Latest Advances on Bacterial Cellulose‐Based Materials for Wound Healing,Delivery Systems,and Tissue Engineering

Bacterial cellulose (BC) is a nanocellulose form produced by some nonpathogenic bacteria. BC presents unique physical, chemical, and biological properties that make it a very versatile material and has found application in several fields, namely in food industry, cosmetics, and biomedicine. This review overviews the latest state‐of‐the‐art usage of BC on three important areas of the biomedical field, namely delivery systems, wound dressing and healing materials, and tissue engineering for regenerative medicine. BC will be reviewed as a promising biopolymer for the design and development of innovative materials for the mentioned applications. Overall, BC is shown to be an effective and versatile carrier for delivery systems, a safe and multicustomizable patch or graft for wound dressing and healing applications, and a material that can be further tuned to better adjust for each tissue engineering application, by using different methods.     

Monitoring the colonization of sugarcane and rice plants by the endophytic diazotrophic bacterium Gluconacetobacter diazotrophicus marked with gfp and gusA reporter genes

Aims: To evaluate the colonization process of sugarcane plantlets and hydroponically grown rice seedlings by Gluconacetobacter diazotrophicus strain PAL5 marked with the gusA and gfp reporter genes. Methods and Results: Sugarcane plantlets inoculated in vitro with PAL5 carrying the gfp::gusA plasmid pHRGFPGUS did not present green fluorescence, but β‐glucuronidase (GUS)‐stained bacteria could be observed inside sugarcane roots. To complement this existing inoculation methodology for micropropagated sugarcane with a more rapid colonization assay, we employed hydroponically grown gnotobiotic rice seedlings to study PAL5–plant interaction. PAL5 could be isolated from the root surface (10⁸ CFU g^?1) and from surface‐disinfected root and stem tissues (10⁴ CFU g^?1) of inoculated plants, suggesting that PAL5 colonized the internal plant tissues. Light microscopy confirmed the presence of bacteria inside the root tissue. After inoculation of rice plantlets with PAL5 marked with the gfp plasmid pHRGFPTC, bright green fluorescent bacteria could be seen colonizing the rice root surface, mainly at the sites of lateral root emergence, at root caps and on root hairs. Conclusion: The plasmids pHRGFPGUS and pHRGFPTC are valid tools to mark PAL5 and monitor the colonization of micropropagated sugarcane and hydroponic rice seedlings. Significance and Impact of the Study: These tools are of use to: (i) study PAL5 mutants affected in bacteria–plant interactions, (ii) monitor plant colonization in real time and (iii) distinguish PAL5 from other bacteria during the study of mixed inoculants.     

Aspartic proteinase in Dugesia tigrina (Girard) planaria

A proteolytic activity was identified in Dugesia tigrina planaria using the chromogenic substrate Phe-Ala-Ala-Phe (4-NO2)-Phe-Val-Leu-O4MP. The activity of the enzyme increased four times during the regeneration and presented a maximum at 120 hr being higher in tail than head regenerating segments. The protease that displays this activity was purified from worms by a single step on pepstatin-agarose followed by gel-filtration high performance liquid chromatography. The purification resulted in a 34-fold increase in specific activity and the final yield was 10%. The active D. tigrina hydrolase appears to be a dimeric protein composed of identical subunits with 34 kDa associated by disulphide bridges similar to vertebrate cathepsin D. By SDS-PAGE several bands were detected but upon gel filtration HPLC one proteolytically active component, termed Asp-68, was detected and isolated. The maximal activity was observed in a range between pH 3.5-5.0 and the enzyme became inactivated at a pH value above 7.2. The purified enzyme was not inhibited by inhibitors from serine (aprotinin, TPCK, PMSF and TLCK), metallo (EDTA) and cysteine proteinase (E-64) classes. In contrast, inhibitors such as pepstatin, EPNP, and 4-beta-PMA efficiently inhibited the activity of the 68-kDa protease.     

Ecological Life Table of Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae)

An ecological life table was constructed, aiming to determine the critical stages and key mortality factors of Tuta absoluta (Lepidoptera: Gelechiidae). The total population mortality of this tomato leafminer was 92.3%. During the egg stage the mortality was 58.7%, mainly due to egg inviability. A total of 8.6% egg parasitism by Trichogramma pretiosum (Riley) (Hymenoptera: Trichogrammatidae) and 5.0% egg predation by Xylocoris sp. (Heteroptera: Anthocoridae), Cycloneda sanguinea (L.) (Coleoptera: Coccinellidae) and members of the family Phlaeothripidae (Thysanoptera) was observed. The mortality of the larval stage was 33.0%. This was considered to be the critical stage as it showed the highest apparent mortality (79.8%). Larval parasitism was low (0.1%), and was only found with Goniozus nigrifemur Ashmead (Hymenoptera: Bethylidae). Predators were responsible for 79.4% of larval mortality. Therefore, their attraction to and maintenance in the target area are important management tactics to be considered for T. absoluta control. The first and second instars were considered to be the most critical, and predation by the above mentioned species was the key mortality factor. The mortality at the pupal stage was low (0.6%) and was due to malformation.     

Conventional sampling plan for the green leafhopper Empoasca kraemeri in common beans

Abstract:  A conventional sampling plan for the leafhopper Empoasca kraemeri Ross & Moore (Auchenorrhyncha: Cicadellidae) in common beans (Phaseolus vulgaris L.) is presented based on samples from 51 commercial fields. The research encompassed three phases. The first phase had the objective of determining the most suitable leaf to be used as a precise and representative sampling unit. In the second phase of the project, the available sampling methods for E. kraemeri were compared, including the approach identified in the first phase. In the third phase, the theoretical frequency distribution of the sampling data was assessed and the number of samples necessary for the sampling plan was established. The best leaves to sample adult leafhoppers were the fourth and the fifth from the apex, while the best leaf for sampling nymphs was the fifth from the apex. The best sampling technique for nymphs and adults was the beating of the apical leaves against a plastic tray. The sampling data obtained with this technique were fit to a negative binomial distribution with common aggregation parameter for adults ( K _common = 1.04) and nymphs ( K _common = 0.47). This sampling plan required 16.8 and 31.2 min to be carried out at an expense of US$ 0.31 and US$ 0.54 for sampling adults and nymphs in a field, respectively.     

Vaccinia virus Transmission through Experimentally Contaminated Milk Using a Murine Model

Bovine vaccinia (BV) is a zoonosis caused by Vaccinia virus (VACV), which affects dairy cattle and humans. Previous studies have detected the presence of viable virus particles in bovine milk samples naturally and experimentally contaminated with VACV. However, it is not known whether milk contaminated with VACV could be a route of viral transmission. However, anti-Orthopoxvirus antibodies were detected in humans from BV endemic areas, whom had no contact with affected cows, which suggest that other VACV transmission routes are possible, such as consumption of contaminated milk and dairy products. Therefore, it is important to study the possibility of VACV transmission by contaminated milk. This study aimed to examine VACV transmission, pathogenesis and shedding in mice orally inoculated with experimentally contaminated milk. Thirty mice were orally inoculated with milk containing 10⁷ PFU/ml of VACV, and ten mice were orally inoculated with uncontaminated milk. Clinical examinations were performed for 30 consecutive days, and fecal samples and oral swabs (OSs) were collected every other day. Mice were euthanized on predetermined days, and tissue and blood samples were collected. Nested-PCR, plaque reduction neutralization test (PRNT), viral isolation, histopathology, and immunohistochemistry (IHC) methods were performed on the collected samples. No clinical changes were observed in the animals. Viral DNA was detected in feces, blood, OSs and tissues, at least in one of the times tested. The lungs displayed moderate to severe interstitial lymphohistiocytic infiltrates, and only the heart, tonsils, tongue, and stomach did not show immunostaining at the IHC analysis. Neutralizing antibodies were detected at the 20^th and 30^th days post infection in 50% of infected mice. The results revealed that VACV contaminated milk could be a route of viral transmission in mice experimentally infected, showing systemic distribution and shedding through feces and oral mucosa, albeit without exhibiting any clinical signs.     

Risk Factors for Death from Influenza A(H1N1)pdm09, State of S?o Paulo,Brazil, 2009

This case-control study aimed to assess the risk factors for death from influenza A(H1N1)pdm09 in patients with laboratory confirmation, who had severe acute respiratory illness-SARI and were hospitalized between June 28^th and August 29^th 2009, in the metropolitan regions of São Paulo and Campinas, Brazil. Medical charts of all the 193 patients who died (cases) and the 386 randomly selected patients who recovered (controls) were investigated in 177 hospitals. Household interviews were conducted with those who had survived and the closest relative of those who had died. 73.6% of cases and 38.1% of controls were at risk of developing influenza-related complications. The 18-to-59-year age group (OR = 2.31, 95%CI: 1.31–4.10 (reference up to 18 years of age)), presence of risk conditions for severity of influenza (OR = 1.99, 95%CI: 1.11–3.57, if one or OR = 6.05, 95%CI: 2.76–13.28, if more than one), obesity (OR = 2.73, 95%CI: 1.28–5.83), immunosuppression (OR = 3.43, 95%CI: 1.28–9.19), and search for previous care associated with the hospitalization (OR = 3.35, 95%CI: 1.75–6.40) were risk factors for death. Antiviral treatment performed within 72 hours of the onset of symptoms (OR = 0.17, 95%CI: 0.08–0.37, if within 48hours, and OR = 0.30, 95%CI: 0.11–0.81, if between 48 and 72 hours) was protective against death. The identification of high-risk patients and early treatment are important factors for reducing morbi-mortality from influenza.     

Yellow Fever 17DD Vaccine Virus Infection Causes Detectable Changes in Chicken Embryos

The yellow fever (YF) 17D vaccine is one of the most effective human vaccines ever created. The YF vaccine has been produced since 1937 in embryonated chicken eggs inoculated with the YF 17D virus. Yet, little information is available about the infection mechanism of YF 17DD virus in this biological model. To better understand this mechanism, we infected embryos of Gallus gallus domesticus and analyzed their histopathology after 72 hours of YF infection. Some embryos showed few apoptotic bodies in infected tissues, suggesting mild focal infection processes. Confocal and super-resolution microscopic analysis allowed us to identify as targets of viral infection: skeletal muscle cells, cardiomyocytes, nervous system cells, renal tubular epithelium, lung parenchyma, and fibroblasts associated with connective tissue in the perichondrium and dermis. The virus replication was heaviest in muscle tissues. In all of these specimens, RT-PCR methods confirmed the presence of replicative intermediate and genomic YF RNA. This clearer characterization of cell targets in chicken embryos paves the way for future development of a new YF vaccine based on a new cell culture system.