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Petroleum-based plastics levy significant environmental and economic costs that can be alleviated with sustainably sourced, biodegradable, and bio-based polymers such as polyhydroxyalkanoates (PHAs). However, industrial-scale production of PHAs faces barriers stemming from insufficient product yields and high costs. To address these challenges, we must look beyond the current suite of microbes for PHA production and investigate non-model organisms with versatile metabolisms. In that vein, we assessed PHA production by the photosynthetic purple non-sulfur bacteria (PNSB) Rhodomicrobium vannielii and Rhodomicrobium udaipurense. We show that both species accumulate PHA across photo-heterotrophic, photo-hydrogenotrophic, photo-ferrotrophic, and photo-electrotrophic growth conditions, with either ammonium chloride (NH 4 Cl) or dinitrogen gas (N 2 ) as nitrogen sources. Our data indicate that nitrogen source plays a significant role in dictating PHA synthesis, with N 2 fixation promoting PHA production during photoheterotrophy and photoelectrotrophy but inhibiting production during photohydrogenotrophy and photoferrotrophy. We observed the highest PHA titres (up to 44.08 mg/L, or 43.61% cell dry weight) when cells were grown photoheterotrophically on sodium butyrate with N 2 , while production was at its lowest during photoelectrotrophy (as low as 0.04 mg/L, or 0.16% cell dry weight). We also find that photohydrogenotrophically grown cells supplemented with NH 4 Cl exhibit the highest electron yields - up to 58.89% - while photoheterotrophy demonstrated the lowest (0.27%-1.39%). Finally, we highlight superior electron conversion and PHA production compared to a related PNSB, Rhodopseudomonas palustris TIE-1. This study illustrates the value of studying non-model organisms like Rhodomicrobium for sustainable PHA production and indicates future directions for exploring PNSB metabolisms., (© 2024 The Author(s). Microbial Biotechnology published by John Wiley & Sons Ltd.)

As one of the most commonly used biocidal cationic surfactants, benzalkonium chlorides (BACs) have been an increasing concern as emerging contaminants. Wastewater has been claimed the main point for BACs to enter into the environment, but to date, it is still largely unknown how the BACs affect the microbes (especially microalgae) in the practical wastewater and how to cost-effectively remove them. In this study, the inhibitory effects of a typical BACs, dodecyl dimethyl benzyl ammonium chloride (DDBAC), on a green microalga Chlorella sp. in oxidation pond wastewater were investigated. The results showed that though a hermetic effect at the first 2 days was observed with the DDBAC at low concentration (<6 mg/L), the algal growth and photosynthesis were significantly inhibited by the DDBAC at all the tested concentrations (3 to 48 mg/L). Fortunately, a new microbial consortium (MC) capable of degrading DDBAC was screened through a gradient domestication method. The MC mainly composed of Wickerhamomyces sp., Purpureocillium sp., and Achromobacter sp., and its maximum removal efficiency and removal rate of DDBAC (48 mg/L) respectively reached 98.1 % and 46.32 mg/L/d. Interestingly, a microbial-microalgal system (MMS) was constructed using the MC and Chlorella sp., and a synergetic effect between the two kinds of microorganisms was proposed: microalga provided oxygen and extracellular polysaccharides as co-metabolic substrates to help the MC to degrade DDBAC, while the MC helped to eliminate the DDBAC-induced inhibition on the alga. Further, by observing the seven kinds of degradation products (mainly including CH 5 O 3 P, C 6 H 5 CH 2 -, and C 8 H 11 N), two possible chemical pathways of the DDBAC degradation were proposed. In addition, the metagenomic sequencing results showed that the main functional genes of the MMS included antibiotic-resistant genes, ABC transporter genes, quorum sensing genes, two-component regulatory system genes, etc. This study provided some theoretical and application findings for the cost-effective pollution prevention of BACs in wastewater., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Ammonia poses a significant challenge in the contemporary intensive breeding industry, resulting in substantial economic losses. Despite this, there is a dearth of research investigating efficacious strategies to prevent ammonia poisoning in poultry. Consequently, the objective of this study was to investigate the molecular mechanisms through which Luteolin (Lut) safeguards mitochondria and restores equilibrium to energy metabolism disorders, thereby shielding chicken spleen lymphocytes from the detrimental effects of ammonia poisoning. Chicken spleen lymphocytes were categorized into 3 distinct groups: the control group, the ammonia group (with the addition of 1 mmol/L of ammonium chloride), and the Lut group (with the treatment of 0.5 μg/mL of Lut for 12 h followed by the addition of 1 mmol/L of ammonium chloride). These groups were then cultured for a duration of 24 h. To investigate the potential protective effect of Lut on lymphocytes exposed to ammonia, various techniques were employed, including CCK-8 analysis, ultrastructural observation, reagent kit methodology, fluorescence microscopy, and quantitative real-time PCR (qRT-PCR). The findings indicate that Lut has the potential to mitigate the morphological damage of mitochondria caused by ammonia poisoning. Additionally, it can counteract the decline in mitochondrial membrane potential, ATP content, and ATPase activities (specifically Na + /K + -ATPase, Ca 2+ -ATPase, Mg 2+ -ATPase, and Ca/Mg 2+ -ATPase) following exposure to ammonia in lymphocytes. Lut also has the ability to regulate the expression of genes involved in mitochondrial fusion (Opa1, Mfn1, and Mfn2) and division (Drp1 and Mff) in spleen lymphocytes after ammonia exposure. This regulation leads to a balanced energy metabolism (HK1, HK2, LDHA, LDHB, PFK, PK, SDHB, and ACO2) and provides protection against ammonia poisoning., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Ammonium (NH 4 + ), a breakdown product of amino acids that can be toxic at high levels, is detected by taste systems of organisms ranging from C. elegans to humans and has been used for decades in vertebrate taste research. Here we report that OTOP1, a proton-selective ion channel expressed in sour (Type III) taste receptor cells (TRCs), functions as sensor for ammonium chloride (NH 4 Cl). Extracellular NH 4 Cl evoked large dose-dependent inward currents in HEK-293 cells expressing murine OTOP1 (mOTOP1), human OTOP1 and other species variants of OTOP1, that correlated with its ability to alkalinize the cell cytosol. Mutation of a conserved intracellular arginine residue (R292) in the mOTOP1 tm 6-tm 7 linker specifically decreased responses to NH 4 Cl relative to acid stimuli. Taste responses to NH 4 Cl measured from isolated Type III TRCs, or gustatory nerves were strongly attenuated or eliminated in an Otop1 -/- mouse strain. Behavioral aversion of mice to NH 4 Cl, reduced in Skn-1a -/- mice lacking Type II TRCs, was entirely abolished in a double knockout with Otop1. These data together reveal an unexpected role for the proton channel OTOP1 in mediating a major component of the taste of NH 4 Cl and a previously undescribed channel activation mechanism., (© 2023. Springer Nature Limited.)

Hydrocarbon contamination is continuing to be a serious environmental problem because of their toxicity. Hydrocarbon components have been known to be carcinogens and neurotoxic organic pollutants. The physical and chemical methods of petroleum removal have become ineffective and also are very costly. Therefore, bioremediation is considered the promising technology for the treatment of these contaminated sites since it is cost-effective and will lead to complete mineralization.The current study also concentrates on bioremediation of petroleum products by bacterium isolated from petroleum hydrocarbon contaminated soil. The current work shows that bacterial strains obtained from a petroleum hydrocarbon contaminated environment may degrade petroleum compounds. Two strains Bacillus licheniformis ARMP2 and Pseudomonas aeruginosa ARMP8 were identified as petroleum-degrading bacteria of the isolated bacterial colonies. The best growth conditions for the ARMP2 strain were determined to be pH 9, temperature 29 °C with sodium nitrate as its nitrogen source, whereas for the ARMP8 strain the optimal growth was found at pH 7, temperature 39 °C, and ammonium chloride as the nitrogen source. Both strains were shown to be effective at degrading petroleum chemicals confirmed by GCMS. Overall petroleum product degradation efficiency of the strains ARMP2 and ARMP8 was about 88 % and 73 % respectively in 48 h.The strains Bacillus licheniformis ARMP2 and Pseudomonas aeruginosa ARMP8 were shown to be effective at degrading petroleum compounds in the current study. Even greater results might be obtained if the organisms were utilised in consortia or the degradation time period was extended., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have influenced the work reported in this paper., (Copyright © 2022 Elsevier GmbH. All rights reserved.)

A mab cluster associated with 3-aminobenzoic acid (3AB) degradation was identified in Comamonas sp. QT12. However, the cellular response of Comamonas sp. QT12 to 3-aminobenzoic acid remains unclear. In this study, label-free quantitative proteome analysis based on LC-MS/MS was used to study the protein expression difference of strain QT12 under the condition of using 3AB (3AB) and citric acid/ammonium chloride as substrates (3ABCon). A total of 2068 proteins were identified, of which 239 were significantly up-regulated in 3AB group, 124 were significantly down-regulated in 3AB group, 624 were expressed only in 3AB group, and 216 were expressed only in 3ABCon group in 3AB group. KEGG pathway analysis found that 83 pathways were up-regulated and 49 pathways were down-regulated, In GO analysis, 315 paths were up-regulated and 156 paths were down-regulated. There were 6 genes in the mab cluster that were only detected in the 3AB group.The mab cluster was found to be related to degradation of 3AB. By knockout, it was found that the growth rate of the mutant △orf7 and △orf9 were slowed down. HPLC results showed that the mutant △orf7 and △orf9 could still degrade 3AB, it was found that orf7, orf9 were not key genes about 3AB degradation and they could be replaced by other genes in strain QT12. These findings improve our understanding of the molecular mechanisms underlying the cellular response of 3AB degradation in Comamonas bacterium., (© 2022. The Author(s).)

Background: Pathophysiological consequences of traumatic brain injury (TBI) mediated secondary injury remain incompletely understood. In particular, the impact of TBI on the differentiation and maintenance of dendritic cells (DCs), which are regarded as the most professional antigen presenting cells of the immune system, remains completely unknown. Here, we report that DC-differentiation, maintenance and functions are altered on day 3 and day 7 after TBI., Methods: Long bones, spleen, peripheral lymph nodes (pLNs), mesenteric lymph nodes (mLNs), liver, lungs, skin and blood were collected from mice with either moderate-level cortical impact (CCI) or sham on day 1, day 3 or day 7 after TBI. Bone marrow cells were isolated from the tibias and femurs of hind limb through flushing. Tissues were digested with Collagenase-D and DNase I. Skin biopsies were digested in the presence of liberase + DNase I. Single cell suspensions were made, red blood cells were lysed with Ammonium chloride (Stem Cell Technology) and subsequently filtered using a 70 μM nylon mesh. DC subsets of the tissues and DC progenitors of the BM were identified through 10-color flow cytometry-based immunophenotyping studies. Intracellular reactive oxygen species (ROS) were identified through H2DCFDA staining., Results: Our studies identify that; (1) frequencies and absolute numbers of DCs in the spleen and BM are altered on day 3 and day 7 after TBI; (2) surface expression of key molecules involved in antigen presentation of DCs were affected on day 3 and day 7 after TBI; (3) distribution and functions of tissue-specific DC subsets of both circulatory and lymphatic systems were imbalanced following TBI; (4) early differentiation program of DCs, especially the commitment of hematopoietic stem cells to common DC progenitors (CDPs), were deregulated after TBI; and (5) intracellular ROS levels were reduced in DC progenitors and differentiated DCs on day 3 and day 7 after TBI., Conclusions: Our data demonstrate, for the first time, that TBI affects the distribution pattern of DCs and induces an imbalance among DC subsets in both lymphoid and non-lymphoid organs. In addition, the current study demonstrates that TBI results in reduced levels of ROS in DCs on day 3 and day 7 after TBI, which may explain altered DC differentiation paradigm following TBI. A deeper understanding on the molecular mechanisms that contribute to DC defects following TBI would be essential and beneficial in treating infections in patients with acute central nervous system (CNS) injuries, such as TBI, stroke and spinal cord injury., (© 2022. The Author(s).)

Background: We found through previous research that hyperammonemia can cause secondary liver damage. However, whether hepatocytes are target cells of ammonia toxicity and whether hyperammonemia affects hepatocyte metabolism remain unknown., Aims: The purpose of the current study is to examine whether the hepatocyte is a specific target cell of ammonia toxicity and whether hyperammonemia can interfere with hepatocyte metabolism., Methods: Cell viability and apoptosis were analyzed in primary hepatocytes and other cells that had been exposed to ammonium chloride. Western blotting was adopted to examine the expression of proteins related to ammonia transport. We also established a metabolomics method based on gas chromatography-mass spectrometry to understand the characteristics of the hepatocyte metabolic spectrum in a hyperammonemia microenvironment, to screen and identify differential metabolites, and to determine the differential metabolic pathway. Different technologies were used to verify the differential metabolic pathways., Results: Hepatocytes are target cells of ammonia toxicity. The mechanism is related to the ammonia transporter. Hyperammonemia interferes with hepatocyte metabolism, which leads to TCA cycle, urea cycle, and RNA synthesis disorder., Conclusions: This study demonstrates that hepatocyte growth and metabolism are disturbed in a hyperammonemia microenvironment, which further deteriorates hepatocyte function., (Copyright © 2021 Elsevier Inc. All rights reserved.)

An extracellular electron transfer (EET) process between an electroactive biofilm and an electrode is a crucial step for the performance of microbial fuel cells (MFCs), which is highly related to the enrichment of exoelectrogens and the electrocatalytic activity of the electrode. Herein, an efficient N- and Fe-abundant carbon cloth (CC) electrode with the comodification of iron porphyrin (FePor) and polyquaternium-7 (PQ) was synthesized using a facile solvent evaporation and immersion method and developed as an anode (named FePor-PQ) in MFCs. The surface structural characterizations confirmed the successful introduction of N and Fe atoms, whereas FePor-PQ achieved the N content of 9.59 at %, which may offer various active sites for EET. The introduction of PQ contributed to improving the surface hydrophilicity, providing the composite electrode good biocompatibility for bacterial attachment and colonization as well as substrate diffusion. Based on the advantages, the MFC with the FePor-PQ anode produced a maximum power density of 2165.7 mW m -2 , strikingly higher than those of CC (1124.0 mW m -2 ), PQ (1668.8 mW m -2 ), and FePor (1978.9 mW m -2 ). Furthermore, with the EET mediated by the binding of flavins and c-type cytochromes on the outer membrane was enhanced prominently, the typical exoelectrogen Geobacter was enriched up to 55.84% in the FePor-PQ anode biofilm. This work reveals a synergistic effect from heteroatom coating and surface properties tailoring to boost both the EET efficiency and exoelectrogen enrichment for enhancing MFC performance, which also provides valuable insights for designing electrodes in other bio-electrochemical systems.

The extreme halophilic archaeon, Haloferax mediterranei can accumulate polyhydroxyalkanoate (PHA) from different renewable resources. To enhance the biosynthesis and quality of PHA, H. mediterranei cultivation media was optimized at different C/N ratios using glucose as the main carbon source. Three sets of media (yeast extract [YE], NH 4 Cl and combination of YE and NH 4 Cl) were prepared at different nitrogen concentrations to achieve C/N ratios of 9, 20, and 35, respectively. The media containing YE (organic nitrogen source) produced a higher growth rate of H. mediterranei than NH 4 Cl (inorganic source) at all tested C/N ratios. The highest PHA accumulation (18.4% PHA/cell dry mass) was achieved in a media that combined YE with NH 4 Cl at a C/N ratio of 20. Analysis of the produced polymers revealed the production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBHV) with different 3-hydroxyvalerate (3HV) content. The polymers produced from YE and the combined media have greater 3HV content (10 mol%) than those polymers recovered from NH 4 Cl (1.5 mol%). Resultingly, PHBHV from YE and the combined media displayed reduced melting points at 144°C. The nitrogen type/concentration was found to also have an impact on the molecular weights and polydispersity indices of the produced biopolymers. Furthermore, the tensile strengths were found to vary with the best tensile strength (14.4 MPa) being recorded for the polymer recovered from YE at C/N = 9. Interestingly, the tensile strength of PHBHV was significantly higher than petroleum-based polyethylene (13.5 MPa), making it a much more suitable bioplastic for industrial application., (© 2020 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

Various Monascus bioactive metabolites used as food or food additives in Asia for centuries are subjected to constant physical and chemical changes and different Monascus genus. With the aim to identify enzymes that participate in or indirectly regulate the pigments and citrinin biosynthesis pathways of Monascus purpureus cultured under high ammonium chloride, the changes of the proteome profile were examined using sequential window acquisition of all theoretical mass spectra-mass spectrometry-based quantitative proteomics approach in combination with bioinformatics analysis . A total of 292 proteins were confidently detected and quantified in each sample, including 163 that increased and 129 that decreased ( t -tests, p ≤ 0.05). Pathway analysis indicated that high ammonium chloride in the present study accelerates the carbon substrate utilization and promotes the activity of key enzymes in glycolysis and β-oxidation of fatty acid catabolism to generate sufficient acetyl-CoA. However, the synthesis of the monascus pigments and citrinin was not enhanced because of inhibition of the polyketide synthase activity. All results demonstrated that the cause of initiation of pigments and citrinin synthesis is mainly due to the apparent inhibition of acyl and acetyl transfer by some acyltransferase and acetyltransferase, likely malony-CoA:ACP transacylase.

This study investigated the effect of different nitrogen sources, namely, ammonium chloride and glutamate, on photoheterotrophic metabolism of Rhodobacter capsulatus grown on acetate as the carbon source. Genes that were significantly differentially expressed according to Affymetrix microarray data were categorized into Clusters of Orthologous Groups functional categories and those in acetate assimilation, hydrogen production, and photosynthetic electron transport pathways were analyzed in detail. Genes related to hydrogen production metabolism were significantly downregulated in cultures grown on ammonium chloride when compared to those grown on glutamate. In contrast, photosynthetic electron transport and acetate assimilation pathway genes were upregulated. In detail, aceA encoding isocitrate lyase, a unique enzyme of the glyoxylate cycle and ccrA encoding the rate limiting crotonyl-CoA carboxylase/reductase enzyme of ethylmalonyl-coA pathway were significantly upregulated. Our findings indicate for the first time that R. capsulatus can operate both glyoxylate and ethylmalonyl-coA cycles for acetate assimilation.

Aims: Improvements in cancer treatment have significantly extended the lifespan of patients. However, due to the adverse effects of cancer treatment, cancer survivors are at increased risk of cardiovascular complications. Doxorubicin is a widely used spectrum antitumor drug, but the life-threatening side-effect of cardiotoxicity limits its clinical application. Ammonium chloride (NH 4 Cl), as a heteropolar compound with pH value regulation, can cause intracellular alkalization and metabolic acidosis thus effecting enzymatic activity and influencing the process of biological system. The underlying effect of NH 4 CL in DOX-induced cardiomyocyte apoptosis and hypertrophy in mice has never been reported before., Main Methods: This study we used DOX to induce cardiac remodeling and dysfunction in mice. Myocardial histology was performed using HE staining. Myocardial cell size was measured by wheat germ agglutinin (WGA) staining. Echocardiographic evaluation of cardiac function, qPCR detection of the mRNA expression of cardiac hypertrophy and inflammation markers. Apoptosis was detected by TUNEL method. Transmission electron microscopy (TEM) was used to detect autophagy., Key Findings: We found that NH 4 CL effectively improved DOX-induced cardiomyocyte apoptosis and cardiac dysfunction in mice. Our results showed that NH 4 CL significantly improved DOX-induced contractile dysfunction, inflammation, apoptosis and autophagy in mice., Significance: Our results indicate that NH 4 CL is effective in improving DOX-induced cardiac dysfunction and remodeling. It may therefore be a therapeutic entry point to limit doxorubicin-mediated adverse cardiac reactions., (Copyright © 2019. Published by Elsevier Inc.)

Melanoma cells preserve intracellular pH (pH i ) within a viable range despite an acidic ambient pH that typically falls below pH 7.0. The molecular mechanisms underlying this form of acidic preservation in melanoma remain poorly understood. Previous studies had demonstrated that proton transporters including the monocarboxylate transporter (MCT), the sodium hydrogen exchanger (NHE), and V-Type ATPase mediate acid extrusion to counter intracellular acidification in melanoma cells. In this report, the expression and function of the Sodium-Coupled Bicarbonate Transporter (NCBT) family of base loaders were further characterized in melanoma cell lines. NCBT family members were found to be expressed in three different melanoma cell lines - A375, MeWo, and HS695T - and included the electrogenic sodium-bicarbonate cotransporter isoforms 1 and 2 (NBCe1 and NBCe2), the electroneutral sodium-bicarbonate cotransporter (NBCn1), and the sodium-dependent chloride-bicarbonate exchanger (NDCBE). These transporters facilitated 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS)-dependent pH i recovery in melanoma cells, in response to intracellular acidification induced by ammonium chloride prepulse. Furthermore, the expression of NCBTs were upregulated via chronic exposure to extracellular acidification. Given the current research interest in the NCBTs as a molecular driver of tumourigenesis, characterising NCBT in melanoma provides impetus for developing novel therapeutic targets for melanoma treatment.

The complexity of chromatin dynamics is orchestrated by several active processes. In fission yeast, the centromeres are clustered around the spindle pole body (SPB) and oscillate in a microtubule- and adenosine triphosphate (ATP)-dependent manner. However, whether and how SPB oscillation are affected by different environmental conditions remain poorly understood. In this study, we quantitated movements of the SPB component, which colocalizes with the centromere in fission yeast. We found that SPB movement was significantly reduced at low glucose concentrations. Movement of the SPB was also affected by the presence of ammonium chloride. Power spectral analysis revealed that periodic movement of the SPB is disrupted by low glucose concentrations. Measurement of ATP levels in living cells by quantitative single-cell imaging suggests that ATP levels are not the only determinant of SPB movement. Our results provide novel insight into how SPB movement is regulated by cellular energy status and additional factors such as the medium nutritional composition., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Previous studies using citrin/mitochondrial glycerol-3-phosphate (G3P) dehydrogenase (mGPD) double-knockout mice have demonstrated that increased dietary protein reduces the extent of carbohydrate-induced hyperammonemia observed in these mice. This study aimed to further elucidate the mechanisms of this effect. Specific amino acids were initially found to decrease hepatic G3P, or increase aspartate or citrulline levels, in mGPD-knockout mice administered ethanol. Unexpectedly, oral glycine increased ammonia in addition to lowering G3P and increasing citrulline. Subsequently, simultaneous glycine-plus-sucrose (Gly + Suc) administration led to a more severe hyperammonemic state in double-KO mice compared to sucrose alone. Oral arginine, ornithine, aspartate, alanine, glutamate and medium-chain triglycerides all lowered blood ammonia following Gly + Suc administration, with combinations of ornithine-plus-aspartate (Orn + Asp) or ornithine-plus-alanine (Orn + Ala) suppressing levels similar to wild-type. Liver perfusion and portal vein-arterial amino acid differences suggest that oral aspartate, similar to alanine, likely activated ureagenesis from ammonia and lowered the cytosolic NADH/NAD + ratio through conversion to alanine in the small intestine. In conclusion, Gly + Suc administration induces a more severe hyperammonemic state in double-KO mice that Orn + Asp or Orn + Ala both effectively suppress. Aspartate-to-alanine conversion in the small intestine allows for effective oral administration of either, demonstrating a pivotal role of inter-organ aspartate metabolism for the treatment of citrin deficiency.

To investigate the potential of application of marine cyanobacterium for concurrent biomass production and ammonium removal, Synechococcus sp. VDW was cultured under different conditions in medium containing varying concentrations of NH 4 Cl. Response surface methodology (RSM) was then used to build a predictive model of the combined effects of independent variables (pH, inoculum size, ammonium concentration). At the optimum conditions of initial pH 7.4, inoculum size 0.17 (OD730) and ammonium concentration 10.5 mg L -1 , the maximum ammonium removal and biomass productivity were about 95% and 34 mg L -1 d -1 , respectively, after seven days of cultivation. The result of fatty acid methyl ester (FAME) analysis showed that the major fatty acids were palmitic acid (C16:0), linoleic acid (C18:2 n6 cis), palmitoleic acid (C16:1) and oleic acid (C18:1 n9 cis), which accounted for more than 80% weight of total fatty acids. Further, analysis of neutral lipid accumulation using flow cytometry revealed that the mean of the fluorescence intensity increased under optimal conditions. These results indicate that Synechococcus sp. VDW has the potential for use for concurrent water treatment and production of biomass that can be applied as biofuel feedstock.

SLC4A11 mutations are associated with Fuchs' endothelial corneal dystrophy (FECD), congenital hereditary endothelial dystrophy (CHED) and Harboyan syndrome (endothelial dystrophy with auditory deficiency). Mice with genetically ablated Slc4a11 recapitulate CHED, exhibiting significant corneal edema and altered endothelial morphology. We recently demonstrated that SLC4A11 functions as an NH 3 sensitive, electrogenic H + transporter. Here, we investigated the properties of five clinically relevant SLC4A11 mutants: R125H, W240S, C386R, V507I and N693A, relative to wild type, expressed in a PS120 fibroblast cell line. The effect of these mutations on the NH 4 Cl-dependent transporter activity was investigated by intracellular pH and electrophysiology measurements. Relative to plasma membrane expression of NaK ATPase, there were no significant differences in plasma membrane SLC4A11 expression among each mutant and wild type. All mutants revealed a marked decrease in acidification in response to NH 4 Cl when compared to wild type, indicating a decreased H + permeability in mutants. All mutants exhibited significantly reduced H + currents at negative holding potentials as compared to wild type. Uniquely, the C386R and W240S mutants exhibited a different inward current profile upon NH 4 Cl challenges, suggesting an altered transport mode. Thus, our data suggest that these SLC4A11 mutants, rather than having impaired protein trafficking, show altered H + flux properties., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Fission yeast 'cut' mutants show defects in temporal coordination of nuclear division with cytokinesis, resulting in aberrant mitosis and lethality. Among other causes, the 'cut' phenotype can be triggered by genetic or chemical perturbation of lipid metabolism, supposedly resulting in shortage of membrane phospholipids and insufficient nuclear envelope expansion during anaphase. Interestingly, penetrance of the 'cut' phenotype in mutants of the transcription factor cbf11 and acetyl-coenzyme A carboxylase cut6, both related to lipid metabolism, is highly dependent on growth media, although the specific nutrient(s) affecting 'cut' occurrence is not known. In this study, we set out to identify the growth media component(s) responsible for 'cut' phenotype suppression in Δcbf11 and cut6-621 cells. We show that mitotic defects occur rapidly in Δcbf11 cells upon shift from the minimal EMM medium ('cut' suppressing) to the complex YES medium ('cut' promoting). By growing cells in YES medium supplemented with individual EMM components, we identified ammonium chloride, an efficiently utilized nitrogen source, as a specific and potent suppressor of the 'cut' phenotype in both Δcbf11 and cut6-621. Furthermore, we found that ammonium chloride boosts lipid droplet formation in wild-type cells. Our findings suggest a possible involvement of nutrient-responsive signaling in 'cut' suppression.

Nano- and microfibers obtained by electrospinning have attracted great attention due to its versatility and potential for applications in diverse technological fields. Polyhydroxyalkanoates (PHAs) are biopolymers synthesized by microorganisms such as the bacterium Burkholderia xenovorans LB400. In particular, LB400 cells are capable to synthesize poly(3-hydroxybutyrate) (PHB) from glucose. The aim of this study was to produce and characterize electrospun fibers obtained from bacterial PHBs. Bacterial strain LB400 was grown in M9 minimal medium using xylose and mannitol (10gL -1 ) as the sole carbon sources and NH 4 Cl (1gL -1 ) as the sole nitrogen source. Biopolymer-based films obtained were used to produce fibers by electrospinning. Diameter and morphology of the microfibers were analyzed by scanning electron microscopy (SEM) and their thermogravimetric properties were investigated. Bead-free fibers using both PHBs were obtained with diameters of less than 3μm. The surface morphology of the microfibers based on PHBs obtained from both carbon sources was different, even though their thermogravimetric properties are similar. The results indicate that the carbon source may determine the fiber structure and properties. Further studies should be performed to analyze the physicochemical and mechanical properties of these PHB-based microfibers, which may open up novel applications., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: The use of enriched stable isotopes is of outstanding importance in chemical metrology as it allows the application of isotope dilution mass spectrometry (IDMS). Primary methods based on IDMS ensure the quality of the analytical measurements and traceability of the results to the international system of units. However, the synthesis of isotopically labelled molecules from enriched stable isotopes is an expensive and a difficult task. Either chemical and biochemical methods to produce labelled molecules have been proposed, but so far, few cost-effective methods have been described., Results: The aim of this study was to use the microalgae Chlamydomonas reinhardtii to produce, at laboratory scale, 15 N-labelled amino acids with a high isotopic enrichment. To do that, a culture media containing 15 NH 4 Cl was used. No kinetic isotope effect (KIE) was observed. The labelled proteins biosynthesized by the microorganism were extracted from the biomass and the 15 N-labelled amino acids were obtained after a protein hydrolysis with HCl. The use of the wall deficient strain CC503 cw92 mt+ is fit for purpose, as it only assimilates ammonia as nitrogen source, avoiding isotope contamination with nitrogen from the atmosphere or the reagents used in the culture medium, and enhancing the protein extraction efficiency compared to cell-walled wild type Chlamydomonas. The isotopic enrichment of the labelled amino acids was calculated from their isotopic composition measured by gas chromatography mass spectrometry (GC-MS). The average isotopic enrichment for the 16 amino acids characterized was 99.56 ± 0.05% and the concentration of the amino acids in the hydrolysate ranged from 18 to 90 µg/mL., Conclusions: Previously reported biochemical methods to produce isotopically labelled proteins have been applied in the fields of proteomics and fluxomics. For these approaches, low amounts of products are required and the isotopic enrichment of the molecules has never been properly determined. So far, only 13 C-labelled fatty acids have been isolated from labelled microalga biomass as valuable industrial products. In this study, we propose Chlamydomonas reinhardtii CC503 as a feasible microorganism and strain to produce labelled biomass from which a standard containing sixteen 15 N-labelled amino acids could be obtained.

Eutrophication promotes massive growth of cyanobacteria and algal blooms, which can poison other algae and reduce biodiversity. To investigate the differences in multiple nitrogen (N) sources in eutrophicated water on the emission of volatile organic compounds (VOCs) from cyanobacteria, and their toxic effects on other algal growth, we analyzed VOCs emitted from Microcystis flos-aquae with different types and concentrations of nitrogen, and determined the effects under Normal-N and Non-N conditions on Chlorella vulgaris. M. flos-aquae released 27, 22, 20, 27, 19, 25 and 17 compounds, respectively, with NaNO 3 , NaNO 2 , NH 4 Cl, urea, Ser, Lys and Arg as the sole N source. With the reduction in N amount, the emission of VOCs was increased markedly, and the most VOCs were found under Non-N condition. C. vulgaris cell propagation, photosynthetic pigment and Fv/Fm declined significantly following exposure to M. flos-aquae VOCs under Non-N condition, but not under Normal-N condition. When C. vulgaris cells were treated with two terpenoids, eucalyptol and limonene, the inhibitory effects were enhanced with increasing concentrations. Therefore, multiple N sources in eutrophicated water induce different VOC emissions from cyanobacteria, and reduction in N can cause nutrient competition, which can result in emissions of more VOCs. Those VOCs released from M. flos-aquae cells under Non-N for nutrient competition can inhibit other algal growth. Among those VOCs, eucalyptol and limonene are the major toxic agents., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Ammonia is a biologically potent molecule, and the regulation of ammonia levels in the mammalian body is, therefore, strictly controlled. The molecular paths of ammonia permeation across plasma membranes remain ill-defined, but the structural similarity of water and NH3 has pointed to the aquaporins as putative NH3-permeable pores. Accordingly, a range of aquaporins from mammals, plants, fungi, and protozoans demonstrates ammonia permeability. Aquaporin 4 (AQP4) is highly expressed at perivascular glia end-feet in the mammalian brain and may, with this prominent localization at the blood-brain-interface, participate in the exchange of ammonia, which is required to sustain the glutamate-glutamine cycle. Here we observe that AQP4-expressing Xenopus oocytes display a reflection coefficient <1 for NH4Cl at pH 8.0, at which pH an increased amount of the ammonia occurs in the form of NH3 Taken together with an NH4Cl-mediated intracellular alkalization (or lesser acidification) of AQP4-expressing oocytes, these data suggest that NH3 is able to permeate the pore of AQP4. Exposure to NH4Cl increased the membrane currents to a similar extent in uninjected oocytes and in oocytes expressing AQP4, indicating that the ionic NH4 (+) did not permeate AQP4. Molecular dynamics simulations revealed partial pore permeation events of NH3 but not of NH4 (+) and a reduced energy barrier for NH3 permeation through AQP4 compared with that of a cholesterol-containing lipid bilayer, suggesting AQP4 as a favored transmembrane route for NH3 Our data propose that AQP4 belongs to the growing list of NH3-permeable water channels., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Background: Because the model yeast Yarrowia lipolytica can synthesize and store lipids in quantities up to 20 % of its dry weight, it is a promising microorganism for oil production at an industrial scale. Typically, optimization of the lipid production process is performed in the laboratory and later scaled up for industrial production. However, the scale-up process can be complicated by genetic modifications that are optimized for one set of growing conditions can confer a less-than-optimal phenotype in a different environment. To address this issue, small cultivation systems have been developed that mimic the conditions in benchtop bioreactors. In this work, we used one such microbioreactor system, the BioLector, to develop high-throughput fermentation procedures that optimize growth and lipid accumulation in Y. lipolytica. Using this system, we were able to monitor lipid and biomass production in real time throughout the culture duration., Results: The BioLector can monitor the growth of Y. lipolytica in real time by evaluating scattered light; this produced accurate measurements until cultures reached an equivalent of OD600nm = 115 and a cell dry weight of 100 g L(-1). In addition, a lipid-specific fluorescent probe was applied which reliably monitored lipid production up to a concentration of 12 g L(-1). Through screening various growing conditions, we determined that a carbon/nitrogen ratio of 35 was the most efficient for lipid production. Further screening showed that ammonium chloride and glycerol were the most valuable nitrogen and carbon sources, respectively, for growth and lipid production. Moreover, a carbon concentration above 1 M appeared to impair growth and lipid accumulation. Finally, we used these optimized conditions to screen engineered strains of Y. lipolytica with high lipid-accumulation capability. The growth and lipid content of the strains cultivated in the BioLector were compared to those grown in benchtop bioreactors., Conclusion: To our knowledge, this is the first time that the BioLector has been used to track lipid production in real time and to monitor the growth of Y. lipolytica. The present study also showed the efficacy of the BioLector in screening growing conditions and engineered strains prior to scale-up. The method described here could be applied to other oleaginous microorganisms.

Trypanosoma cruzi, like other trypanosomatids analyzed so far, can use both glucose and amino acids as carbon and energy source. In these parasites, glycolysis is compartmentalized in glycosomes, authentic but specialized peroxisomes. The major part of this pathway, as well as a two-branched glycolytic auxiliary system, are present in these organelles. The first enzyme of one branch of this auxiliary system is the PPi-dependent pyruvate phosphate dikinase (PPDK) that converts phosphoenolpyruvate (PEP), inorganic pyrophosphate (PPi) and AMP into pyruvate, inorganic phosphate (Pi) and ATP, thus contributing to the ATP/ADP balance within the glycosomes. In this work we cloned, expressed and purified the T. cruzi PPDK. It kinetic parameters were determined, finding KM values for PEP, PPi and AMP of 320, 70 and 17 μM, respectively. Using molecular exclusion chromatography, two native forms of the enzyme were found with estimated molecular weights of 200 and 100 kDa, corresponding to a homodimer and monomer, respectively. It was established that T. cruzi PPDK's specific activity can be enhanced up to 2.6 times by the presence of ammonium in the assay mixture. During growth of epimastigotes in batch culture an apparent decrease in the specific activity of PPDK was observed. However, when its activity is normalized for the presence of ammonium in the medium, no significant modification of the enzyme activity per cell in time was found., (Copyright © 2016 Elsevier Inc. All rights reserved.)

As a promising candidate for biodiesel production, the green alga Chlorella protothecoides can efficiently produce oleaginous biomass and the lipid biosynthesis is greatly influenced by the availability of nitrogen source and corresponding nitrogen assimilation pathways. Based on isotope-assisted kinetic flux profiling (KFP), the fluxes through the nitrogen utilization pathway were quantitatively analyzed. We found that autotrophic C. protothecoides cells absorbed ammonium mainly through glutamate dehydrogenase (GDH), and partially through glutamine synthetase (GS), which was the rate-limiting enzyme of nitrogen assimilation process with rare metabolic activity of glutamine oxoglutarate aminotransferase (GOGAT, also known as glutamate synthase); whereas under heterotrophic conditions, the cells adapted to GS-GOGAT cycle for nitrogen assimilation in which GS reaction rate was associated with GOGAT activity. The fact that C. protothecoides chooses the adenosine triphosphate-free and less ammonium-affinity GDH pathway, or alternatively the energy-consuming GS-GOGAT cycle with high ammonium affinity for nitrogen assimilation, highlights the metabolic adaptability of C. protothecoides exposed to altered nitrogen conditions., (© 2015 Phycological Society of America.)

Poly-β-hydroxybutyrate (PHB) has attracted a great deal of attention in recent years due to its potential use for production of fully degradable bioplastics, however, high cost of PHB production is the major bottleneck for its wide range industrial applications. In the current study rice straw hydrolysate (RSH) was employed as a cost-effective substrate for PHB production. RSH was prepared based on biphasic acid-pretreatment of rice straw i.e. first phase treatment with 1% sulphuric acid at 121 °C for 45 min, followed by second phase treatment using 5% sulphuric acid at 121 °C for 60 min (solid:liquid ratio, 1:10). RSH turned out be an efficient substrate for PHB production from a recently isolated Bacillus cereus PS 10, and yielded higher PHB amount than that obtained with glucose (8.6g/L in glucose based medium vs 10.61 g/L in RSH based medium) after response surface methodology (RSM) based optimization. Design of experiments based on RSM was used to optimize three process variables i.e. amount of RSH and NH4Cl, and medium pH, and enhanced PHB yield (23.3%) was obtained. PHB produced was investigated by differential scanning calorimetry and X-ray diffraction powder analysis., (Copyright © 2015 Elsevier B.V. All rights reserved.)

To assess the role of open-ocean ecosystems in global CO2 fixation, we investigated how picophytoplankton, which dominate primary production, responded to episodic increases in nutrient availability. Previous experiments have shown nitrogen alone, or in combination with phosphorus or iron, to be the proximate limiting nutrient(s) for total phytoplankton grown over several days. Much less is known about how nutrient upshift affects picophytoplankton CO2 fixation over the duration of the light period. To address this issue, we performed a series of small volume (8-60 ml) - short term (10-11 h) nutrient addition experiments in different regions of the Atlantic Ocean using NH4 Cl, FeCl3 , K medium, dust and nutrient-rich water from 300 m depth. We found no significant nutrient stimulation of group-specific CO2 fixation rates of two taxonomically and size-distinct groups of plastidic protists. The above was true regardless of the region sampled or nutrient added, suggesting that this is a generic phenomenon. Our findings show that at least in the short term (i.e. daylight period), nutrient availability does not limit CO2 fixation by the smallest plastidic protists, while their taxonomic composition does not determine their response to nutrient addition., (© 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.)

In this study, levan production by Bacillus licheniformis NS032 isolated from a petroleum sludge sample was investigated. High levan yield was obtained in a wide range of sucrose concentrations (up to 400 g/L) and, contrary to most levan-producing strains, using ammonium chloride as the sole N source. Interaction between sucrose, ammonium chloride, and initial pH of the medium in a low sucrose (60-200 g/L) and a high sucrose (300-400 g/L) system was analyzed by response surface methodology. According to the calculated model in the low sucrose system, maximum predicted levan yield was 47.8 g/L (sucrose 196.8 g/L, ammonium chloride 2.4 g/L, pH 7.0), while in the high sucrose system, levan yield was 99.2 g/L (sucrose 397.6 g/L, ammonium chloride 4.6 g/L, pH 7.4). In addition, protective effect of microbial levan against copper toxicity to Daphnia magna is observed for the first time. The acute toxicity (48 h EC50) of copper decreased from 0.14 to 0.44 mg/L by levan in concentration of 50 ppm.

One of the mechanisms of healthy hepatocytes to detoxify ammonia in the liver consists of converting it into urea. When liver function is impaired, this detoxification capacity decreases and may cause severe pathologies, such as hepatic encephalopathy. Consequently, urea synthesis is a parameter that can be used to monitor liver functionality. In this chapter, a protocol for the measurement of urea synthesis in the culture medium of cultured rat hepatocytes is described. The procedure relies on a chromogenic reagent that specifically forms a colored complex with urea. The latter can be measured colorimetrically and is directly proportional to the urea concentration in the sample.

Plants form symbiotic associations with endophytic bacteria within tissues of leaves, stems, and roots. It is unclear whether or how plants obtain nitrogen from these endophytic bacteria. Here we present evidence showing nitrogen flow from endophytic bacteria to plants in a process that appears to involve oxidative degradation of bacteria. In our experiments we employed Agave tequilana and its seed-transmitted endophyte Bacillus tequilensis to elucidate organic nitrogen transfer from (15)N-labeled bacteria to plants. Bacillus tequilensis cells grown in a minimal medium with (15)NH4Cl as the nitrogen source were watered onto plants growing in sand. We traced incorporation of (15)N into tryptophan, deoxynucleosides and pheophytin derived from chlorophyll a. Probes for hydrogen peroxide show its presence during degradation of bacteria in plant tissues, supporting involvement of reactive oxygen in the degradation process. In another experiment to assess nitrogen absorbed as a result of endophytic colonization of plants we demonstrated that endophytic bacteria potentially transfer more nitrogen to plants and stimulate greater biomass in plants than heat-killed bacteria that do not colonize plants but instead degrade in the soil. Findings presented here support the hypothesis that some plants under nutrient limitation may degrade and obtain nitrogen from endophytic microbes.

Spore suspensions of Aspergillus oryzae NRRL 3484 were subjected to mutagenesis using ultraviolet-irradiation followed by chemical treatments to improve the biosynthesis of cellulase. Ten mutant strains namely UEAC7, UEAR5, UNAC4, UNAC16, UNAR19, UNBC7, UNBR3, UNBR10, UNBR23 and UNBR25 were selected and their extracellular cellulase activities were assayed. Mutant UNAC4 gave the highest cellulase production [2,455 ± 28 U/g-dry substrate (ds) for filter paper-ase (FP-ase)] in a yield 4-fold exceeding that of the wild type strain (578 ± 5.0 U/g-ds for FP-ase). Rice straw (RS) was used as a sole carbon source for the enzyme production at a concentration of 10 % (w/v). Maximum cellulase production was achieved at initial medium pH 5.5, initial moisture content 77 % and an incubation temperature 28 °C on the fifth day of growth. NH4Cl proved to be the suitable added nitrogen source for maximum enzyme production followed by peptone. These results clearly indicate the cost-effectiveness of solid state fermentation technology in the economic production of extracellular cellulase. The hyper-production of cellulase by mutant strain UNAC4 has potential for industrial processes that convert lignocellulosic material (e.g. RS) into products of commercial value such as glucose and biofuels.

The reproductive toxicity potential of Ammonium Salt of Perfluorinated Hexanoic Acid (PFHxA Ammonium Salt) in pregnant Crl: CD1(ICR) mice was investigated. Twenty females/group were administered the test substance or vehicle once daily from gestation day 6 through 18. Phase 1 doses: 0, 100, 350, and 500 mg/kg/d; phase 2: 0, 7, 35, and 175 mg/kg/d. Parameters evaluated include mortality, viability, body weights, clinical signs, abortions, premature deliveries, pregnancy and fertility, litter observations, maternal behavior, and sexual maturity in the F1 generation. The level of PFHxA Ammonium Salt was measured in the liver of F0 and F1 mice. At doses of 350 and 500 mg/kg/d maternal mortalities, excess salivation and changes in body weight gains occurred. Pup body weights were reduced on postpartum day (PPD) 0 in all the dosage groups, but persisted only in the 350 and 500 mg/kg/d groups. Additional effects at 300 and 500 mg/kg/d included stillbirths, reductions in viability indices, and delays in physical development. Levels of PFHxA Ammonium Salt in the livers of the 100 mg/kg/d dams were all below the lower limit of quantization (0.02 µg/mL); in the 350 mg/kg/d group, 3 of the 8 samples had quantifiable analytical results. In phase 2 no PFHxA Ammonium Salt was found in the liver. Adverse effects occurred only in the 175 mg/kg/d group and consisted of increased stillborn pups, pups dying on PPD 1, and reduced pup weights on PPD 1. Based on these data, the maternal and reproductive no observable adverse effect level of PFHxA Ammonium Salt is 100 mg/kg/d., (© The Author(s) 2014.)

A process for the conversion of post consumer (agricultural) polyethylene (PE) waste to the biodegradable polymer medium chain length polyhydroxyalkanoate (mcl-PHA) is reported here. The thermal treatment of PE in the absence of air (pyrolysis) generated a complex mixture of low molecular weight paraffins with carbon chain lengths from C8 to C32 (PE pyrolysis wax). Several bacterial strains were able to grow and produce PHA from this PE pyrolysis wax. The addition of biosurfactant (rhamnolipids) allowed for greater bacterial growth and PHA accumulation of the tested strains. Some strains were only capable of growth and PHA accumulation in the presence of the biosurfactant. Pseudomonas aeruginosa PAO-1 accumulated the highest level of PHA with almost 25 % of the cell dry weight as PHA when supplied with the PE pyrolysis wax in the presence of rhamnolipids. The change of nitrogen source from ammonium chloride to ammonium nitrate resulted in faster bacterial growth and the earlier onset of PHA accumulation. To our knowledge, this is the first report where PE is used as a starting material for production of a biodegradable polymer.

Background: 2-phenylethanl (2-PE) and its derivatives are important chemicals, which are widely used in food materials and fine chemical industries and polymers and it's also a potentially valuable alcohol for next-generation biofuel. However, the biosynthesis of 2-PE are mainly biotransformed from phenylalanine, the price of which barred the production. Therefore, it is necessary to seek more sustainable technologies for 2-PE production., Results: A new strain which produces 2-PE through the phenylpyruvate pathway was isolated and identified as Enterobacter sp. CGMCC 5087. The strain is able to use renewable monosaccharide as the carbon source and NH4Cl as the nitrogen source to produce 2-PE. Two genes of rate-limiting enzymes, chorismate mutase p-prephenate dehydratase (PheA) and 3-deoxy-d-arabino-heptulosonic acid 7-phosphate synthase (DAHP), were cloned from Escherichia coli and overexpressed in E. sp. CGMCC 5087. The engineered E. sp. CGMCC 5087 produces 334.9 mg L⁻¹ 2-PE in 12 h, which is 3.26 times as high as the wild strain., Conclusions: The phenylpyruvate pathway and the substrate specificity of 2-keto-acid decarboxylase towards phenylpyruvate were found in E. sp. CGMCC 5087. Combined with the low-cost monosaccharide as the substrate, the finding provides a novel and potential way for 2-PE production.

Slc4a11, a member of the solute linked cotransporter 4 family that is comprised predominantly of bicarbonate transporters, was described as an electrogenic 2Na(+)-B(OH)4(-) (borate) cotransporter and a Na(+)-2OH(-) cotransporter. The goal of the current study was to confirm and/or clarify the function of SLC4A11. In HEK293 cells transfected with SLC4A11 we tested if SLC4A11 is a: 1) Na(+)-HCO3(-) cotransporter, 2) Na(+)-OH(-)(H(+)) transporter, and/or 3) Na(+)-B(OH)4(-) cotransporter. CO2/HCO3(-) perfusion yielded no significant differences in rate or extent of pHi changes or Na(+) flux in SLC4A11-transfected compared with control cells. Similarly, in CO2/HCO3(-), acidification on removal of Na(+) and alkalinization on Na(+) add back were not significantly different between control and transfected indicating that SLC4A11 does not have Na(+)-HCO3(-) cotransport activity. In the absence of CO2/HCO3(-), SLC4A11-transfected cells showed higher resting intracelllular Na(+) concentration ([Na(+)]i; 25 vs. 17 mM), increased NH4(+)-induced acidification and increased acid recovery rate (160%) after an NH4 pulse. Na(+) efflux and influx were faster (80%) following Na(+) removal and add back, respectively, indicative of Na(+)-OH(-)(H(+)) transport by SLC4A11. The increased alkalinization recovery was confirmed in NHE-deficient PS120 cells demonstrating that SLC4A11 is a bonafide Na(+)-OH(-)(H(+)) transporter and not an activator of NHEs. SLC4A11-mediated H(+) efflux is inhibited by 5-(N-ethyl-N-isopropyl) amiloride (EIPA; EC50: 0.1 μM). The presence of 10 mM borate did not alter dpHi/dt or ΔpH during a Na(+)-free pulse in SLC4A11-transfected cells. In summary our results show that SLC4A11 is not a bicarbonate or borate-linked transporter but has significant EIPA-sensitive Na(+)-OH(-)(H(+)) and NH4(+) permeability.

The fungal organisms, especially pathogens, change their vegetative (Y, unicellular yeast and H, hypha) morphology reversibly for survival and proliferation in the host environment. NAD-dependent glutamate dehydrogenase (NAD-GDH, EC 1.4.1.2) from a non-pathogenic dimorphic zygomycete Benjaminiella poitrasii was previously reported to be an important biochemical correlate of the transition process. The enzyme was purified to homogeneity and characterized. It is a 371 kDa native molecular weight protein made up of four identical subunits. Kinetic studies showed that unlike other NAD-GDHs, it may act as an anabolic enzyme and has more affinity towards 2-oxoglutarate than L-glutamate. Chemical modifications revealed the involvement of single histidine and lysine residues in the catalytic activity of the enzyme. The phosphorylation and dephosphorylation study showed that the NAD-GDH is present in active phosphorylated form in hyphal cells of B. poitrasii. Two of the 1,2,3 triazole linked β-lactam-bile acid conjugates synthesized in the laboratory (B18, B20) were found to be potent inhibitors of purified NAD-GDH which also significantly affected Y-H transition in B. poitrasii. Furthermore, the compound B20 inhibited germ tube formation during Y-H transition in Candida albicans strains and Yarrowia lipolytica. The possible use of NAD-GDH as a target for antifungal agents is discussed.

Biofilters degrade a small fraction of the natural organic matter (NOM) contained in seawater which is the leading cause of biofouling in downstream processes. This work studies the effects of chemical additions on NOM biodegradation by biofilters. In this work, biofiltration of seawater with an empty bed contact time (EBCT) of 6 min and a hydraulic loading rate of 10 mh(-1) reduces the biological oxygen demand (BOD7) by 8%, the dissolved organic carbon (DOC) by 6% and the UV absorbance at 254 nm (A₂₅₄) by 7%. Different amounts of ammonium chloride are added to the seawater (up to twice the total dissolved nitrogen in untreated seawater) to study its possible effect on the removal of NOM by a pilot-scale biofilter. Seawater is amended with different amounts of easily biodegradable dissolved organic carbon (BDOC) supplied as sodium acetate (up to twice the DOC) for the same purpose. The results of this work reveal that the ammonium chloride additions do not significantly affect NOM removal and the sodium acetate is completely consumed by the biofiltration process. For both types of chemical additions, the BOD₇, DOC and A₂₅₄ in the outlet stream of the biofilter are similar to the values for the untreated control. These results indicate that this biofilter easily removes the BDOC from the seawater when the EBCT is not above 6 min. Furthermore, nitrogen does not limit the NOM biodegradation in seawater under these experimental conditions., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Although nitrate (NO3(-)) but not ammonium (NH4(+)) improves plant tolerance to oxygen deficiency, the mechanisms involved in this phenomenon are just beginning to be understood. By using gas chromatography-mass spectrometry, we investigated the metabolic fate of (15)NO3(-) and (15)NH4(+) in soybean plants (Glycine max L. Merril cv. IAC-23) subjected to root hypoxia. This stress reduced the uptake of (15)NO3(-) and (15)NH4(+) from the medium and decreased the overall assimilation of these nitrogen sources into amino acids in roots and leaves. Root (15)NO3(-) assimilation was more affected by hypoxia than that of (15)NH4(+), resulting in enhanced nitrite and nitric oxide release in the solution. However, (15)NO3(-) was translocated in substantial amounts by xylem sap and considerable (15)NO3(-) assimilation into amino acids also occurred in the leaves, both under hypoxia and normoxia. By contrast, (15)NH4(+) assimilation occurred predominantly in roots, resulting in accumulation of mainly (15)N-alanine in this tissue during hypoxia. Analysis of lactate levels suggested higher fermentation in roots from NH4(+)-treated plants compared to the NO3(-) treatment. Thus, foliar NO3(-) assimilation may be relevant to plant tolerance to oxygen deficiency, since it would economize energy expenditure by hypoxic roots. Additionally, the involvement of nitric oxide synthesis from nitrite in the beneficial effect of NO3(-) is discussed., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

Helicobacter pylori increased the gamma-glutamyltranspeptidase (GGT) production under low-pH (maximal at pH 4) and appropriate pCO2 conditions, while the production of GGT mRNA correlated with increased total enzyme activity. At pH 4, the bacterium augmented enzyme production in the presence of glutamine (~10 mM) in the medium, which predominantly occurred after a 6-min time-lag. Monovalent salts such as NaCl or NH4Cl facilitated enzymatic activation in acidic solutions of approximately pH 4.5. In addition, glutathione's gamma-glutamyl moiety cysteinylglycine appeared to be taken up readily by the intact H. pylori, but not by the one pretreated with a potent GGT inhibitor, acivicin, suggesting that the GGT may partake in glutathione uptake by the cell.

Piperazine is widely used as an intermediate in the manufacture of insecticides, rubber chemicals, corrosion inhibitors, and urethane. In this study, a highly effective piperazine-degrading bacteria strain, TOH, was isolated from the acclimated activated sludge of a pharmaceutical plant. This strain, identified as Paracoccus sp., utilises piperazine as the sole source of carbon, nitrogen and energy for growth. The optimal pH and temperature for the growth of TOH were 8.0 and 30°C, respectively. The effects of co-substrates and heavy metals on the degradation efficiency of piperazine were investigated. The results indicated that exogenously supplied glucose promoted the degradation of piperazine, while the addition of ammonium chloride slightly inhibited piperazine degradation. Metal ions such as Ni(2+) and Cd(2+) inhibited the degradation of piperazine, whereas Mg(2+) increased it. In addition, metabolic intermediates were identified by mass spectrometry, allowing a degradation pathway for piperazine to be proposed for the first time., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

In this study, Na(+) uptake and acid-base balance in the euryhaline pupfish Cyprinodon variegatus variegatus were characterized when fish were exposed to pH 4.5 freshwater (7mM Na(+)). Similar to the related cyprinodont, Fundulus heteroclitus, Na(+) uptake was significantly inhibited when exposed to low pH water. However, it initially appeared that C. v. variegatus increased apparent net acid excretion at low pH relative to circumneutral pH. This result is opposite to previous observations for F. heteroclitus under similar conditions where fish were observed to switch from apparent net H(+) excretion at circumneutral pH to apparent net H(+) uptake at low pH. Further investigation revealed disparate observations between these studies were the result of using double endpoint titrations to measure titratable alkalinity fluxes in the current study, while the earlier study utilized single endpoint titrations to measure these fluxes (i.e.,. Cyprinodon acid-base transport is qualitatively similar to Fundulus when characterized using single endpoint titrations). This led to a comparative investigation of these two methods. We hypothesized that either the single endpoint methodology was being influenced by a change in the buffer capacity of the water (e.g., mucus being released by the fish) at low pH, or the double endpoint methodology was not properly accounting for ammonia flux by the fish. A series of follow-up experiments indicated that buffer capacity of the water did not change significantly, that excretion of protein (a surrogate for mucus) was actually reduced at low pH, and that the double endpoint methodology does not properly account for NH(3) excretion by fish under low pH conditions. As a result, it overestimates net H(+) excretion during low pH exposure. After applying the maximum possible correction for this error (i.e., assuming that all ammonia is excreted as NH(3)), the double endpoint methodology indicates that net H(+) transport was reduced to effectively zero in both species at pH 4.5. However, significant differences between the double endpoint (no net H(+) transport at low pH) and single endpoint titrations (net H(+) uptake at low pH) remain to be explained., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Sacramento Valley (California, USA) soils and sediments have high concentrations of Cr(III) because they are partially derived from ultramafic material. Some Cr(III) is oxidized to more toxic and mobile Cr(VI) by soil Mn oxides. Valley soils typically have neutral to alkaline pH at which Cr(III) is highly immobile. Much of the valley is under cultivation and is both fertilized and irrigated. A series of laboratory incubation experiments were conducted to assess how cultivation might impact Cr cycling in shallow vadose zone material from the valley. The first experiments employed low (7.1 mmol N per kg soil) and high (35 mmol Nkg(-1)) concentrations of applied (NH(4))(2)SO(4). Initially, Cr(VI) concentrations were up to 45 and 60% greater than controls in low and high incubations, respectively. After microbially-mediated oxidation of all NH(4)(+), Cr(VI) concentrations dropped below control values. Increased nitrifying bacterial populations (estimated by measurement of phospholipid fatty acids) may have increased the Cr(VI) reduction capacity of the vadose zone material resulting in the observed decreases in Cr(VI). Another series of incubations employed vadose zone material from a different location to which low (45 meq kg(-1)) and high (128 meq kg(-1)) amounts of NH(4)Cl, KCl, and CaCl(2) were applied. All treatments, except high concentration KCl, resulted in mean soil Cr(VI) concentrations that were greater than the control. High concentrations of water-leachable Ba(2+) (mean 38 μmol kg(-1)) in this treatment may have limited Cr(VI) solubility. A final set of incubations were amended with low (7.1 mmol Nkg(-1)) and high (35 mmol Nkg(-1)) concentrations of commercial liquid ammonium polyphosphate (APP) fertilizer which contained high concentrations of Cr(III). Soil Cr(VI) in the low APP incubations increased to a concentration of 1.8 μmol kg(-1) (5× control) over 109 days suggesting that Cr(III) added with the APP fertilizer was more reactive than naturally-occurring soil Cr(III)., (Published by Elsevier B.V.)

This study aimed to clone and sequence the cystic fibrosis transmembrane conductance regulator (cftr) from, and to determine the effects of seawater acclimation or exposure to 100 mmol l⁻¹ NH₄Cl in freshwater on its mRNA and protein expressions in, the gills of Anabas testudineus. There were 4,530 bp coding for 1,510 amino acids in the cftr cDNA sequence from A. testudineus. The branchial mRNA expression of cftr in fish kept in freshwater was low (<50 copies of transcript per ng cDNA), but significant increases were observed in fish acclimated to seawater for 1 day (92-fold) or 6 days (219-fold). Branchial Cftr expression was detected in fish acclimated to seawater but not in the freshwater control, indicating that Cl⁻ excretion through the apical Cftr of the branchial epithelium was essential to seawater acclimation. More importantly, fish exposed to ammonia also exhibited a significant increase (12-fold) in branchial mRNA expression of cftr, with Cftr being expressed in a type of Na⁺/K⁺-ATPase-immunoreactive cells that was apparently different from the type involved in seawater acclimation. It is probable that Cl⁻ excretion through Cftr generated a favorable electrical potential across the apical membrane to drive the excretion of NH₄⁺ against a concentration gradient through a yet to be determined transporter, but it led to a slight loss of endogenous Cl⁻. Since ammonia exposure also resulted in significant decreases in blood pH, [HCO₃⁻] and [total CO₂] in A. testudineus, it can be deduced that active NH₄⁺ excretion could also be driven by the exit of HCO₃⁻ through the apical Cftr. Furthermore, A. testudineus uniquely responded to ammonia exposure by increasing the ambient pH and decreasing the branchial bafilomycin-sensitive V-type H⁺-ATPase activity, which suggests that its gills might have low NH₃ permeability.

Hypokalemia is associated with increased ammoniagenesis and stimulation of net acid excretion by the kidney in both humans and experimental animals. The molecular mechanisms underlying these effects remain unknown. Toward this end, rats were placed in metabolic cages and fed a control or K(+)-deficient diet (KD) for up to 6 days. Rats subjected to KD showed normal acid-base status and serum electrolytes composition. Interestingly, urinary NH(4)(+) excretion increased significantly and correlated with a parallel decrease in urine K(+) excretion in KD vs. control animals. Molecular studies showed a specific upregulation of the glutamine transporter SN1, which correlated with the upregulation of glutaminase (GA), glutamate dehydrogenase (GDH), and phosphoenolpyruvate carboxykinase. These effects occurred as early as day 2 of KD. Rats subjected to a combined KD and 280 mM NH(4)Cl loading (to induce metabolic acidosis) for 2 days showed an additive increase in NH(4)(+) excretion along with an additive increment in the expression levels of ammoniagenic enzymes GA and GDH compared with KD or NH(4)Cl loading alone. The incubation of cultured proximal tubule cells NRK 52E or LLC-PK(1) in low-K(+) medium did not affect NH(4)(+) production and did not alter the expression of SN1, GA, or GDH in NRK cells. These results demonstrate that K(+) deprivation stimulates ammoniagenesis through a coordinated upregulation of glutamine transporter SN1 and ammoniagenesis enzymes. This effect is developed before the onset of hypokalemia. The signaling pathway mediating these events is likely independent of KD-induced intracellular acidosis. Finally, the correlation between increased NH(4)(+) production and decreased K(+) excretion indicate that NH(4)(+) synthesis and transport likely play an important role in renal K(+) conservation during hypokalemia.

In this study, we describe the presence of apoptosis, associated with a mitochondrial dysfunction in the hippocampus of animals in an experimental model defined as minimal hepatic encephalopathy (MHE). This experimental model was studied after 10 days of induced portal vein calibrated stricture, leading to portal hypertension and to a moderate hyperammonemia, without the presence of other evident central nervous system changes. The molecular mechanisms here proposed indicate the presence of apoptotic intrinsic pathways that point to hippocampal mitochondria as an important mediator of apoptosis in this experimental model. In this model of MHE, the presence of DNA fragmentation is documented by 2.3-times increased number of TUNEL-positive cells. These findings together with a higher ratio of the Bcl-2 family members Bax/Bcl-xL in the outer mitochondrial membrane of the MHE animals together with 11% of cytochrome c release indicate the presence of apoptosis in this experimental model. A detailed analysis of the hippocampal mitochondrial physiology was performed after mitochondrial isolation. The determination of the respiratory rate in the presence of malate plus glutamate and ADP showed a 45% decrease in respiratory control in MHE animals as compared with the sham group. A marked decrease of cytochrome oxidase (complex IV of the electron transport chain) was also observed, showing 46% less activity in hippocampal mitochondria from MHE animals. In addition, mitochondria from these animals showed less ability to maintain membrane potential (ΔΨ (m)) which was 13% lower than the sham group. Light scattering experiments showed that mitochondria from MHE animals were more sensitive to swell in the presence of increased calcium concentrations as compared with the sham group. In addition, in vitro studies performed in mitochondria from sham animals showed that mitochondrial permeability transition (MPT) could be a mitochondrial mediator of the apoptotic signaling in the presence of NH(4) (+) and calcium.

The chromatophore membrane of the photosynthetic diazotroph Rhodospirillum rubrum is of vital importance for a number of central processes, including nitrogen fixation. Using a novel amphiphile, we have identified protein complexes present under different nitrogen availability conditions by the use of two-dimensional Blue Native/SDS-PAGE and NSI-LC-LTQ-Orbitrap mass spectrometry. We have identified several membrane protein complexes, including components of the ATP synthase, reaction center, light harvesting, and NADH dehydrogenase complexes. Additionally, we have identified differentially expressed proteins, such as subunits of the succinate dehydrogenase complex and other TCA cycle enzymes that are usually found in the cytosol, thus hinting at a possible association to the membrane in response to nitrogen deficiency. We propose a redox sensing mechanism that can influence the membrane subproteome in response to nitrogen availability.

Background: Foot-and-mouth disease virus (FMDV) is able to cause persistent infection in ruminants besides acute infection and disease. Since the mechanisms of viral persistence and the determining factors are still unknown, in vitro systems help explore and reveal mechanisms of persistence in vivo by providing useful models for the study of RNA genome mutations and evolution. Ammonium chloride, a lysosomotropic agent that raises intralysosomal pH, reduces the yield of FMDV during infection of BHK-21 cells., Results: The persistent infection with FMDV serotype O in BHK-21 cells was selected and established readily after treatment of ammonium chloride that acts primarily on the cells. Intact virions were observed located inside the endosomes. Viral genome RNAs and specific proteins were detected in the persistent cells to validate the establishment of viral persistence. Infection of the persistent viruses could not form plaques in host cells but virulence was enhanced. Basing on analysis and comparison of cDNA sequences of resident viruses and wild type viruses, 15 amine acid mutations were found, all of which were located in nonstructural proteins rather than in structural proteins., Conclusions: Therefore, persistent infection of cell cultures with FMDV is successfully established with some distinctive features. It would be worthwhile to further investigate the mechanisms of viral persistence.

HCV has two envelop proteins named as E1 and E2 which play an important role in cell entry through two main pathways: direct fusion at the plasma membrane and receptor-mediated endocytosis. Fusion of the HCV envelope proteins is triggered by low pH within the endosome. Lysosomotropic agents (LA) such as Chloroquine and Ammonium chloride (NH₄Cl) are the weak bases and penetrate in lysosome as protonated form and increase the intracellular pH. To investigate the antiviral effect of LA (Chloroquine and NH₄Cl) on pH dependent endocytosis, HCV pseudoparticles (HCVpp) of 1a and 3a genotype were produced and used to infect liver cells. The toxicological effects of Chloroquine and NH₄Cl were tested in liver cells through MTT cell proliferation assay. For antiviral screening of Chloroquine and NH₄Cl, liver cells were infected with HCVpp of 3a and 1a genotype in the presence or absence of different concentrations of Chloroquine and NH₄Cl and there luciferase activity was determined by using a luminometer. The results demonstrated that Chloroquine and NH₄Cl showed more than 50% reduction of virus infectivity at 50 μM and 10 mM concentrations respectively. These results suggest that inhibition of HCV at fusion step by increasing the lysosomal pH will be better option to treat chronic HCV.