Assessing the effect of mercury pollution on cultured benthic foraminifera community using morphological and eDNA metabarcoding approaches.

Mercury (Hg) is a highly toxic element for living organisms and is known to bioaccumulate and biomagnify. Here, we analyze the response of benthic foraminifera communities cultured in mesocosm and exposed to different concentrations of Hg. Standard morphological analyses and environmental DNA metabarcoding show evidence that Hg pollution has detrimental effects on benthic foraminifera. The molecular analysis provides a more complete view of foraminiferal communities including the soft-walled single-chambered monothalamiids and small-sized hard-shelled rotaliids and textulariids than the morphological one. Among these taxa that are typically overlooked in morphological studies we found potential bioindicators of Hg pollution. The mesocosm approach proves to be an effective method to study benthic foraminiferal responses to various types and concentrations of pollutants over time. This study further supports foraminiferal metabarcoding as a complementary and/or alternative method to standard biomonitoring program based on the morphological identification of species communities.

The effect of the aqueous extract Peumus boldus on the proliferation of hepatocytes and liver function in rats submitted to expanded hepatectomy.

PURPOSE:: To assess the effect of aqueous extract of Peumus Boldus (AEPB) on the liver proliferative response after parcial hepatectomy of 70% (PH) in rodents. METHODS:: Twenty Wistar rats were divided in two groups: AEPB100 (whose rats received 100mg/Kg of AEPB, once a day, orally, in 4 days prior to the first surgical procedure) and Vehicle (whose rats were treated similarly with distilled water). Both groups underwent PH. After 24 hours the remaining livers were removed for studying the proliferation of hepatocytes by Ki-67 and 2mL of blood were collected for serological assessment: cholesterol, glucose, triglycerides, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, and total, direct and indirect bilirubin. All data were analyzed by Gaussian distribution. Statistically significant differences between mean values were analyzed using T Student's test. Non-Gaussian data were analyzed using Mann-Whitney's test. RESULTS:: The liver of all these rats presented positive staining of Ki-67, indicating liver proliferation. Laboratory results showed no significant difference in serum values between the analyzed groups. The analysis of Ki-67 was significantly more positive in AEPB100 group than in Vehicle group. CONCLUSION:: Aqueous extract of Peumus Boldus acute administration exerts significant positive effect on liver regeneration after 24h in rats that underwent parcial hepatectomy, while maintaining unchanged hepatic function.

The effect of the aqueous extract Peumus boldus on the proliferation of hepatocytes and liver function in rats submitted to expanded hepatectomy

ABSTRACT PURPOSE: To assess the effect of aqueous extract of Peumus Boldus (AEPB) on the liver proliferative response after parcial hepatectomy of 70% (PH) in rodents. METHODS: Twenty Wistar rats were divided in two groups: AEPB100 (whose rats received 100mg/Kg of AEPB, once a day, orally, in 4 days prior to the first surgical procedure) and Vehicle (whose rats were treated similarly with distilled water). Both groups underwent PH. After 24 hours the remaining livers were removed for studying the proliferation of hepatocytes by Ki-67 and 2mL of blood were collected for serological assessment: cholesterol, glucose, triglycerides, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, and total, direct and indirect bilirubin. All data were analyzed by Gaussian distribution. Statistically significant differences between mean values were analyzed using T Student's test. Non-Gaussian data were analyzed using Mann-Whitney's test. RESULTS: The liver of all these rats presented positive staining of Ki-67, indicating liver proliferation. Laboratory results showed no significant difference in serum values between the analyzed groups. The analysis of Ki-67 was significantly more positive in AEPB100 group than in Vehicle group. CONCLUSION: Aqueous extract of Peumus Boldus acute administration exerts significant positive effect on liver regeneration after 24h in rats that underwent parcial hepatectomy, while maintaining unchanged hepatic function.

Occurrence of enterotoxin-encoding genes in Staphylococcus aureus causing mastitis in lactating goats / Ocorrência de genes codificadores de enterotoxinas em Staphylococcus aureus associados a mastite em cabras em lactação

Staphylococcal enterotoxins are the leading cause of human food poisoning worldwide. Staphylococcus spp. are the main mastitis-causing agents in goats and frequently found in high counts in goat milk. This study aimed to investigate the occurrence of enterotoxin-encoding genes in Staphylococcus aureus associated with mastitis in lactating goats in Paraiba State, Brazil. Milk samples (n=2024) were collected from 393 farms. Staphylococcus aureus was isolated in 55 milk samples. Classical (sea, seb, sec, sed, see) and novel (seg, seh, sei) enterotoxin-encoding genes were investigated by means of polymerase chain reaction (PCR). From thirty-six tested isolates, enterotoxin-encoding genes were detected in 7 (19.5 percent) S. aureus. The gene encoding enterotoxin C (seC) was identified in six isolates, while seiwas observed in only one isolate. The genes sea, seb, sed, see, seg and seh were not observed amongst the S. aureus investigated in this study. In summary, S. aureus causing mastitis in goats can harbor enterotoxin-encoding genes and seC was the most frequent gene observed amongst the investigated isolates. This finding is important for surveillance purposes, since enterotoxin C should be investigated in human staphylococcal food poisoning outbreaks caused by consumption of goat milk and dairy products.

As enterotoxinas estafilocócicas são as principais causas de intoxicação alimentar em humanos em todo o mundo. O principal agente causador da mastite caprina são os Staphylococcus spp., frequentemente encontrado em altas contagens no leite caprino. Este estudo objetivou investigar a ocorrência de genes codificadores de enterotoxinas em Staphylococus aureus associados com mastite em cabras em lactação no estado da Paraíba, Brasil. As amostras de leite (n=2024) foram coletadas em 393 propriedades. Foram isolados 55 S. aureus em amostras de leite. Os genes codificadores de enterotoxinas clássicas (sea, seb, sec, sed, see) e as novas (seg, seh, sei) foram investigadas por meio de reação em cadeia de polimerase (PCR). Foram testados trinta e seis isolados, foram detectados 7 (19,5 por cento) genes codificadores de enterotoxinas de S. aureus. O gene codificador da enterotoxina C (sec) foi identificado em seis isolados, enquanto sei foi observado em apenas um isolado. Os genes sea, seb, sed, see, seg e seh não foram observados entre os S. aureus investigados neste estudo. Em síntese, a mastite caprina causada por S. aureus pode abrigar genes codificadores de enterotoxinas e sec foi o gene mais frequentemente observado entre os isolados investigados. Essa descoberta é importante para fins de vigilância e a enterotoxina C deve ser investigada em surtos de intoxicações alimentares estafilocócicas em humanos causadas pelo consumo de leite caprino e seus derivados.

Absorption spectroscopy.

Absorption spectroscopy is one of the most widely used techniques employed for determining the concentrations of absorbing species (chromophores) in solutions. It is a nondestructive technique which biologists and biochemists and now systems biologists use to quantify the cellular components and characteristic parameters of functional molecules. This quantification is most relevant in the context of systems biology. For creating a quantitative depiction of a metabolic pathway, a number of parameters and variables are important and these need to be determined experimentally. This chapter describes the UV-visible absorption spectroscopy used to produce experimental data for bottom-up modeling approaches of systems biology which uses concentrations and kinetic parameters (K(m) and V(max)) of enzymes of metabolic/signaling pathways, intracellular concentrations of metabolites and fluxes. It also briefly describes the application of this technique for quantification of biomolecules and investigating biomolecular interactions.

Systems biochemistry in practice: experimenting with modelling and understanding, with regulation and control.

Biology and medicine have become 'big science', even though we may not always like this: genomics and the subsequent analysis of what the genomes encode has shown that interesting living organisms require many more than 300 gene products to interact. We once thought that somewhere in this jungle of interacting macromolecules was hidden the molecule that constitutes the secret of Life, and therewith of health and disease. Now we know that, somehow, the secret of Life is the jungle of interactions. Consequently, we need to find the Rosetta Stones, i.e. interpretations of this jungle of systems biology. We need to find, perhaps convoluted, paths of understanding and intervention. Systems biochemistry is a good place to start, as it has the foothold that what goes in must come out. In the present paper, we review two strategies, which look at control and regulation. We discuss the difference between control and regulation and prove a relationship between them.

Reconfiguration of yeast 40S ribosomal subunit domains by the translation initiation multifactor complex.

In the process of protein synthesis, the small (40S) subunit of the eukaryotic ribosome is recruited to the capped 5' end of the mRNA, from which point it scans along the 5' untranslated region in search of a start codon. However, the 40S subunit alone is not capable of functional association with cellular mRNA species; it has to be prepared for the recruitment and scanning steps by interactions with a group of eukaryotic initiation factors (eIFs). In budding yeast, an important subset of these factors (1, 2, 3, and 5) can form a multifactor complex (MFC). Here, we describe cryo-EM reconstructions of the 40S subunit, of the MFC, and of 40S complexes with MFC factors plus eIF1A. These studies reveal the positioning of the core MFC on the 40S subunit, and show how eIF-binding induces mobility in the head and platform and reconfigures the head-platform-body relationship. This is expected to increase the accessibility of the mRNA channel, thus enabling the 40S subunit to convert to a recruitment-competent state.

Unwinding single RNA molecules using helicases involved in eukaryotic translation initiation.

The small (40 S) subunit of the eukaryotic ribosome may have to scan more than 2000 nucleotides (>600 nm) from its 5'cap recruiting point on an mRNA molecule before initiating on a translation start codon. As with many other processes in living cells, including transcription, editing, mRNA splicing, pre-rRNA processing, RNA transport and RNA decay, scanning is facilitated by helicase activity. However, precise quantitative data on the molecular mechanism of scanning, including the roles of helicases, are lacking. Here, we describe a novel atomic force microscopy (AFM)-based procedure to examine the roles of two yeast helicases, eIF4A and Ded1, previously implicated in translation initiation by genetic and biochemical studies. Our results show that eIF4A, especially in the presence of its "cofactor" eIF4B, promotes ATP-dependent unwinding of localised secondary structure in long RNA molecules under tensional loading, albeit only at high protein:RNA ratios. Thus eIF4A can act to separate only a limited number of base-pairs, possibly via a steric unwinding mechanism. In contrast, Ded1 is more effective in reducing (by up to 50 pN at an AFM loading rate of 14 nNs(-1)) the force necessary to disrupt an RNA stem-loop, and thus shows significant kinetic competence to facilitate fast unwinding. These single molecule experiments indicate that Ded1 is likely to act as the more potent unwinding factor on natural mRNA substrates.

Modulation of the hepatitis C virus RNA-dependent RNA polymerase activity by the non-structural (NS) 3 helicase and the NS4B membrane protein.

The hepatitis C virus (HCV) nonstructural protein 5B (NS5B) is believed to be the central catalytic enzyme responsible for HCV replication but there are many unanswered questions about how its activity is controlled. In this study we reveal that two other HCV proteins, NS3 (a protease/helicase) and NS4B (a hydrophobic protein of unknown function), physically and functionally interact with the NS5B polymerase. We describe a new procedure for generating highly pure NS4B, and use this protein in biochemical studies together with NS5B and NS3. To study the functional effects of the protein-protein interactions, we have developed an in vitro replication assay using the natural noncoding 3' regions of the respective positive ((+)-3'-untranslated region) and negative ((-)-3'-terminal region) RNA strands of the HCV genome. Our studies show that NS3 dramatically modulates template recognition by NS5B and changes the synthetic products generated by this enzyme. The use of an NTPase-deficient mutant form of NS3 demonstrates that the NTPase activity (and thus helicase activity) of this protein is specifically required for these effects. Moreover, NS4B is found to be a negative regulator of the NS3-NS5B replication complex. Overall, these results reveal that NS3, NS4B, and NS5B can interact to form a regulatory complex that could feature in the process of HCV replication.