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.

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.

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).

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.

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.

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.

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.

Geranylgeranylation signals to the Hippo pathway for breast cancer cell proliferation and migration.

Protein geranylgeranylation (GGylation) is an important biochemical process for many cellular signaling molecules. Previous studies have shown that GGylation is essential for cell survival in many types of cancer. However, the molecular mechanism mediating the cell survival effect remains elusive. In this report, we show that the Hippo pathway mediates GGylation-dependent cell proliferation and migration in breast cancer cells. Blockade of GGylation enhanced phosphorylation of Mst1/2 and Lats1, and inhibited YAP and TAZ activity and the Hippo-YAP/TAZ pathway-dependent transcription. The effect of GGylation blockade on inhibition of breast cancer cell proliferation and migration is dependent on the Hippo-YAP/TAZ signaling, in which YAP appears to regulate cell proliferation and TAZ to regulate cell migration. Furthermore, GGylation-dependent cell proliferation is correlated with the activity of YAP/TAZ in breast cancer cells. Finally, Gγ and RhoA are the GGylated proteins that may transduce GGylation signals to the Hippo-YAP/TAZ pathway. Taken together, our studies have demonstrated that the Hippo-YAP/TAZ pathway is essential for GGylation-dependent cancer cell proliferation and migration.

An evolutionary shift in the regulation of the Hippo pathway between mice and flies.

The Hippo pathway plays a key role in controlling organ growth in many animal species and its deregulation is associated with different types of cancer. Understanding the regulation of the Hippo pathway and discovering upstream regulators is thus a major quest. Interestingly, while the core of the Hippo pathway contains a highly conserved kinase cascade, different components have been identified as upstream regulators in Drosophila and vertebrates. However, whether the regulation of the Hippo pathway is indeed different between Drosophila and vertebrates or whether these differences are due to our limited analysis of these components in different organisms is not known. Here we show that the mouse Fat4 cadherin, the ortholog of the Hippo pathway regulator Fat in Drosophila, does not apparently regulate the Hippo pathway in the murine liver. In fact, we uncovered an evolutionary shift in many of the known upstream regulators at the base of the arthropod lineage. In this evolutionary transition, Fat and the adaptor protein Expanded gained novel domains that connected them to the Hippo pathway, whereas the cell-adhesion receptor Echinoid evolved as a new protein. Subsequently, the junctional adaptor protein Angiomotin (Amot) was lost and the downstream effector Yap lost its PDZ-binding motif that interacts with cell junction proteins. We conclude that fundamental differences exist in the upstream regulatory mechanisms of Hippo signaling between Drosophila and vertebrates.

ATM kinase enables the functional axis of YAP, PML and p53 to ameliorate loss of Werner protein-mediated oncogenic senescence.

Werner syndrome (WS) results from dysfunction of the WRN protein, and is associated with premature aging and early death. Here we report that loss of WRN function elicits accumulation of the Yes-associated protein (YAP protein), a major effector of the Hippo tumor suppressor pathway, both experimentally and in WS-derived fibroblasts. YAP upregulation correlates with slower cell proliferation and accelerated senescence, which are partially mediated by the formation of a complex between YAP and the PML protein, whose activity promotes p53 activation. The ATM kinase is necessary for YAP and PML accumulation in WRN-depleted cells. Notably, the depletion of either YAP or PML partially impairs the induction of senescence following WRN loss. Altogether, our findings reveal that loss of WRN activity triggers the activation of an ATM-YAP-PML-p53 axis, thereby accelerating cellular senescence. The latter has features of SASP (senescence-associated secretory phenotype), whose protumorigenic properties are potentiated by YAP, PML and p53 depletion.

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.

Opposing roles of angiomotin-like-1 and zona occludens-2 on pro-apoptotic function of YAP.

YAP (Yes-associated protein) oncogene has been found to form a stable complex with members of the Angiomotin (Amot) family of proteins, which bind WW domains of YAP and sequester the protein in the cytoplasm and junctional complexes. The Amot-mediated retention of YAP in the cytoplasm results in the inhibition of its proliferative function. Using apoptotic 'read-out' of YAP in HEK293 cells, we confirmed the molecular mode by which Amot regulates YAP. We showed that a representative member of the Amot family, AmotL1 (Angiomotin-like-1), uses its PPxY motifs to bind WW domains of YAP and inhibit YAP's nuclear translocation and pro-apoptotic function. Recently we also showed that YAP uses its PDZ-binding motif to interact with zona occludens-2 (ZO-2) protein, which promotes YAP's translocation to the nucleus. We also asked if AmotL1, YAP and ZO-2 signal together. We report here that AmotL1 and ZO-2 form a tripartite complex with YAP and regulate its function in HEK293 cells in opposite directions. AmotL1 inhibits pro-apoptotic function of YAP, whereas ZO-2 enhances it. As YAP is a potent oncogene, the identification and characterization of its regulators is important. AmotL1 and ZO-2 are two candidates that could be harnessed to control the oncogenic function of YAP.

From Rous sarcoma virus to plasminogen activator, src oncogene and cancer management.

Plasminogen activator (PLAU) is a serine protease that converts plasminogen to plasmin, a general protease, which promotes fibrinolysis and degradation of extracellular matrix. PLAU was reported in 1970s as one of the robustly induced enzymatic activities in Rous sarcoma virus (RSV)-transformed chicken cells. More than three decades later, with the completion of the sequencing of the chicken genome and the subsequent availability of Affymetrix GeneChip genome arrays, several laboratories have surveyed the transcriptional program affected by the RSV transformation. Interestingly, the PLAU gene was shown to be the most highly upregulated transcript. The induction of PLAU was a transformation-dependent process because viruses with deleted Src gene did not induce the transcription of the PLAU gene. Both Src and PLAU genes are associated with and contribute to the complex phenotype of human cancer. Although the activity and structures of these two enzymes are well characterized, the precise molecular function of these gene products in signaling networks is still not fully understood. Yet, the knowledge of their association with cancer is already translated into the clinical setting. Src kinase inhibitors are being tested in clinical trials of cancer therapy, and PLAU gene and its inhibitor have been included as biomarkers with strong prognostic and therapeutic predictive values. This vignette reviews the history of PLAU and Src discovery, and illuminates the complexity of their relationship, but also points to their emerging impact on public health.

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.

YAP dysregulation by phosphorylation or ΔNp63-mediated gene repression promotes proliferation, survival and migration in head and neck cancer subsets.

Overexpression of the Yes-associated protein (YAP), and TP53 family members ΔNp63 and p73, have been independently detected in subsets of head and neck squamous cell carcinomas (HNSCCs). YAP may serve as a nuclear cofactor with ΔNp63 and p73, but the functional role of YAP and their potential relationship in HNSCCs are unknown. In this study, we show that in a subset of HNSCC lines and tumors, YAP expression is increased but localized in the cytoplasm in association with increased AKT and YAP phosphorylation, and with decreased expression of ΔNp63 and p73. In another subset, YAP expression is decreased but detectable in the nucleus in association with lower AKT and YAP phosphorylation, and with increased ΔNp63 and p73 expression. Inhibiting AKT decreased serine-127 phosphorylation and enhanced nuclear translocation of YAP. ΔNp63 bound to the YAP promoter and suppressed its expression. Transfection of a YAP-serine-127-alanine phosphoacceptor-site mutant or ΔNp63 knockdown significantly increased nuclear YAP and cell death. Conversely, YAP knockdown enhanced cell proliferation, survival, migration and cisplatin chemoresistance. Thus, YAP function as a tumor suppressor may alternatively be dysregulated by AKT phosphorylation at serine-127 and cytoplasmic sequestration, or by transcriptional repression by ΔNp63, in different subsets of HNSCC. AKT and/or ΔNp63 are potential targets for enhancing YAP-mediated apoptosis and chemosensitivity in HNSCCs.

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)).

EGR-1 forms a complex with YAP-1 and upregulates Bax expression in irradiated prostate carcinoma cells.

In this study, we investigated the functional role of early growth response-1 (Egr1 gene) in the regulation of radiation-induced clonogenic inhibition and apoptosis in p53 wild-type and mutant prostate cancer cells 22Rv1 and DU145, respectively. 22Rv1 cells were more sensitive to irradiation compared with DU145 cells, and the sensitivity was enhanced by overexpression of EGR-1 in both cells. Dominant-negative EGR-1 mutant (dnEGR-1) or repressor of EGR-1, NGFIA binding protein 1 (NAB1), increased radioresistance of these cells. Significant activation of caspases 3 and 9 and Bcl2-associated X (Bax) with increased poly(ADP-ribose) polymerase (PARP) cleavage and cytochrome c release was observed in radiation-exposed EGR-1 overexpressing cells. Gel shift analysis and chloramphenicol acetyl transferase (CAT) reporter assays indicate that EGR-1 transactivates the promoter of the Bax gene. Interaction of EGR-1 and Yes kinase-associated protein 1 (YAP-1) through the WW domain of YAP-1 enhances the transcriptional activity of EGR-1 on the Bax promoter as shown by chromatin immunoprecipitation and reporter assays. Irradiation of PC3 cell xenografts that were treated with adenoviral EGR-1 showed significant regression in tumor volume. These findings establish the radiation-induced pro-apoptotic action of EGR-1, in a p53-independent manner, by directly transactivating Bax, and prove that alters the B-cell CLL/lymphoma 2 (Bcl-2)/Bax ratio as one of the mechanisms resulting in significant activation of caspases, leading to cell death through the novel interaction of EGR-1 with YAP-1.

Probing WW Domains to Uncover and Refine Determinants of Specificity in Ligand Recognition.

Understanding the specificity of protein-protein interaction mediated by domains and their ligands will have strong impact on basic and applied research. Visual inspection of WW domain sequences prompted a general classification of the domains into two large subfamilies. One subfamily contains two consecutive aromatic residues in the beta 2 strand of the domain whereas the other contains three or four consecutive aromatic residues in the same position. In the recent past, we proposed a rule of 'two vs. three aromatics' in the beta 2 strand of WW domains as a molecular discriminator between Class I and Class II WW domains, which recognize PPxY or PPLP motifs, respectively. Using phage display libraries expressing WW domains with random sequences replacing a part of the beta 2 strand, we provided additional evidence supporting our rule. We conclude that three consecutive aromatic amino acids within the beta 2 strand of WW domain are required but not always sufficient for the WW domain to belong to Class II.