Generation and characterization of monoclonal antibody against Advanced Glycation End Products in chronic kidney disease

Advanced Glycation End Products (AGEs) are toxins that are involved in structural and functional alterations of several organs and tissues, resulting in various pathologies. Several types of AGEs have been described but carboxymethyllysine (CML) is the major antigenic AGE compound. In this study, three different immunogenic carrier proteins (KLH, keyhole limpet hemocyanin; BSA, bovine serum albumin; and HSA, human serum albumin) were modified by glycation. The glycated molecules were used to produce epitope-specific monoclonal antibodies able to recognize the CML domain and to detect uremic toxins in the serum of patients with chronic kidney disease (CKD). A competitive ELISA was standardized in order to quantify CML in the sera of CKD patients. An increase in uremic toxins can compromise the clinical condition of these patients, thus, the detection and quantification of these toxins should contribute to a better management and understanding of this disease.     

Nucleocytoplasmic Shuttling Activity of Ataxin-3

Spinocerebellar ataxia type-3, also known as Machado-Joseph Disease (MJD), is one of many inherited neurodegenerative disorders caused by polyglutamine-encoding CAG repeat expansions in otherwise unrelated genes. Disease protein misfolding and aggregation, often within the nucleus of affected neurons, characterize polyglutamine disorders. Several evidences have implicated the nucleus as the primary site of pathogenesis for MJD. However, the molecular determinants for the nucleocytoplasmic transport of human ataxin-3 (Atx3), the protein which is mutated in patients with MJD, are not characterized.In order to characterize the nuclear shuttling activity of Atx3, we performed yeast nuclear import assays and found that Atx3 is actively imported into the nucleus, by means of a classical nuclear localizing sequence formed by a cluster of lysine and arginine residues. On the other hand, when active nuclear export was inhibited using leptomycin B, a specific inhibitor of the nuclear export receptor CRM1, both endogenous Atx3 and transfected GFP-Atx3 accumulated inside the nucleus of a subpopulation of COS-7 cells, whereas both proteins are normally predominant in the cytoplasm.Additionally, using a Rev(1.4)-GFP nuclear export assay, we performed an extensive analysis of six putative aliphatic nuclear export motifs identified in Atx3 amino acid sequence. Although none of the tested peptide sequences were found to drive nuclear export when isolated, we have successfully mapped the region of Atx3 responsible for its CRM1-independent nuclear export activity. Curiously, the N-terminal Josephin domain alone is exported into the cytoplasm, but the nuclear export activity of Atx3 is significantly enhanced in a longer construct that is truncated after the two ubiquitin interaction motifs, upstream from the polyQ tract.Our data show that Atx3 is actively imported to and exported from the cell nucleus, and that its nuclear export activity is dependent on a motif located at its N-terminal region. Since pathological Atx3 aggregates in the nucleus of affected neurons in MJD, and there is in vivo evidence that nuclear localization of Atx3 is required for the manifestation of symptoms in MJD, defects in the nucleocytoplasmic shuttling activity of the protein may be involved in the nuclear accumulation and aggregation of expanded Atx3.     

Comparison of Interferon-γ Release Assay to Two Cut-Off Points of Tuberculin Skin Test to Detect Latent Mycobacterium tuberculosis Infection in Primary Health Care Workers

Background

An interferon-γ release assay, QuantiFERON-TB (QFT) test, has been introduced an alternative test for the diagnosis of latent Mycobacterium tuberculosis infection (LTBI). Here, we compared the performance of QFT with tuberculin skin test (TST) measured at two different cut-off points among primary health care work (HCW) in Brazil.

Methods

A cross-sectional study was carried out among HCWs in four Brazilian cities with a known history of high incidence of TB. Results of the QFT were compared to TST results based on both ≥5 mm and ≥10 mm as cut-off points.

Results

We enrolled 632 HCWs. When the cut-off value of ≥10 mm was used, agreement between QFT and TST was 69% (k = 0.31), and when the cut-off of ≥5 mm was chosen, the agreement was 57% (k = 0.22). We investigated possible factors of discordance of TST vs QFT. Compared to the TST−/QFT− group, risk factors for discordance in the TST+/QFT− group with TST cut-off of ≥5 mm included age between 41–45 years [OR = 2.70; CI 95%: 1.32–5.51] and 46–64 years [OR = 2.04; CI 95%: 1.05–3.93], BCG scar [OR = 2.72; CI 95%: 1.40–5.25], and having worked only in primary health care [OR = 2.30; CI 95%: 1.09–4.86]. On the other hand, for the cut-off of ≥10 mm, BCG scar [OR = 2.26; CI 95%: 1.03–4.91], being a household contact of a TB patient [OR = 1.72; CI 95%: 1.01–2.92] and having had a previous TST [OR = 1.66; CI 95%: 1.05–2.62], were significantly associated with the TST+/QFT− group. No statistically significant associations were found among the TST−/QFT+ discordant group with either TST cut-off value.

Conclusions

Although we identified BCG vaccination to contribute to the discordance at both TST cut-off measures, the current Brazilian recommendation for the initiation of LTBI treatment, based on information gathered from medical history, TST, chest radiograph and physical examination, should not be changed.     

Lactate metabolism in the muscle of the crab Chasmagnathus granulatus during hypoxia and post-hypoxia recovery

The present study showed that the lactate/glucose ratio in the hemolymph of Chasmagnathus granulatus maintained in normoxia (controls) was 4.9, suggesting that lactate is an important substrate for this crab. Periods of hypoxia are part of the biological cycle of this crab, and lactate is the main end product of anaerobiosis in this crab. Our hypothesis was that this lactate would be, therefore, used by gluconeogenic pathway or can be oxidized or excreted to the aquatic medium during hypoxia and post-hypoxia periods in C. granulatus. The concentrations of hemolymphatic lactate in animals in normoxia are high, and are used as an energy substrate. In hypoxia, muscle gluconeogenesis and excretion of lactate to the aquatic medium would contribute significantly in regulating the concentration of circulating lactate. Utilization of these pathways would serve the objective of maintaining the acid-base equilibrium of the organism. Muscle gluconeogenesis participates, during the recovery process, in metabolizing the lactate produced during the period of hypoxia. Lactate excretion to the external medium, was one of the strategies used to decrease the higher hemolymphatic lactate levels. However, oxidation of lactate in the muscle is not a main strategy used by this crab to metabolize lactate in the recovery periods.     

Daily variation of melatonin content in the optic lobes of the crab Neohelice granulata

Melatonin is a biogenic amine, known from almost all phyla of living organisms. In vertebrates melatonin is produced rhythmically in the pinealocytes of the pineal gland, relaying information of the environmental light/dark cycle to the organism. With regard to crustaceans only a handful of studies exist that has attempted to identify the presence and possible daily variation of this substance. We set out to investigate whether in the crab Neohelice granulata melatonin was produced in the optic lobes of these animals and underwent rhythmic fluctuations related to the daily light/dark cycle. Our experimental animals were divided into three groups exposed to different photoperiods: normal photoperiod (12L:12D), constant dark (DD), and constant light (LL). The optic lobes were collected every 4 hours over a 24-h period for melatonin quantification by radioimmunoassay (RIA). N. granulata kept under 12 L:12D and DD conditions, showed daily melatonin variations with two peaks of abundance (p<0.05), one during the day and another, more extensive one, at night. Under LL-conditions no significant daily variations were noticeable (p>0.05). These results demonstrate the presence of a daily biphasic fall and rise of melatonin in the eyestalk of N. granulata and suggest that continuous exposure to light inhibits the production of melatonin synthesis.     

Anti-plasmodium activity of angiotensin II and related synthetic peptides

Plasmodium species are the causative agents of malaria, the most devastating insect-borne parasite of human populations. Finding and developing new drugs for malaria treatment and prevention is the goal of much research. Angiotensins I and II (ang I and ang II) and six synthetic related peptides designated Vaniceres 1-6 (VC1-VC6) were assayed in vivo and in vitro for their effects on the development of the avian parasite, Plasmodium gallinaceum. Ang II and VC5 injected into the thoraces of the insects reduced mean intensities of infection in the mosquito salivary glands by 88% and 76%, respectively. Although the mechanism(s) of action is not completely understood, we have demonstrated that these peptides disrupt selectively the P.gallinaceum cell membrane. Additionally, incubation in vitro of sporozoites with VC5 reduced the infectivity of the parasites to their vertebrate host. VC5 has no observable agonist effects on vertebrates, and this makes it a promising drug for malaria prevention and chemotherapy.     

Increased quality of in natura and cryopreserved semen of water buffaloes supplemented with saturated and unsaturated fatty acids from the palm oil industry

Ruminant energy supplementation with vegetable oils or fats has been standing out worldwide and oil palm processing has been receiving growing interest. This study assessed the effect of supplementation with saturated and unsaturated fatty acids from the palm oil industry on the lipid profile of seminal plasma and of the sperm membrane, as well as on the morphological and functional characteristics of raw and cryopreserved buffalo semen. Twelve purebred Murrah bulls (Bubalus bubalis) were assigned to the experimental groups and fed diets for 120 days with no added lipids (CONT, four bulls), or with an extra amount of 3% lipids from crude palm oil (PALM, four bulls), or from palm oil deodorizer distillate (PODD, four bulls). Semen was collected and cryopreserved every 15 days. The lipid composition of membranes and semen quality were determined after collections. Lipid supplementation did not impact feed intake (P>0.05). Diet enrichment with PALM increased the linoleic acid (C18:2,ω6) in seminal plasma. Lipid supplementation did not increase the polyunsaturated fatty acids in the sperm membrane composition, but significantly increased the lignoceric acid (C24:0). Cryopreserved semen of the supplemented bulls presented higher progressive motility (60.2 vs. 67.9 vs. 65.2%; P<0.05) and sperm viability detected by eosin-nigrosin staining (61.1 vs. 69.4 vs. 67.8%; P<0.05). Palm oil reduced major sperm defects in both raw (12.2 vs. 9.3 vs. 13.2%; P<0.0001) and cryopreserved semen (12.4 vs. 9.4 vs. 11.2%; P<0.0001). The lipids added to the diet did not impact the population of spermatozoa with intact plasma and acrosomal membranes (PI-/PSA-), but significantly increased the percentage of spermatozoa with high mitochondrial potential (25.6 vs. 31.5 vs. 32.0%; P=0.008). The results suggest that lipid supplementation based on crude palm oil or palm oil deodorizer distillate can be safely used to feed buffalo bulls and may increase sperm attributes related to male fertility.     

Theory does not meet experiment: transient dynamics changes patterns of exclusion in an intraguild predation system

Population Ecology - Although empirical verifications of ecological theory are essential for the advance of our understanding of ecosystems functioning, they are often hard to obtain or even...     

Snakebites in “Invisible Populations”: A cross-sectional survey in riverine populations in the remote western Brazilian Amazon

In the Brazilian Amazon, long distances, low healthcare coverage, common use of ineffective or deleterious self-care practices, and resistance to seeking medical assistance have an impact on access to antivenom treatment. This study aimed to estimate snakebite underreporting, and analyze barriers that prevent victims from obtaining healthcare in communities located in 15 municipalities on the banks of the Solimões, Juruá and Purus Rivers, in the remote Western Brazilian Amazon. Information on the participants’ demographics, previous snakebites, access to healthcare, time taken to reach medical assistance, use of self-care practices, and the reason for not accessing healthcare were collected through semi-structured interviews. In the case of deaths, information was collected by interviewing parents, relatives or acquaintances. A total of 172 participants who reported having suffered snakebites during their lifetime were interviewed. A total of 73 different treatment procedures was reported by 65.1% of the participants. Participants living in different river basins share few self-care procedures that use traditional medicine, and 91 (52.9%) participants reported that they had access to healthcare. Living in communities along the Juruá River [OR = 12.6 (95% CI = 3.2–49.7; p<0.001)] and the use of traditional medicine [OR = 11.6 (95% CI = 3.4–39.8; p<0.001)] were variables that were independently associated to the lack of access to healthcare. The main reasons for not accessing healthcare were the pprioritization of traditional treatments (70.4%), and the failure to recognize the situation as being potentially severe (50.6%). Four deaths from complications arising from the snakebite were reported, and three of these were from communities on the banks of the Juruá River. Only one of these received medical assistance. We found an unexpectedly high underreporting of snakebite cases and associated deaths. Snakebite victims utilized three main different healing systems: 1) self-care using miscellaneous techniques; 2) official medical healthcare generally combined with traditional practices; and 3) self-care using traditional practices combined with Western medicines. To mitigate snakebite burden in the Brazilian Amazon, an innovative intervention that would optimize timely delivery of care, including antivenom distribution among existing community healthcare centers, is needed.     

Glass transition temperature of hard chairside reline materials after post‐polymerisation treatments

doi:10.1111/j.1741‐2358.2009.00312.x
Glass transition temperature of hard chairside reline materials after post‐polymerisation treatments Objective: This study evaluated the effect of post‐polymerisation treatments on the glass transition temperature (T_g) of five hard chairside reline materials (Duraliner II‐D, Kooliner‐K, New Truliner‐N, Ufi Gel hard‐U and Tokuso Rebase Fast‐T). Materials and methods: Specimens (10 × 10 × 1 mm) were made following the manufacturers’ instructions and divided into three groups (n = 5). Control group specimens were left untreated. Specimens from the microwave group were irradiated with pre‐determined power/time combinations, and specimens from the water‐bath group were immersed in hot water at 55°C for 10 min. Glass transition (°C) was performed by differential scanning calorimetry. Data were analysed using anova, followed by post hoc Tukey’s test (α = 0.05). Results: Both post‐polymerisation treatments promoted a significant (p < 0.05) increase in the T_g of reline material K. Materials K, D and N showed the lowest T_g (p < 0.05). No significant difference between T and U specimens was observed. Conclusion: Post‐polymerisation treatments improved the glass transition of material Kooliner, with the effect being more pronounced for microwave irradiation.