Elimination of inhibitory effects of dodecyl dimethyl benzyl ammonium chloride on microalgae in wastewater by cocultivation with a newly screened microbial consortium.

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 CH5O3P, C6H5CH2-, and C8H11N), 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.

The proton channel OTOP1 is a sensor for the taste of ammonium chloride.

Ammonium (NH4+), 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 (NH4Cl). Extracellular NH4Cl 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 NH4Cl relative to acid stimuli. Taste responses to NH4Cl measured from isolated Type III TRCs, or gustatory nerves were strongly attenuated or eliminated in an Otop1-/- mouse strain. Behavioral aversion of mice to NH4Cl, 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 NH4Cl and a previously undescribed channel activation mechanism.

The protective effect of Luteolin on chicken spleen lymphocytes from ammonia poisoning through mitochondria and balancing energy metabolism disorders.

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, Ca2+-ATPase, Mg2+-ATPase, and Ca/Mg2+-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.

Traumatic brain injury alters dendritic cell differentiation and distribution in lymphoid and non-lymphoid organs.

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.

Quantitative proteomic analysis of the microbial degradation of 3-aminobenzoic acid by Comamonas sp. QT12.

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.

Characterization of petroleum degrading bacteria and its optimization conditions on effective utilization of petroleum hydrocarbons.

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.

Disturbance of hepatocyte growth and metabolism in a hyperammonemia microenvironment.

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.

Tailoring Surface Properties of Electrodes for Synchronous Enhanced Extracellular Electron Transfer and Enriched Exoelectrogens in Microbial Fuel Cells.

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.

Optimization of nitrogen source supply for enhanced biosynthesis and quality of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by extremely halophilic archaeon Haloferax mediterranei.

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], NH4 Cl and combination of YE and NH4 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 NH4 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 NH4 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 NH4 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.

Quantitative Proteomics Analysis by Sequential Window Acquisition of All Theoretical Mass Spectra-Mass Spectrometry Reveals Inhibition Mechanism of Pigments and Citrinin Production of Monascus Response to High Ammonium Chloride Concentration.

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 ß-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.

Acidic Stress Triggers Sodium-Coupled Bicarbonate Transport and Promotes Survival in A375 Human Melanoma Cells.

Melanoma cells preserve intracellular pH (pHi) 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 pHi 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.

NH4Cl treatment prevents doxorubicin-induced myocardial dysfunction in vivo.

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 (NH4Cl), 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 NH4CL 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 NH4CL effectively improved DOX-induced cardiomyocyte apoptosis and cardiac dysfunction in mice. Our results showed that NH4CL significantly improved DOX-induced contractile dysfunction, inflammation, apoptosis and autophagy in mice. SIGNIFICANCE: Our results indicate that NH4CL 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.

Spindle pole body movement is affected by glucose and ammonium chloride in fission yeast.

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.

Pivotal role of inter-organ aspartate metabolism for treatment of mitochondrial aspartate-glutamate carrier 2 (citrin) deficiency, based on the mouse model.

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.

Optimization of Synechococcus sp. VDW Cultivation with Artificially Prepared Shrimp Wastewater for Ammonium Removal and Its Potential for Use As a Biofuel Feedstock.

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 NH4Cl. 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-1d-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.

Transcriptome analysis of Rhodobacter capsulatus grown on different nitrogen sources.

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.

R125H, W240S, C386R, and V507I SLC4A11 mutations associated with corneal endothelial dystrophy affect the transporter function but not trafficking in PS120 cells.

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 NH3 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 NH4Cl-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 NH4Cl 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 NH4Cl 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.

Mitotic defects in fission yeast lipid metabolism 'cut' mutants are suppressed by ammonium chloride.

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.

Bacterial polyhydroxybutyrate for electrospun fiber production.

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 NH4Cl (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.

A cost-effective approach to produce 15N-labelled amino acids employing Chlamydomonas reinhardtii CC503.

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, 15N-labelled amino acids with a high isotopic enrichment. To do that, a culture media containing 15NH4Cl was used. No kinetic isotope effect (KIE) was observed. The labelled proteins biosynthesized by the microorganism were extracted from the biomass and the 15N-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 13C-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 15N-labelled amino acids could be obtained.