Nitrification kinetics, N2O emission, and energy use in intermittently aerated hybrid reactor under different organic loading rates.

This study investigated the impact of intermittent aeration strategies and reduction in the reactor's organic and nitrogen loading rates on the course of particular stages of the nitrification process, as well as energy consumption and N2O emissions in a hybrid reactor with nitrification/denitrification. Each of the analysed series revealed the greatest ammonia oxidation activity in activated sludge flocs. The highest activity of nitrite nitrogen oxidation was demonstrated in the case of biofilm. A reduction in the reactor's organic and nitrogen loading rate value had a greater effect on changes in the activity of ammonia-oxidizing bacteria than nitrite-oxidizing bacteria. In a system where the operation of air pumps was controlled through switching them and off according to the adopted ratio between non-aerated and aerated sub-phase times and the assumed oxygen concentration, a reduction in the duration of aerated sub-phases caused no decrease in energy use for aeration. Lower N2O emission was recorded when the reactor operated with a longer duration of aerated sub-phases. Supplementary Information: The online version contains supplementary material available at 10.1007/s13762-022-04715-6.

Carbon source recovery from excess sludge by mechanical disintegration for biological denitrification.

The goal of the study was to evaluate the possibility of carbon source recovery from excess sludge by mechanical disintegration for biological denitrification. The total efficiency of denitrification, unit demand for organic compounds for denitrification, unit volume of disintegrated sludge and unit cost of nitrogen removal as a function of energy density used for excess sludge disintegration (70, 140 and 210 kJ/L) were analyzed. In the study a full-scale disc disintegrator was used (motor power: 30 kWh, motor speed: 2,950 rpm). It was shown that the amounts of organic compounds released from the activated sludge flocs at all tested levels of energy density are high enough to be used to intensify the removal of nitrogen compounds from wastewater. It was also documented that the energy density provided during process of disintegration was an important factor determining the characteristics of organic compounds obtained under the disintegration for their use in order to intensify the process of denitrification. The highest value of total efficiency of denitrification (50.5 ± 3.1 mg N/L) was obtained for carbon source recovery from excess sludge at 70 kJ/L, but the lowest unit cost of nitrogen removal occurred for 140 kJ/L (0.0019 ± 0.0011 EUR/g N).

The rate of denitrification using hydrodynamically disintegrated excess sludge as an organic carbon source.

This study investigates the potential of hydrodynamically disintegrated excess activated sludge when used as a supplementary carbon source for denitrification. Two objectives constituted this study: (i) to analyse the denitrification rate by using excess sludge subjected to hydrodynamic disintegration (HD), performed at different energy densities, as an organic carbon source, and (ii) to analyse the impact of hydrolysis of disintegrated sludge on the denitrification rate. Nitrate reduction tests were conducted to assess the denitrification rate for the following sources of organic carbon: thickened excess sludge disintegrated at three levels of energy density (70, 140 and 210 kJ/L), acetic acid solution and municipal wastewater after mechanical treatment. It was found that the HD of excess sludge conducted at different levels of energy density led to dissolved organic compounds characterised by various properties as donors of H+ in the denitrification process. The susceptibility of disintegrated sludge to anaerobic hydrolysis decreased along with the increasing energy density. The obtained organic carbon contributed to a lower increase in the denitrification rate in comparison to that when disintegrated sludge not subjected to hydrolysis was applied.

In utero gene therapy rescues microcephaly caused by Pqbp1-hypofunction in neural stem progenitor cells.

Human mutations in PQBP1, a molecule involved in transcription and splicing, result in a reduced but architecturally normal brain. Examination of a conditional Pqbp1-knockout (cKO) mouse with microcephaly failed to reveal either abnormal centrosomes or mitotic spindles, increased neurogenesis from the neural stem progenitor cell (NSPC) pool or increased cell death in vivo. Instead, we observed an increase in the length of the cell cycle, particularly for the M phase in NSPCs. Corresponding to the developmental expression of Pqbp1, the stem cell pool in vivo was decreased at E10 and remained at a low level during neurogenesis (E15) in Pqbp1-cKO mice. The expression profiles of NSPCs derived from the cKO mouse revealed significant changes in gene groups that control the M phase, including anaphase-promoting complex genes, via aberrant transcription and RNA splicing. Exogenous Apc4, a hub protein in the network of affected genes, recovered the cell cycle, proliferation, and cell phenotypes of NSPCs caused by Pqbp1-cKO. These data reveal a mechanism of brain size control based on the simple reduction of the NSPC pool by cell cycle time elongation. Finally, we demonstrated that in utero gene therapy for Pqbp1-cKO mice by intraperitoneal injection of the PQBP1-AAV vector at E10 successfully rescued microcephaly with preserved cortical structures and improved behavioral abnormalities in Pqbp1-cKO mice, opening a new strategy for treating this intractable developmental disorder.

Efficiency of wastewater treatment in SBR and IFAS-MBSBBR systems in specified technological conditions.

The objective of this study is to compare wastewater treatment effectiveness in sequencing batch reactor (SBR) and integrated fixed-film activated sludge-moving-bed sequencing batch biofilm reactor (IFAS-MBSBBR) systems in specific technological conditions. The comparison of these two technologies was based on the following assumptions, shared by both series, I and II: the reactor's active volume was 28 L; 8-hour cycle of reactor's work, with the same sequence and duration of its consecutive phases; and the dissolved oxygen concentration in the aerobic phases was maintained at a level of 3.0 mg O2/L. For both experimental series (I and II), comparable effectiveness of organic compound (chemical oxygen demand (COD)) removal, nitrification and biological phosphorus removal has been obtained at levels of 95.1%, 97% and 99%, respectively. The presence of the carrier improved the efficiency of total nitrogen removal from 86.3% to 91.7%. On the basis of monitoring tests, it has been found that the ratio of simultaneous denitrification in phases with aeration to the total efficiency of denitrification in the cycle was 1.5 times higher for IFAS-MBSBBR.

Impact of multiple wastewater feedings on the efficiency of nutrient removal in an IFAS-MBSBBR: number of feedings vs. efficiency of nutrient removal.

This article presents the results of research into the influence of one, two and three wastewater feedings in a cycle on efficiency and performance of combined biological nitrogen and phosphorus removal in an integrated fixed-film activated sludge and moving-bed sequencing batch biofilm reactor (IFAS-MBSBBR). The experiment lasted 158 days and was conducted in two laboratory models of the IFAS-MBSBBR with an active volume of 28 L. It was found that along with an increase in the number of wastewater feedings, an increase in nitrogen removal efficiency was observed (from 56.9 ± 2.30% for a single feeding to 91.4 ± 1.77% for three feedings). Moreover, the contribution of simultaneous nitrification/denitrification in nitrogen removal increased (from 2.58% for a single feeding to 69.5% for three feedings). Systems with a greater number of feedings stimulated the process of denitrifying phosphorus removal. Regardless of the way in which wastewater feeding was applied to the IFAS-MBSBBR, highly efficient chemical oxygen demand (COD) removal (94.8 ± 1.80%) and biological phosphorus removal (98.9 ± 0.87%) were achieved.

Nitrogen and phosphorus removal in a denitrifying phosphorus removal process in a sequencing batch reactor with a forced anoxic phase.

The objective of this study was to establish such operating conditions in a sequencing batch reactor (SBR) that will enable the achievement of the highest possible share of denitrifying P removal in nutrient elimination. Two different operating strategies for SBRs were analysed. Both of these strategies used a forced anoxic phase in the SBR treatment cycle. The first one was based on an intermittent aeration, which led to periodic occurrence of anoxic conditions when the uptake of P-PO4(3-) could occur. The second strategy was based on mimicking the A2O process and forcing an anoxic phase straight after an anaerobic phase. The experiments were performed in a laboratory reactor operating at a maximum fill of 26.8-27.7 litres and a constant temperature of 18 degrees C. It was found that a SBR configuration with intermittent aeration did not allow the achievement of significant denitrifying P removal, despite the DPAO/PAO ratio being equal to 50.5%. Almost the entire load of orthophosphates was being removed in aerobic conditions right after the anaerobic phase, even though this aerobic period lasted only 20 minutes. However, a SBR with a forced anoxic phase occurring after an anaerobic phase and created by an introduction of NO(x) rich stream of wastewater guaranteed the highest DPAO/PAO ratio of 82.8% and the highest share of denitrifying P removal (above 80%) in the total removal of phosphorus.

Evaluation of deammonification process performance at different aeration strategies.

In a deammonification process applied in the moving bed biofilm reactor (MBBR) oxygen is a crucial parameter for the process performance and efficiency. The objective of this study was to investigate different aeration strategies, characterised by the ratio between non-aerated and aerated phase times (R) and dissolved oxygen concentrations (DO). The series of batch tests were conducted with variable DO concentrations (2, 3, 4 mg L(-1)) and R values (0-continuous aeration; 1/3, 1, 3-intermittent aeration) but with the same initial ammonium concentration, volume of the moving bed and temperature. It was found that the impact of DO on deammonification was dependent on the R value. At R=0 and R=1/3, an increase of DO caused a significant increase in nitrogen removal rate, whereas for R=1 and R=3 similar rates of the process were observed irrespectively of the DO. The highest nitrogen removal rate of 3.33 g N m(-2) d(-1) (efficiency equal to 69.5%) was obtained at R=1/3 and DO=4 mg L(-1). Significantly lower nitrogen removal rates (1.17-1.58 g N m(-2) d(-1)) were observed at R=1 and R=3 for each examined DO. It was a consequence reduced aerated phase duration times and lesser amounts of residual nitrite in non-aerated phases as compared to R=1/3.

Signal transducing molecules and glycosyl-phosphatidylinositol-linked proteins form a caveolin-rich insoluble complex in MDCK cells.

GPI-linked protein molecules become Triton-insoluble during polarized sorting to the apical cell surface of epithelial cells. These insoluble complexes, enriched in cholesterol, glycolipids, and GPI-linked proteins, have been isolated by flotation on sucrose density gradients and are thought to contain the putative GPI-sorting machinery. As the cellular origin and molecular protein components of this complex remain unknown, we have begun to characterize these low-density insoluble complexes isolated from MDCK cells. We find that these complexes, which represent 0.4-0.8% of the plasma membrane, ultrastructurally resemble caveolae and are over 150-fold enriched in a model GPI-anchored protein and caveolin, a caveolar marker protein. However, they exclude many other plasma membrane associated molecules and organelle-specific marker enzymes, suggesting that they represent microdomains of the plasma membrane. In addition to caveolin, these insoluble complexes contain a subset of hydrophobic plasma membrane proteins and cytoplasmically-oriented signaling molecules, including: (a) GTP-binding proteins--both small and heterotrimeric; (b) annex II--an apical calcium-regulated phospholipid binding protein with a demonstrated role in exocytic fusion events; (c) c-Yes--an apically localized member of the Src family of non-receptor type protein-tyrosine kinases; and (d) an unidentified serine-kinase activity. As we demonstrate that caveolin is both a transmembrane molecule and a major phospho-acceptor component of these complexes, we propose that caveolin could function as a transmembrane adaptor molecule that couples luminal GPI-linked proteins with cytoplasmically oriented signaling molecules during GPI-membrane trafficking or GPI-mediated signal transduction events. In addition, our results have implications for understanding v-Src transformation and the actions of cholera and pertussis toxins on hetero-trimeric G proteins.

Yes-associated protein 65 localizes p62(c-Yes) to the apical compartment of airway epithelia by association with EBP50.

We recently showed that the COOH terminus of the cystic fibrosis transmembrane conductance regulator associates with the submembranous scaffolding protein EBP50 (ERM-binding phosphoprotein 50 kD; also called Na(+)/H(+) exchanger regulatory factor). Since EBP50 associates with ezrin, this interaction links the cystic fibrosis transmembrane conductance regulator (CFTR) to the cortical actin cytoskeleton. EBP50 has two PDZ domains, and CFTR binds with high affinity to the first PDZ domain. Here, we report that Yes-associated protein 65 (YAP65) binds with high affinity to the second EBP50 PDZ domain. YAP65 is concentrated at the apical membrane in airway epithelia and interacts with EBP50 in cells. The COOH terminus of YAP65 is necessary and sufficient to mediate association with EBP50. The EBP50-YAP65 interaction is involved in the compartmentalization of YAP65 at the apical membrane since mutant YAP65 proteins lacking the EBP50 interaction motif are mislocalized when expressed in airway epithelial cells. In addition, we show that the nonreceptor tyrosine kinase c-Yes is contained within EBP50 protein complexes by association with YAP65. Subapical EBP50 protein complexes, containing the nonreceptor tyrosine kinase c-Yes, may regulate apical signal transduction pathways leading to changes in ion transport, cytoskeletal organization, or gene expression in epithelial cells.

Assessment of the propensity of biofilm growth on newfloat carrier media through process and biological experiments.

This paper presents the results of research on biomass growth on Newfloat carrier elements and the implications of this growth on the wastewater treatment process. Supervision of the experiment comprised of the analysis of treatment efficiency (dynamic experiments), the estimation of the content of nitrifying bacteria in the biofilm (batch tests) and biological investigations of the biofilm structure and composition. It has been demonstrated that the biofilm growing on the carrier elements was rich in nitrifying bacteria and that this in turn guaranteed the highly efficient oxidation of ammoniacal nitrogen. After the full growth of biofilm had been established, average removal efficiencies were as follows: organic C removal-88.8% (effluent COD below 60 mg O2 l(-1)), nitrification-97.9% (effluent ammoniacal N below 1 mg N-NH4+ l(-1)), denitrification (after the COD loading rate increased to over 0.53 kg COD m(-3) d(-1))-95.7% (total N in the effluent below 8 mg N l(-1)).

Cellular proteins homologous to the viral yes gene product.

We raised antibodies in rabbits against the amino-terminal portion of the viral yes protein produced in bacteria with the use of an expression vector based on the lac operon. The anti-yes serum thus obtained precipitated P90gag-yes from Yamaguchi 73 virus-transformed chicken embryo fibroblasts, and this immunoprecipitation was blocked by the purified antigen. The anti-yes serum did not recognize viral src, fps, or fgr proteins. Affinity-purified anti-yes immunoglobulin G (IgG) precipitated two proteins of 59 and 62 kilodaltons from lysates of normal chicken embryo fibroblasts. Two-dimensional tryptic peptide mapping showed that these proteins are closely related to P90gag-yes and that they are different from pp60c-src. Similar to P90gag-yes, the 59- and 62-kilodalton proteins were phosphorylated exclusively on tyrosine in an in vitro kinase reaction, whereas in vivo they were phosphorylated on serine and, to a lesser extent, on tyrosine as well. Expression of the 59- and 62-kilodalton proteins, determined by the immune complex kinase assay, was relatively high in brain, retina, kidney, and liver. The presence in normal chicken embryo fibroblasts and in chicken kidney of two transcripts, 3.7 and 3.9 kilobases in length, that hybridize with a yes-specific DNA probe, as well as the two proteins recognized by anti-yes IgG, suggests either differential splicing of cellular yes gene transcripts or the existence of another yes-related gene.

Inactivation of c-Yes tyrosine kinase by elevation of intracellular calcium levels.

We have previously shown that the c-Src tyrosine kinase is activated four- to fivefold when cultured keratinocytes differentiate following the elevation of intracellular calcium levels. In contrast to c-Src, another Src family tyrosine kinase, c-Yes, was rapidly inactivated in these same cells, despite its marked similarity in structure and enzymatic activity to c-Src. The inactivation of c-Yes was independent of the protein kinase C pathway, which is usually activated by elevation of intracellular calcium levels. The protein levels of c-Src and c-Yes were not altered, but the phosphotyrosine content of both proteins was greatly reduced. As has been demonstrated for c-Src, in vitro dephosphorylation of c-Yes by incubation with protein tyrosine phosphatases also resulted in its activation, not inactivation. In vitro reconstitution experiments showed that c-Yes can be inactivated by preincubation with a Ca(2+)-supplemented cell extract and that this inhibition was reversed by the addition of EGTA [ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid]. Gradient sedimentation of cell lysates showed that in cells treated with calcium and ionophore, c-Yes formed complexes with two distinct cellular proteins, whereas similar complexes were not seen in c-Src immunoprecipitates. One of these two proteins has the ability to inhibit c-Yes kinase activity in vitro. Finally, the Ca(2+)-dependent inactivation of c-Yes was observed in kidney tubular cells and fibroblasts, suggesting that the Ca(2+)-dependent regulation of c-Yes tyrosine kinase is not unique to keratinocytes. We postulate that c-Yes is inactivated through a Ca2+ -dependent association with cellular proteins, which seems to override its activation resulting from tyrosine dephosphorylation.

Expression of the yes proto-oncogene in cerebellar Purkinje cells.

To identify the kinds of cells in the brain that express the yes proto-oncogene, we examined chicken brains by using immunofluorescent staining and in situ hybridization. Both approaches showed that the highest level of the yes gene product was in cerebellar Purkinje cells. In addition, we analyzed Purkinje cell degeneration (pcd) mutant mice. The level of yes mRNA in cerebella of pcd mutants was four times lower than that found in cerebella of normal littermates. Our studies point to Purkinje cells as an attractive model for functional studies of the yes protein.

RUNX3 is a novel negative regulator of oncogenic TEAD-YAP complex in gastric cancer.

Runt-related transcription factor 3 (RUNX3) is a well-documented tumour suppressor that is frequently inactivated in gastric cancer. Here, we define a novel mechanism by which RUNX3 exerts its tumour suppressor activity involving the TEAD-YAP complex, a potent positive regulator of proliferative genes. We report that the TEAD-YAP complex is not only frequently hyperactivated in liver and breast cancer, but also confers a strong oncogenic activity in gastric epithelial cells. The increased expression of TEAD-YAP in tumour tissues significantly correlates with poorer overall survival of gastric cancer patients. Strikingly, RUNX3 physically interacts with the N-terminal region of TEAD through its Runt domain. This interaction markedly reduces the DNA-binding ability of TEAD that attenuates the downstream signalling of TEAD-YAP complex. Mutation of RUNX3 at Arginine 122 to Cysteine, which was previously identified in gastric cancer, impairs the interaction between RUNX3 and TEAD. Our data reveal that RUNX3 acts as a tumour suppressor by negatively regulating the TEAD-YAP oncogenic complex in gastric carcinogenesis.

Yes-associated protein (YAP65) is a proline-rich phosphoprotein that binds to the SH3 domain of the Yes proto-oncogene product.

Yes belongs to the Src family of protein-tyrosine kinases. In order to understand molecular aspects of its signaling, we decided to isolate proteins that bind to the modulatory region of the Yes molecule. By generating anti-idiotypic antibodies against the aminoterminal domain of the Yes protein, we have identified, characterized and cloned a cDNA for a novel protein that binds to the Src homology domain 3 (SH3) of the Yes proto-oncogene product. The protein is of 65 kiloDalton (kDa) molecular mass, it is phosphorylated in vivo on serine and is particularly rich in proline. We named it YAP65 for Yes-Associated Protein of 65 kDa. Within the YAP65 sequence, we identified a motif, PVKQPPPLAP, similar to that found in proteins that bind to the SH3 domain of the Abl kinase. Competition assays with synthetic peptides showed the involvement of the predicted proline-rich sequence in binding between YAP65 and the Yes kinase. The YAP65 protein was also shown to bind to other signaling molecules that contain SH3 domains including Nck, Crk and Src. At lower stoichiometry, YAP65 was also shown to bind to the SH3 domains of Abl and guanosine triphosphatase activating protein (GAP). Further characterization of YAP65 should illuminate Yes signaling pathways and could also identify a novel link between protein-tyrosine and serine kinases.

From Src Homology domains to other signaling modules: proposal of the 'protein recognition code'.

The study of oncogenes has illuminated many aspects of cellular signaling. The delineation and characterization of protein modules exemplified by Src Homology domains has revolutionized our understanding of the molecular events underlying signal transduction pathways. Several well characterized intracellular modules which mediate protein-protein interactions, namely SH2, SH3, PH, PTB, EH, PDZ, EVH1 and WW domains, are directly involved in the multitude of membrane, cytoplasmic and nuclear processes in multicellular and/or unicellular organisms. The modular character of these protein domains and their cognate motifs, the universality of their molecular function, their widespread occurrence, and the specificity as well as the degeneracy of their interactions have prompted us to propose the concept of the 'protein recognition code'. By a parallel analogy to the universal genetic code, we propose here that there will be a finite set of precise rules to govern and predict protein-protein interactions mediated by modules. Several rules of the 'protein recognition code' have already emerged.

c-yes protein kinase is associated with a 38 kD protein in cerebellum.

p62c-yes, the protein product of the yes proto-oncogene, was found in association with a cellular protein of 38 kD in chicken cerebella. The complex was detected by immunoprecipitation of cerebellar membranes with affinity purified anti-yes IgG followed by in vitro phosphorylation of the immunocomplex. Both proteins were found to be phosphorylated exclusively on tyrosine. The sedimentation profile of the yes kinase indicated that a fraction of p62c-yes was complexed with the 38 kD protein and comigrated in the gradient with a molecular mass of approximately 150 kD. We have previously described the association of p60c-src with a 38 kD protein, referred to as p38 [Grandori, C. and Hanafusa, H., J. Cell Biol. (1988), 107: 2125-2135]. Comparison of the src-associated p38 with the yes-associated 38 kD protein indicates that they are indistinguishable by one-dimensional peptide mapping. Association of p38 with more than one member of the src-family of tyrosine kinases makes this protein an attractive probe to study the structural and functional aspects of these enzymes.

Expression of cellular-yes protein in mammalian tissues.

Expression of the c-yes proto-oncogene in normal mammalian tissues was studied by immunohistochemistry and by immunoprecipitation assays using affinity purified anti-yes IgG. The antibody was raised against the amino-terminal domain of the human c-yes protein expressed in bacteria as a trpE-yes fusion protein. We detected p62c-yes in a variety of rat and human tissues but its relative level of expression varied significantly among different cell types. Certain epithelial cells, platelets and spermatid acrosomes showed the highest levels of p62c-yes. Intracellular localization of p62c-yes in epithelial cells differed among organs performing different physiological functions. The overall pattern of expression suggests that p62c-yes may be involved in numerous cellular pathways.

Localization of p62c-yes protein in mammalian neural tissues.

Expression of the c-yes proto-oncogene in mammalian brain, retina and adrenal gland was studied by immunohistochemistry and immune assays with affinity-purified anti-yes IgG. Immunohistochemical staining with anti-yes IgG showed that in the central nervous system p62c-yes is highly expressed in mitral cells of the olfactory bulb, Purkinje cells of the cerebellum, hippocampal neurons and granule neurons of the dentate gyrus and ependymal cells lining central nervous system ventricles. Less intense labeling by anti-yes IgG was observed in most neuronal cell bodies in the brain. In addition, we observed intense c-yes immunoreactivity in the ganglion cells of the retina, the inner segment layer of rods and cones and medullary cells of the adrenal gland. Mapping p62c-yes expression to specific areas of mammalian neural tissues points to attractive experimental systems which could be used to investigate the function of the proto-oncogene product in neural processes.