Mathematical kinetic modelling followed by in vitro and in vivo assays reveal the bifunctional rice GTPCHII/DHBPS enzymes and demonstrate the key roles of OsRibA proteins in the vitamin B2 pathway.

BACKGROUND: Riboflavin is the precursor of several cofactors essential for normal physical and cognitive development, but only plants and some microorganisms can produce it. Humans thus rely on their dietary intake, which at a global level is mainly constituted by cereals (> 50%). Understanding the riboflavin biosynthesis players is key for advancing our knowledge on this essential pathway and can hold promise for biofortification strategies in major crop species. In some bacteria and in Arabidopsis, it is known that RibA1 is a bifunctional protein with distinct GTP cyclohydrolase II (GTPCHII) and 3,4-dihydroxy-2-butanone-4-phosphate synthase (DHBPS) domains. Arabidopsis harbors three RibA isoforms, but only one retained its bifunctionality. In rice, however, the identification and characterization of RibA has not yet been described. RESULTS: Through mathematical kinetic modeling, we identified RibA as the rate-limiting step of riboflavin pathway and by bioinformatic analysis we confirmed that rice RibA proteins carry both domains, DHBPS and GTPCHII. Phylogenetic analysis revealed that OsRibA isoforms 1 and 2 are similar to Arabidopsis bifunctional RibA1. Heterologous expression of OsRibA1 completely restored the growth of the rib3∆ yeast mutant, lacking DHBPS expression, while causing a 60% growth improvement of the rib1∆ mutant, lacking GTPCHII activity. Regarding OsRibA2, its heterologous expression fully complemented GTPCHII activity, and improved rib3∆ growth by 30%. In vitro activity assays confirmed that both OsRibA1 and OsRibA2 proteins carry GTPCHII/DHBPS activities, but that OsRibA1 has higher DHBPS activity. The overexpression of OsRibA1 in rice callus resulted in a 28% increase in riboflavin content. CONCLUSIONS: Our study elucidates the critical role of RibA in rice riboflavin biosynthesis pathway, establishing it as the rate-limiting step in the pathway. By identifying and characterizing OsRibA1 and OsRibA2, showcasing their GTPCHII and DHBPS activities, we have advanced the understanding of riboflavin biosynthesis in this staple crop. We further demonstrated that OsRibA1 overexpression in rice callus increases its riboflavin content, providing supporting information for bioengineering efforts.

Influence of Brainstem's Area A5 on Sympathetic Outflow and Cardiorespiratory Dynamics.

Area A5 is a noradrenergic cell group in the brain stem characterised by its important role in triggering sympathetic activity, exerting a profound influence on the sympathetic outflow, which is instrumental in the modulation of cardiovascular functions, stress responses and various other physiological processes that are crucial for adaptation and survival mechanisms. Understanding the role of area A5, therefore, not only provides insights into the basic functioning of the sympathetic nervous system but also sheds light on the neuronal basis of a number of autonomic responses. In this review, we look deeper into the specifics of area A5, exploring its anatomical connections, its neurochemical properties and the mechanisms by which it influences sympathetic nervous system activity and cardiorespiratory regulation and, thus, contributes to the overall dynamics of the autonomic function in regulating body homeostasis.

Gender-Specific Effects on the Cardiorespiratory System and Neurotoxicity of Intermittent and Permanent Low-Level Lead Exposures.

Lead exposure is a significant health concern, ranking among the top 10 most harmful substances for humans. There are no safe levels of lead exposure, and it affects multiple body systems, especially the cardiovascular and neurological systems, leading to problems such as hypertension, heart disease, cognitive deficits, and developmental delays, particularly in children. Gender differences are a crucial factor, with women's reproductive systems being especially vulnerable, resulting in fertility issues, pregnancy complications, miscarriages, and premature births. The globalization of lead exposure presents new challenges in managing this issue. Therefore, understanding the gender-specific implications is essential for developing effective treatments and public health strategies to mitigate the impact of lead-related health problems. This study examined the effects of intermittent and permanent lead exposure on both male and female animals, assessing behaviours like anxiety, locomotor activity, and long-term memory, as well as molecular changes related to astrogliosis. Additionally, physiological and autonomic evaluations were performed, focusing on baro- and chemoreceptor reflexes. The study's findings revealed that permanent lead exposure has more severe health consequences, including hypertension, anxiety, and reactive astrogliosis, affecting both genders. However, males exhibit greater cognitive, behavioural, and respiratory changes, while females are more susceptible to chemoreflex hypersensitivity. In contrast, intermittent lead exposure leads to hypertension and reactive astrogliosis in both genders. Still, females are more vulnerable to cognitive impairment, increased respiratory frequency, and chemoreflex hypersensitivity, while males show more reactive astrocytes in the hippocampus. Overall, this research emphasizes the importance of not only investigating different types of lead exposure but also considering gender differences in toxicity when addressing this public health concern.

OsTH1 is a key player in thiamin biosynthesis in rice.

Thiamin is a vital nutrient that acts as a cofactor for several enzymes primarily localized in the mitochondria. These thiamin-dependent enzymes are involved in energy metabolism, nucleic acid biosynthesis, and antioxidant machinery. The enzyme HMP-P kinase/thiamin monophosphate synthase (TH1) holds a key position in thiamin biosynthesis, being responsible for the phosphorylation of HMP-P into HMP-PP and for the condensation of HMP-PP and HET-P to form TMP. Through mathematical kinetic model, we have identified TH1 as a critical player for thiamin biofortification in rice. We further focused on the functional characterization of OsTH1. Sequence and gene expression analysis, along with phylogenetic studies, provided insights into OsTH1 bifunctional features and evolution. The indispensable role of OsTH1 in thiamin biosynthesis was validated by heterologous expression of OsTH1 and successful complementation of yeast knock-out mutants impaired in thiamin production. We also proved that the sole OsTH1 overexpression in rice callus significantly improves B1 concentration, resulting in 50% increase in thiamin accumulation. Our study underscores the critical role of OsTH1 in thiamin biosynthesis, shedding light on its bifunctional nature and evolutionary significance. The significant enhancement of thiamin accumulation in rice callus upon OsTH1 overexpression constitutes evidence of its potential application in biofortification strategies.

Computer-aided design and implementation of efficient biosynthetic pathways to produce high added-value products derived from tyrosine in Escherichia coli.

Developing efficient bioprocesses requires selecting the best biosynthetic pathways, which can be challenging and time-consuming due to the vast amount of data available in databases and literature. The extension of the shikimate pathway for the biosynthesis of commercially attractive molecules often involves promiscuous enzymes or lacks well-established routes. To address these challenges, we developed a computational workflow integrating enumeration/retrosynthesis algorithms, a toolbox for pathway analysis, enzyme selection tools, and a gene discovery pipeline, supported by manual curation and literature review. Our focus has been on implementing biosynthetic pathways for tyrosine-derived compounds, specifically L-3,4-dihydroxyphenylalanine (L-DOPA) and dopamine, with significant applications in health and nutrition. We selected one pathway to produce L-DOPA and two different pathways for dopamine-one already described in the literature and a novel pathway. Our goal was either to identify the most suitable gene candidates for expression in Escherichia coli for the known pathways or to discover innovative pathways. Although not all implemented pathways resulted in the accumulation of target compounds, in our shake-flask experiments we achieved a maximum L-DOPA titer of 0.71 g/L and dopamine titers of 0.29 and 0.21 g/L for known and novel pathways, respectively. In the case of L-DOPA, we utilized, for the first time, a mutant version of tyrosinase from Ralstonia solanacearum. Production of dopamine via the known biosynthesis route was accomplished by coupling the L-DOPA pathway with the expression of DOPA decarboxylase from Pseudomonas putida, resulting in a unique biosynthetic pathway never reported in literature before. In the context of the novel pathway, dopamine was produced using tyramine as the intermediate compound. To achieve this, tyrosine was initially converted into tyramine by expressing TDC from Levilactobacillus brevis, which, in turn, was converted into dopamine through the action of the enzyme encoded by ppoMP from Mucuna pruriens. This marks the first time that an alternative biosynthetic pathway for dopamine has been validated in microbes. These findings underscore the effectiveness of our computational workflow in facilitating pathway enumeration and selection, offering the potential to uncover novel biosynthetic routes, thus paving the way for other target compounds of biotechnological interest.

Monensina sódica sobre vacas em fase inicial de lactação: produção de leite e peso vivo / Sodium monensin on early lactation cows: milk production and live weight

Avaliou-se o efeito do fornecimento de monensina sódica no desempenho de vacas leiteiras em fase inicial de lactação, cuja alimentação, em sua dieta total no cocho, compôs-se em 81,4% de silagem de milho e 18,6% de concentrado, em que se fez uso de maior proporção de silagem de milho de que concentrado. Foram utilizadas 14 vacas multíparas 7/8 Holandês, aos 20±10 dias pós-parto aproximadamente, com média de 22±0,5kg vaca-1 dia-1 de leite, em delineamento de blocos casualizados, adotando-se o esquema de parcelas subdivididas no tempo, em modelo de medidas repetidas. Os tratamentos nas parcelas foram diferenciados pela presença (200mg animal-1dia-1) ou ausência da monensina na ração e, nas subparcelas, pelos diferentes períodos de avaliação. Não se observaram diferenças no consumo em matéria seca da dieta, conversão alimentar, produção de leite in natura e corrigida e peso vivo nas médias diárias de cada período. No entanto, a produção de sólidos totais foi superior para os tratamentos com o ionóforo, sinalizando que a administração da monensina sódica para vacas no início de lactação modifica o rendimento em produção de leite.

The effect on the performance of early lactation cows when supplying them with sodium monensin was evaluated, whose power in their overall diet in the trough, was composed of 81.4% of corn silage and 18.6% of concentrate in which use was made of a higher proportion of corn silage to concentrate. Fourteen multiparous 7/8 Dutch cows were used, approximately 20±10 days post-labor, with the average weight of 22±0.5kg cow-1 day-1 of lactation, in randomized blocks, adopting the scheme of subdivided portions in time and repetitive measurement model. The treatments in portions were differentiated by the presence (200mg.animal-1day-1) or absence of monensin in the food and, in the sub-portions, different evaluation periods were used. The animals were fed with their total diet in the feeder composed of 81.4% of corn silage and 18.6% of concentrate. No difference was found in the consumption of diet dry matter, alimentary conversion, in natural and corrected production of milk and live weight in the daily averages of each period. However, the production of total solids was superior for treatments with ionophore, signalizing that the administration of sodium monensin in early lactation cows changes the milk production performance.

Registros de Collembola (Arthropoda, Hexapoda) no Estado do Rio Grande do Norte, Brasil / Records of Collembolans (Arthropoda, Hexapoda) from Rio Grande do Norte State, Brazil

O estado de conhecimento sobre Collembola no Brasil é deficitário, sendo a maior parte dos biomas pobremente amostrados. Este trabalho tem por objetivo fornecer uma lista de espécies de colêmbolos para o Estado do Rio Grande do Norte. As coletas foram realizadas nos municípios de Extremoz, Natal, Parnamirim e Serra Negra do Norte, por meio da utilização de armadilhas de queda, armadilha luminosa e coleta manual diretamente do substrato. Foram registradas 16 espécies, distribuídas em 12 gêneros e sete famílias. A família com maior riqueza de espécies foi Entomobryidae, com sete espécies. O gênero com maior número de espécies foi Seira, com quatro. As duas únicas espécies registradas na Caatinga também foram observadas nas áreas de Mata Atlântica. Por outro lado, tais espécies são novas para a ciência, o que sugere a urgência de um inventário na Caatinga, domínio ainda subestimado. Foram encontradas cinco espécies novas de colêmbolos no Estado.

The state of knowledge of collembolans in Brazil is deficient and most biomes are poorly sampled. This paper provides a list of species of Collembola from Rio Grande do Norte State. The samplings were carried out in the municipalities of Extremoz, Natal, Parnamirim and Serra Negra do Norte, by using pitfall traps, light traps and direct collections. Sixteen species of springtails were recorded, distributed in 12 genera and seven families. The family with the highest diversity was Entomobryidae, with seven species. The most speciose genus was Seira, with four species. Only two species were recorded in Caatinga and they both were also reported to areas of Brazilian Atlantic Forest. However, these species are new to science, which indicates the urgency of a full inventory in Caatinga, an underestimated domain. Five new species of Collembola were found in the State.