatively long branch length within a stable clade together with the phenotypic data strongly supported that strain RCQ1071(T) represented a novel species. Based on the combination of physiological, phylogenetic and genomic data, strain RCQ1071(T) is suggested to represent a novel species of the genus Streptomyces, for which the name Streptomyces lunalinharesii sp. nov. is proposed. The type strain is RCQ1071(T) (=ATCC BAA-1231(T) =CIP 108852(T) =DSM 41876(T)).

Chitin from Streptomyces lunalinharesii spores, detected on its outermost surface layer, was isolated and characterized by chemical and spectroscopic methods, transmission electron microscopy and flow cytometry analysis. Gold-chitinase- and gold-lectin (Lycopersicum esculentum agglutinin, LEA)-conjugated labels were used in microscopy experiments, whereas a fluorescence-lectin (LEA) conjugate was used in flow cytometry analysis. Chitin isolation consisted of several steps of hot alkali and nitrous acid treatment, and the final material was obtained in the colloidal form. The infrared and the 13C CP/MAS NMR spectra of Streptomyces sp. colloidal chitin and colloidal chitin obtained from commercial crab shell chitin were very similar. Incubation of the spores with gold-labeled lectin, or gold-labeled recombinant chitinase, showed the presence of gold particles around the spore surface, indicating the specific binding of the lectin or the recombinant chitinase with the chitin present on the outermost surface. Flow cytometry analysis, using the fluorescence-lectin conjugate, confirmed these results. According to scanning electron microscopy, S. lunalinharesii presented spore surface ornamentation belonging to the spiny group. This is the first detailed characterization of chitin on the spore's outermost layer from a Streptomyces species.

Protease production by Streptomyces sp. 594 was obtained after submerged fermentation (SF) and solid-state fermentation (SSF) using feather meal (FM) and corn steep liquor (CSL) as sole sources of carbon and nitrogen. Enzyme productions were 13.4 U ml(-1) in SF and 21.5 U g(-1) in SSF; these values were approximately 86% and 39% higher, respectively, than those obtained previously when yeast extract was used in place of CSL. The proteases, which belong to the serine and metalloproteinase classes, were active at high temperatures (55 degrees C to 90 degrees C) and over a wide range of pH values (5.0 to 10.0). Thus, these thermophilic proteases have shown interesting properties for industrial purposes. As far as we are concerned, this is the first contribution toward the microbial production of thermophilic proteases by a streptomycete using a low-cost medium composed of industrial poultry (FM) and corn processing by-products (CSL).

Aims: Protease production by Streptomyces sp. 594 in submerged (SF) and solid-state fermentation (SSF) using feather meal, an industrial poultry residue, and partial characterization of the crude enzyme., Methods and Results: Streptomyces sp. 594 produced proteases in SF (7.2 +/- 0.2 U ml(-1)) and SSF (15.5 +/- 0.41 U g(-1)), with pH increase in both media. Considering protease activity, values obtained in the liquid extract after SSF (6.3 +/- 0.17 U ml(-1)) were lower than those from SF. The proteases, which belong to serine and metalloproteinase classes, were active over a wide range of pH (5.0-10.0) and high temperatures (55-80 degrees C). Strain 594 was also able to degrade feather in agar and liquid media. Keratinase activity (80 U l(-1)) also confirmed the keratin degrading capacity of this streptomycete., Conclusions: Proteases produced using residues from poultry industry have shown interesting properties for industrial purposes., Significance and Impact of the Study: As far as we are concerned, this is the first contribution towards the production of thermophilic protease by a streptomycete in SSF using a keratinous waste.

Streptomyces malaysiensis AMT-3, isolated from a Brazilian cerrado soil, showed proteolytic activities detected by gelatin-sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The optimum proteinase production was obtained when using 2.5% wheat bran and 0.1% yeast extract in the culture medium, after 5 days incubation at 30 degrees C. The enzymatic complex degraded gelatin optimally at pH 7.0, and under these conditions eight proteolytic bands (four serine-proteinases and four metaloproteinases), ranging from 20 to 212 kDa, were detected on the culture supernatant filtrates. In addition, a 35-kDa proteinase was thermostable at 60 degrees C for 120 min. These results point out to the applicability of gelatin zymograms in the characterization of crude enzymatic complexes. According to our results, this enzymatic complex could be used for biotechnological applications.

The present work analyzes the production of endochitinase by Colletotrichum gloeosporioides, a phytopathogenic fungus, using six different carbon sources and two pH values. For quantitative assay of endochitinase activity in solution, the synthetic substrate 4-methylumbelliferyl-beta-D-N,N',N"-triacetylchitotrioside was used. The major productions were obtained at pH 7.0 and 9.0, when colloidal chitin and glucose were used, whereas xylose and lactose were not good carbon sources. When testing different concentrations of colloidal chitin, glucose and glucosamine, colloidal chitin 0.5% was the best substrate, giving values of 2.4 U at the fifth day. When using glucose, best production occurred at 0.3% concentration, after 5 days growth, with values of 1.31 U. Endochitinase production was markedly decreased in high levels of glucose and in all glucosamine concentrations tested. SDS-PAGE co-polymerized with glycol-chitin analysis showed three major activity bands of 200, 100, and 95 kDa, when incubated at 50 degrees C.

1. We have investigated the inhibitory effects of blockers of volume-activated (Cl(vol)) and calcium-activated (Cl(Ca)) chloride channels on hypotonic solution (HS)-induced contractions of rat trachea, comparing their effects with those of the voltage-dependent calcium channel (VDCC) blocker nifedpine. 2. HS elicited large, stable contractions that were partially dependent on the cellular chloride gradient; a reduction to 41.45+/-7.71% of the control response was obtained when extracellular chloride was removed. In addition, HS-induced responses were reduced to 26.8+/-5.6% of the control by 1 microm nifedipine, and abolished under calcium-free conditions, indicating a substantial requirement for extracellular calcium entry, principally via VDCCs. 3. The established Cl(vol) blockers tamoxifen (

The phytopathogenic fungus Colletotrichum gloeosporioides was analyzed for chitinase activity, the best production occurring at the fourth day. A 43 kDa endochitinase with specific activity of 413 U microg(-1) protein was purified corresponding to a 75% yield. The optima of temperature and pH for the enzyme were 50 degrees C and pH 7.0, respectively. The enzyme showed a high stability at 50 degrees C and pH 7.0. Values of pH from 5.0 up to 7.0 gave, at least, 50% of maximum activity, suggesting a biotechnological application. Further studies are in progress to determine the possible use of this endochitinase in biological control.

Streptomyces are important microorganisms because of their capacity to produce numerous bioactive molecules. In the present work protease production, by Streptomyces sp. 594 isolated from a Brazilian Cerrado soil, was maximized by optimizing a low-cost culture medium composition (casitone and sugarcane molasses) using statistical experimental design. The final protease activity (56 U/mL) was 2.8-fold and 58-fold higher than that obtained in the beginning of this study, and in a previous work, using an actinomycete selection medium, respectively. Protease production, not growth associated, appeared to be modulated by an inducer system, whereby the C/N ratio seemed to play a significant role.

Aims: To investigate the effect of the culture supernatants from three newly isolated Streptomyces strains, 221, 235 and 606 on eukaryotic cells., Methods and Results: Cell lines were treated with the culture filtrates and assayed for protein synthesis by metabolic labelling, followed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis analysis. RNA synthesis was investigated by [5-3H]uridine incorporation. The three culture filtrates presented a strong inhibitory activity, reducing total protein synthesis of different eukaryotic cell lines by more than 85%. No effect on cellular RNA synthesis was detected. The culture filtrates did not affect the growth of the prokaryotic cells tested., Conclusions: These new Streptomyces strains, recently isolated from Brazilian tropical soils, produce molecule(s) with inhibitory activity specific to eukaryote protein synthesis., Significance and Impact of the Study: Streptomyces strains 221, 235 and 606, probably representing new species, might produce new bioactive compound(s), and can be used as valuable tools to study the protein synthesis pathway in eukaryotes.

ABSTRACT Three field experiments were conducted in 1997, 1998, and 1999 to investigate the effects of angular leaf spot and rust, separately or combined, on host growth and yield of individual bean plants (Phaseolus vulgaris). In each experiment, three treatments were established by inoculating cv. Carioca with Phaeoisariopsis griseola, Uromyces appendiculatus, or with both pathogens. An additional control treatment was not inoculated, but was sprayed with a fungicide. In the 1997 and 1999 experiments, angular leaf spot reached higher disease levels than rust, whereas in 1998, rust was more severe than angular leaf spot. Host growth, expressed as healthy leaf area duration (HAD), and yield were the highest in 1997 and lowest in 1998. In each experiment, the treatments did not differ significantly to the area under leaf area progress curve, HAD, and healthy leaf area absorption (HAA). All inoculated treatments had significantly more severe disease and less yield than the control treatment. Based on the analysis of 60 plants in each experiment, yield was not related to the areas under disease progress curve for either or both diseases. In 1997 and 1999, yield was related to HAD (R(2) = 0.57 and 0.43) and HAA(R(2) = 0.60 and 0.55). Based on the combined analysis of all 36 plots, angular leaf spot reduced the leaf area because of defoliation, whereas rust did not affect the leaf area. Rust reduced yield more than four times that of angular leaf spot, although the decrease in photosynthesis to angular leaf spot was twice that of rust.

Four hundred and fifteen actinomycete strains were screened for hyaluronidase activity in two plate assays media. In the first one, using hyaluronic acid as substrate and bovine serum albumin (BSA) to help precipitation of the nondegraded substrate, only strain 594 and hyaluronidase control were positive. In the second assay, plates with hyaluronic acid, but not BSA, gave the same results. For plates containing only BSA, proteinase activity was detected in strain 594. When hyaluronic acid was treated with pronase, the only clear zones, in the second assay without BSA, were those around hyaluronidase controls. Protease activity, commonly found in actinomycetes, was detected only in strain 594, among the 415 studied, when tested in hyaluronidase assay using hyaluronate plus BSA. This may be due to the composition of the growth medium, since media with different composition gave different results for protease activity in each of the 15 strains analyzed. These data suggest that proteases can affect an accurate detection of hyaluronidase in media containing proteins, not only from hyaluronate preparations, but also from other medium ingredients. Thus, for a correct interpretation of the method, they must be excluded. Commercial Hyaluronidase used as controls must be also tested for the presence of protease contamination.

Aims: The chitinolytic activity of an actinomycete, isolated from a tropical acidic ferrasol (FAO) under cerrado (savanna) vegetation, is reported., Methods and Results: Selection of the strain was based on spot inoculation on solid colloidal chitin medium. The use of chemotaxonomic, morphological and physiological procedures placed it in the Streptomyces genus, but identification to species level could not be achieved. A protein with endochitinase activity was isolated and purified from the supernatant fluid by concentration, precipitation, hydrophobic interaction, gel filtration and adsorption procedures. The molecular size of the purified chitinase was estimated by gel filtration to be 70 kDa, and its pI was 6.1. The enzyme had temperature and pH optima of 40 degrees C and 8.0, respectively, and showed thermal (30-70 degrees C) and pH (4-9) stabilities. Antifungal activity of the selected strain was observed following in vitro experiments using growing cells, crude extract or the purified endochitinase, and by detecting growth inhibition of the tested phytopathogenic fungi., Conclusion: Strain Streptomyces RC 1071 could not be placed into any known species, suggesting a new taxon. The purified endochitinase presented similar molecular weight, optimum temperature and pH activity, and stability of other endochitinolytic enzymes reported in the literature. In all three in vitro experiments performed, inhibition of growth of the phytopathogenic fungi used as test organisms was observed., Significance and Impact of the Study: Some of the endochitinase characteristics such as thermal stability, as well as pH tolerance, are very interesting for biotechnological purposes. In addition, due to its antifungal activity, Streptomyces RC 1071 seems promising for use in biological control.