To identify new genes involved in acetate uptake in Saccharomyces cerevisiae, an analysis of the gene expression profiles of cells shifted from glucose to acetic acid was performed. The gene expression reprogramming of yeast adapting to a poor non-fermentable carbon source was observed, including dramatic metabolic changes, global activation of translation machinery, mitochondria biogenesis and the induction of known or putative transporters. Among them, the gene ADY2/YCR010c was identified as a new key element for acetate transport, being homologous to the Yarrowia lipolytica GPR1 gene, which has a role in acetic acid sensitivity. Disruption of ADY2 in S. cerevisiae abolished the active transport of acetate. Microarray analyses of ady2 strains showed that this gene is not a critical regulator of acetate response and that its role is directly connected to acetate transport. Ady2p is predicted to be a membrane protein and is a valuable acetate transporter candidate.

In recent years, (bio)electrochemical systems (B)ES have emerged as an energy efficient alternative for the recovery of TAN (total ammonia nitrogen, including ammonia and ammonium) from wastewater. In these systems, TAN is removed or concentrated from the wastewater under the influence of an electrical current and transported to the cathode. Subsequently, it can be removed or recovered through stripping, chemisorption, or forward osmosis. A crucial parameter that determines the energy required to recover TAN is the load ratio: the ratio between TAN loading and applied current. For electrochemical TAN recovery, an energy input is required, while in bioelectrochemical recovery, electric energy can be recovered together with TAN. Bioelectrochemical recovery relies on the microbial oxidation of COD for the production of electrons, which drives TAN transport. Here, the state-of-the-art of (bio)electrochemical TAN recovery is described, the performance of (B)ES for TAN recovery is analyzed, the potential of different wastewaters for BES-based TAN recovery is evaluated, the microorganisms found on bioanodes that treat wastewater high in TAN are reported, and the toxic effect of the typical conditions in such systems (e.g., high pH, TAN, and salt concentrations) are described. For future application, toxicity effects for electrochemically active bacteria need better understanding, and the technologies need to be demonstrated on larger scale.

The Rho-guanine nucleotide exchange factor (RhoGEF) TRIO acts as a key regulator of neuronal migration, axonal outgrowth, axon guidance, and synaptogenesis by activating the GTPase RAC1 and modulating actin cytoskeleton remodeling. Pathogenic variants in TRIO are associated with neurodevelopmental diseases, including intellectual disability (ID) and autism spectrum disorders (ASD). Here, we report the largest international cohort of 24 individuals with confirmed pathogenic missense or nonsense variants in TRIO. The nonsense mutations are spread along the TRIO sequence, and affected individuals show variable neurodevelopmental phenotypes. In contrast, missense variants cluster into two mutational hotspots in the TRIO sequence, one in the seventh spectrin repeat and one in the RAC1-activating GEFD1. Although all individuals in this cohort present with developmental delay and a neuro-behavioral phenotype, individuals with a pathogenic variant in the seventh spectrin repeat have a more severe ID associated with macrocephaly than do most individuals with GEFD1 variants, who display milder ID and microcephaly. Functional studies show that the spectrin and GEFD1 variants cause a TRIO-mediated hyper-or hypo-activation of RAC1, respectively, and we observe a striking correlation between RAC1 activation levels and the head size of the affected individuals. In addition, truncations in TRIO GEFD1 in the vertebrate model X. tropicalis induce defects that are concordant with the human phenotype. This work demonstrates distinct clinical and molecular disorders clustering in the GEFD1 and seventh spectrin repeat domains and highlights the importance of tight control of TRIO-RAC1 signaling in neuronal development.

CREB3 proteins comprise a set of ER-localised bZip transcription factors defined by the presence of a transmembrane domain. They are regulated by inter-compartmental transport, Golgi cleavage and nuclear transport where they promote appropriate transcriptional responses. Although CREB3 proteins play key roles in differentiation, inflammation and metabolism, a general framework relating their defining features to these diverse activities is lacking. We identify unique features of CREB3 organisation including the ATB domain, which we show is essential for transcriptional activity. This domain is absent in all other human bZip factors, but conserved in Drosophila CREBA, which controls secretory pathway genes (SPGs). Furthermore, each of the five human CREB3 factors was capable of activating SPGs in Drosophila, dependent upon the ATB domain. Expression of the CREB3 protein, CREB-H, in 293 cells, upregulated genes involved in secretory capacity, extracellular matrix formation and lipid metabolism and increased secretion of specific cargos. In liver cells, which normally express CREB-H, the active form specifically induced secretion of apolipoproteins, including ApoA-IV, ApoAI, consistent with data implicating CREB-H in metabolic homeostasis. Based on these data and other recent studies, we propose of a general role for the CREB3 family in regulating secretory capacity, with particular relevance to specialised cargos.

The biochemical methane potential (BMP) of raw poultry litter waste was assessed in batch assays. Biological co-treatment with Clostridium cellulolyticum, Caldicellulosiruptor saccharolyticum and Clostridium thermocellum as bioaugmentation strains, and thermochemical pre-treatments with lime and sodium hydroxide performed at different temperatures and pressures were applied as strategies to improve the BMP by favouring the hydrolysis of the cellulolytic material in the waste. Anaerobic digestion of the raw waste allowed a specific methane production of 145 ± 14 LCH(4)kg(-1)VS, with 1% total solids and 0.72 g VS(inoculum)g(-1)VS(waste). The pre- and co-treatments contributed to a significant increase (up to 74%) in the waste solubilisation when using C. saccharolyticum, but methane production did not improve considerably. Therefore, the conversion of soluble organic matter to methane was the limiting step of the anaerobic digestion process of poultry litter waste.

bLipids can be anaerobically digested to methane, but methanogens are often considered to be highly sensitive to the long-chain fatty acids (LCFA) deriving from lipids hydrolysis. In this study, the effect of unsaturated (oleate [C 18:1 ]) and saturated (stearate [C 18:0 ] and palmitate [C 16:0 ]) LCFA toward methanogenic archaea was studied in batch enrichments and in pure cultures. Overall, oleate had a more stringent effect on methanogens than saturated LCFA, and the degree of tolerance to LCFA was different among distinct species of methanogens. Methanobacterium formicicum was able to grow in both oleate-and palmitate-degrading enrichments (OM and PM cultures, respectively), whereas Methanospirillum hungatei only survived in a PM culture. The two acetoclastic methanogens tested, Methanosarcina mazei and Methanosaeta concilii, could be detected in both enrichment cultures, with better survival in PM cultures than in OM cultures. Viability tests using live/dead staining further confirmed that exponential growth-phase cultures of M. hungatei are more sensitive to oleate than are M. formicicum cultures; exposure to 0.5 mM oleate damaged 99% ؎ 1% of the cell membranes of M. hungatei and 53% ؎ 10% of the cell membranes of M. formicicum. In terms of methanogenic activity, M. hungatei was inhibited for 50% by 0.3, 0.4, and 1 mM oleate, stearate, and palmitate, respectively. M. formicicum was more resilient, since 1 mM oleate and >4 mM stearate or palmitate was needed to cause 50% inhibition on methanogenic activity.A naerobic degradation of long-chain fatty acids (LCFA) is essential for efficient biogas production from complex lipidcontaining wastewaters (1-3). However, there is still concern regarding the potential toxic effect of LCFA toward methanogenic communities. Toxicity of LCFA is the main reason for insufficient treatment results of LCFA-containing wastewaters (4-8). Studies of methanogenic inhibition in the rumen confirm the toxicity of these compounds. Soliva et al. (9) showed the antimethanogenic potential of saturated fatty acids of medium chain length, specifically myristate (C 14:0 ) and laurate (C 12:0 ). The adverse effect of fatty acids toward methanogens appears to be more pronounced at longer chain lengths and more unsaturated bonds (10, 11). Moreover, methanogens inhibition by fatty acids is a rapid phenomenon with long recuperation time, as shown by Koster (12): a 50% loss of methanogenic activity was observed after exposing methanogenic sludge to 7.5 mM laurate for only 7.5 min.Sensitivity of microorganisms to LCFA seems to be related to their cell wall structure, with Gram-positive bacteria and methanogens being more easily inhibited than Gram-negative bacteria (13). Inhibition of pure cultures of methanogens by fatty acids is reported in literature (11, 14). Nevertheless, methanogens are ubiquitous in anaerobic bioreactors treating LCFA-rich wastewaters, even over prolonged continuous LCFA loading (15,16). Sousa et al. (16) identified three predominant genera of methanogens in LCFA-degradi...

Yeast Dop1p is an essential protein that is highly conserved in evolution and whose function is largely unknown. Here, we provide evidence that Dop1p localizes to endosomes and exists in a complex with two other conserved proteins: Neo1p, a P 4 -ATPase and putative flippase, and the scaffolding protein Ysl2p/Mon2p. The latter operates during membrane budding at the tubular endosomal network/trans-Golgi network (TEN/TGN) in a process that includes clathrin recruitment via adaptor proteins. Consistent with a role for Dop1p during this process, temperature-sensitive dop1-3 cells accumulate multivesicular, elongated tubular and ring-like structures similar to those displayed by neo1 and ysl2 mutants. In further agreement with the concept of Dop1p-Neo1p-Ysl2p complex formation and co-operation, we show that dop1-3 cells exhibit reduced levels of Neo1p and Ysl2p at steady state. Conversely, mutations or deletions in NEO1 and YSL2 lead to a decrease in Dop1p levels. In addition to binding to Neo1p and Ysl2p, Dop1p can form dimers or multimers. A critical region for dimerization resides in the C-terminus with leucine zipper-like domains. Dop1p's membrane association is largely mediated by its internal region, but Ysl2p might not be crucial for membrane recruitment.

The CREB3 transcription factors are a specialized set of regulatory proteins that are normally anchored in the endoplasmic reticulum and relay perturbation of function back to the nucleus. Phosphorylation and degradation modulate the response of one such factor (CREB-H), which is involved in secretion and metabolic homeostasis.