Hypoxia–Ischemia Alters Nucleotide and Nucleoside Catabolism and Na+,K+-ATPase Activity in the Cerebral Cortex of Newborn Rats

It is well known that the levels of adenosine in the brain increase dramatically during cerebral hypoxic-ischemic (HI) insults. Its levels are tightly regulated by physiological and pathophysiological changes that occur during the injury acute phase. The aim of the present study was to examine the effects of the neonatal HI event on cytosolic and ecto-enzymes of purinergic system––NTPDase, 5′-nucleotidase (5′-NT) and adenosine deaminase (ADA)––in cerebral cortex of rats immediately post insult. Furthermore, the Na⁺/K⁺-ATPase activity, adenosine kinase (ADK) expression and thiobarbituric acid reactive species (TBARS) levels were assessed. Immediately after the HI event the cytosolic NTPDase and 5′-NT activities were increased in the cerebral cortex. In synaptosomes there was an increase in the ecto-ADA activity while the Na⁺/K⁺ ATPase activity presented a decrease. The difference between ATP, ADP, AMP and adenosine degradation in synaptosomal and cytosolic fractions could indicate that NTPDase, 5′-NT and ADA were differently affected after insult. Interestingly, no alterations in the ADK expression were observed. Furthermore, the Na⁺/K⁺-ATPase activity was correlated negatively with the cytosolic NTPDase activity and TBARS content. The increased hydrolysis of nucleotides ATP, ADP and AMP in the cytosol could contribute to increased adenosine levels, which could be related to a possible innate neuroprotective mechanism aiming at potentiating the ambient levels of adenosine. Together, these results may help the understanding of the mechanism by which adenosine is produced following neonatal HI injury, therefore highlighting putative therapeutical targets to minimize ischemic injury and enhance recovery.     

Tigutcystatin, a cysteine protease inhibitor from Triatoma infestans midgut expressed in response to Trypanosoma cruzi

The insect Triatoma infestans is a vector of Trypanosoma cruzi, the etiological agent of Chagas disease. A cDNA library was constructed from T. infestans anterior midgut, and 244 clones were sequenced. Among the EST sequences, an open reading frame (ORF) with homology to a cystatin type 2 precursor was identified. Then, a 288-bp cDNA fragment encoding mature cystatin (lacking signal peptide) named Tigutcystatin was cloned fused to a N-terminal His tag in pET-14b vector, and the protein expressed in Escherichia coli strain Rosetta gami. Tigutcystatin purified and cleaved by thrombin to remove His tag presented molecular mass of 11 kDa and 10,137 Da by SDS–PAGE and MALDI-TOF mass spectrometry, respectively. Purified Tigutcystatin was shown to be a tight inhibitor towards cruzain, a T. cruzi cathepsin L-like enzyme (K_i = 3.29 nM) and human cathepsin L (K_i = 3.78 nM). Tissue specific expression analysis showed that Tigutcystatin was mostly expressed in anterior midgut, although amplification in small intestine was also detected by semi quantitative RT-PCR. qReal time PCR confirmed that Tigutcystatin mRNA is significantly up-regulated in anterior midgut when T. infestans is infected with T. cruzi. Together, these results indicate that Tigutcystatin may be involved in modulation of T. cruzi in intestinal tract by inhibiting parasite cysteine proteases, which represent the virulence factors of this protozoan.     

Secondary forest fragments offer important carbon and biodiversity cobenefits

Tropical forests store large amounts of carbon and high biodiversity, but are being degraded at alarming rates. The emerging global Forest and Landscape Restoration (FLR) agenda seeks to limit global climate change by removing carbon dioxide from the atmosphere through the growth of trees. In doing so, it may also protect biodiversity as a free cobenefit, which is vital given the massive shortfall in funding for biodiversity conservation. We investigated whether natural forest regeneration on abandoned pastureland offers such cobenefits, focusing for the first time on the recovery of taxonomic diversity (TD), phylogenetic diversity (PD) and functional diversity (FD) of trees, including the recovery of threatened and endemic species richness, within isolated secondary forest (SF) fragments. We focused on the globally threatened Brazilian Atlantic Forest, where commitments have been made to restore 1 million hectares under FLR. Three decades after land abandonment, regenerating forests had recovered ~20% (72 Mg/ha) of the above‐ground carbon stocks of a primary forest (PF), with cattle pasture containing just 3% of stocks relative to PFs. Over this period, SF recovered ~76% of TD, 84% of PD and 96% of FD found within PFs. In addition, SFs had on average recovered 65% of threatened and ~30% of endemic species richness of primary Atlantic forest. Finally, we find positive relationships between carbon stock and tree diversity recovery. Our results emphasize that SF fragments offer cobenefits under FLR and other carbon‐based payments for ecosystem service schemes (e.g. carbon enhancements under REDD+). They also indicate that even isolated patches of SF could help to mitigate climate change and the biodiversity extinction crisis by recovering species of high conservation concern and improving landscape connectivity.     

Diversity of indigenous Beauveria and Metarhizium spp. in a commercial banana field and their virulence toward Cosmopolites sordidus (Coleoptera: Curculionidae)

Metarhizium anisopliae and Beauveria bassiana sensu lato were isolated, from 7 and 41 % of soil samples from a commercial banana field, with average fungal density of 4.3 × 10³ and 8.2 × 10³ CFU g^?1 soil, respectively. Twenty-one morphologically distinct B. bassiana and four M. anisopliae sensu lato isolates from different plots within the field were further characterized. ISSR fingerprinting revealed six different clusters for B. bassiana, whereas gene sequencing revealed three M. anisopliae sensu stricto and one unclassified Metarhizium sp. Bioassays with one or more representative isolates from each Metarhizium species and B. bassiana cluster showed that all indigenous isolates had lower virulence and significantly greater ST₅₀s than reference (exotic) isolates. The data suggest that the low virulence of most indigenous isolates toward Cosmopolites sordidus adults and their relatively low density in soil samples, may help explain the low occurrence of epizootics caused by entomopathogenic fungi in populations of this pest, also known to burrow under the soil surface in banana plantations.     

European Ancestry Predominates in Neuromyelitis Optica and Multiple Sclerosis Patients from Brazil

Background

Neuromyelitis optica (NMO) is considered relatively more common in non-Whites, whereas multiple sclerosis (MS) presents a high prevalence rate, particularly in Whites from Western countries populations. However, no study has used ancestry informative markers (AIMs) to estimate the genetic ancestry contribution to NMO patients.

Methods

Twelve AIMs were selected based on the large allele frequency differences among European, African, and Amerindian populations, in order to investigate the genetic contribution of each ancestral group in 236 patients with MS and NMO, diagnosed using the McDonald and Wingerchuck criteria, respectively. All 128 MS patients were recruited at the Faculty of Medicine of Ribeirão Preto (MS-RP), Southeastern Brazil, as well as 108 healthy bone marrow donors considered as healthy controls. A total of 108 NMO patients were recruited from five Neurology centers from different Brazilian regions, including Ribeirão Preto (NMO-RP).

Principal Findings

European ancestry contribution was higher in MS-RP than in NMO-RP (78.5% vs. 68.7%) patients. In contrast, African ancestry estimates were higher in NMO-RP than in MS-RP (20.5% vs. 12.5%) patients. Moreover, principal component analyses showed that groups of NMO patients from different Brazilian regions were clustered close to the European ancestral population.

Conclusions

Our findings demonstrate that European genetic contribution predominates in NMO and MS patients from Brazil.     

Hemostatic Dysfunction Is Increased in Patients with Hepatosplenic Schistosomiasis Mansoni and Advanced Periportal Fibrosis

Background

Schistosomiasis mansoni is an endemic parasitic disease and a public health problem in Northeast Brazil. In some patients, hepatic abnormalities lead to periportal fibrosis and result in the most severe clinical form, hepatosplenic schistosomiasis. This study aimed to evaluate whether abnormal blood coagulation and liver function tests in patients with hepatosplenic schistosomiasis (n = 55) correlate with the severity of their periportal fibrosis.

Methodology/Principal Findings

Blood samples were used for liver function tests, hemogram and prothrombin time (International Normalized Ratio, INR). The blood coagulation factors (II, VII, VIII, IX and X), protein C and antithrombin IIa (ATIIa), plasminogen activator inhibitor 1 (PAI-1) and D-dimer were measured by photometry or enzyme linked immunosorbent assay. Hyperfibrinolysis was defined on the basis of PAI-1 levels and a D-dimer concentration greater than a standard cut-off of 483 ng/mL. Standard liver function tests were all abnormal in the patient group compared to healthy controls (n = 29), including raised serum transaminases (p<0.001) and lower levels of albumin (p = 0.0156). Platelet counts were 50% lower in patients, while for coagulation factors there was a 40% increase in the INR (p<0.001) and reduced levels of Factor VII and protein C in patients compared to the controls (both p<0.001). Additionally, patients with more advanced fibrosis (n = 38) had lower levels of protein C compared to those with only central fibrosis (p = 0.0124). The concentration of plasma PAI-1 in patients was one-third that of the control group (p<0.001), and D-dimer levels 2.2 times higher (p<0.001) with 13 of the 55 patients having levels above the cut-off.

Conclusion/Significance

This study confirms that hemostatic abnormalities are associated with reduced liver function and increased liver fibrosis. Of note was the finding that a quarter of patients with hepatosplenic schistosomiasis and advanced periportal fibrosis have hyperfibrinolysis, as judged by excessive levels of D-dimer, which may predispose them to gastrointestinal bleeding.     

Phage Therapy Is Effective against Infection by Mycobacterium ulcerans in a Murine Footpad Model

Background

Buruli Ulcer (BU) is a neglected, necrotizing skin disease caused by Mycobacterium ulcerans. Currently, there is no vaccine against M. ulcerans infection. Although the World Health Organization recommends a combination of rifampicin and streptomycin for the treatment of BU, clinical management of advanced stages is still based on the surgical resection of infected skin. The use of bacteriophages for the control of bacterial infections has been considered as an alternative or to be used in association with antibiotherapy. Additionally, the mycobacteriophage D29 has previously been shown to display lytic activity against M. ulcerans isolates.

Methodology/Principal findings

We used the mouse footpad model of M. ulcerans infection to evaluate the therapeutic efficacy of treatment with mycobacteriophage D29. Analyses of macroscopic lesions, bacterial burdens, histology and cytokine production were performed in both M. ulcerans-infected footpads and draining lymph nodes (DLN). We have demonstrated that a single subcutaneous injection of the mycobacteriophage D29, administered 33 days after bacterial challenge, was sufficient to decrease pathology and to prevent ulceration. This protection resulted in a significant reduction of M. ulcerans numbers accompanied by an increase of cytokine levels (including IFN-γ), both in footpads and DLN. Additionally, mycobacteriophage D29 treatment induced a cellular infiltrate of a lymphocytic/macrophagic profile.

Conclusions/Significance

Our observations demonstrate the potential of phage therapy against M. ulcerans infection, paving the way for future studies aiming at the development of novel phage-related therapeutic approaches against BU.