Analyses of the microdiversity and physiology of nitrite-oxidizing bacteria in the genus Nitrospira

Der biogeochemische Stickstoff-Kreislauf ist von enormer Bedeutung für die Biosphäre, da das Element Stickstoff für alle Organismen lebensnotwendig ist. Aus diesem Grund heraus wurde seit mehr als einem Jahrhundert intensiv an jenen Mikroorganismen geforscht, die für die Um-wandlung von Stickstoffverbindungen veranwortlich sind. Doch bis heute gibt es noch viele zu klärende Fragen, was diese Prozesse betrifft. Diesbezüglich ist vor allem der Prozess der Nitrifi-kation von besonderem Interesse, da gerade in Bezug darauf innerhalb des letzten Jahrzehnts viele neue Erkenntnisse gewonnen wurden. Die Nitrifikation ist jener Teil des Stickstoff-Kreislaufes, in dem Ammonium/Ammoniak zu Nitrat oxidiert wird. Diese Diplomarbeit fokussierte sich auf die weitere Erforschung der Gattung Nitrospira, einer Gattung von Nitrit-oxidierenden Bakterien (NOB), welche den zweiten Schritt der Nitrifikation – die Oxidation von Nitrit zu Nitrat - durchführen. Ziel dabei war es einerseits, mehr über die Ökophysiologie dieser Mikroorganismen sowie andererseits mehr über ihre temporäre Vertei-lung in der Kläranlage der Veterinärmedizinischen Universität Wien in Erfahrung zu bringen. Das Schlüsselenzym für die Oxidation von Nitrit ist das membranassoziierte Enzym Nitrit-Oxidoreductase (Nxr). Jene Gene, welche dieses Enzym codieren, liegen im Genom von “Can-didatus Nitrospira defluvii“ (Ca. N. defluvii), einer Art der sublineage I der Gattung Nitrospira, in jeweils zwei paralogen Kopien vor. Jene beiden paralogen Gene, welche die alpha-Untereinheiten codieren, unterscheiden sich dabei deutlich voneinander. Da diese Untereinhei-ten für die Substratbindung verantwortlich sind, könnte dies auf unterschiedliche Substrat-Affinititäten hindeuten. Daher fokussierte sich der erste Teil dieser Studie darauf, die Expressi-on dieser alpha-Untereinheiten unter unterschiedlichen Nitritkonzentrationen zu untersuchen, um so eine mögliche Substrat-abhängige Regulierung dieser Gene aufzeigen zu können. Dafür wurde eine qPCR-Methode entwickelt, welche spezifisch die Expression dieser beiden Gene detektieren kann. Erste Experimente stützen die Hypothese, dass eines der beiden Gene konsti-tuitiv exprimiert wird, während die Expression des zweiten Gens bei einer Nitrit-Konzentration von 3 mM hochreguliert wird. Dies entspricht jener Konzentration, bei der Ca. N. defluvii die höchste Nitrit-Oxidationsrate aufweist. Weiters kann mit der entwickelten qPCR-Methode auch getestet werden, ob eines der beiden codierten Nxr-Enzyme auch den reversen Schritt - die Re-duktion von Nitrat zu Nitrit - katalysieren kann. Dies konnte zuvor schon für das Nxr-Enzym von anderen Organismen gezeigt werden. Erste Versuche konnten diese Hypothese jedoch nicht stützen. Eine weitere Arbeit dieser Diplomarbeit beschäftigte sich mit der Untersuchung eines möglichen mixotrophen Lebensstils von Ca. N. defluvii. Um dies zu untersuchen sollte die Expression des Enzyms Citrat-Synthase untersucht werden. Dieses Enzym ist essentiell im oxidativen Tricar-bonsäurezyklus (Krebs-Zyklus, Zitronensäurezyklus). Ob dieser in Ca. N. defluvii funktionell ist, konnte bislang noch nicht gezeigt werden. Deshalb wurde auch für das Gen gltA, welches die Citrat-Synthase codiert, eine qPCR-Methode entwickelt. Diese ermöglicht den Nachweis der Nutzung von organischen Verbindungen über den Tricarbonsäurezyklus, wenn eine erhöhte Ex-pression dieses Gens nachgewiesen werden kann. Aufgrund von Biomasselimitierung konnten diesbezüglich jedoch keine spezifischen Untersuchungen durchgeführt werden. Diese könnten jedoch Gegenstand von zukünftigen Experimenten sein, bei denen Anreicherungskulturen von Ca. N. defluvii unter oxischen Bedingungen mit bestimmten organischen Verbindungen inku-biert werden könnten. Ergebnisse aus diesen Untersuchungen könnten Hinweise für die weite Verbreitung sowie die Dominanz der Gattung Nitrospira in Kläranlagen aufgrund von mögli-chen Präferenzen bezogen auf verschiedene organische Verbindungen liefern. Ein weiterer Teil dieser Diplomarbeit befasste sich mit der Verbreitung von Populationen der Gattung Nitrospira in der Kläranlage der Veterinärmedizinischen Universität Wien. Dabei wur-den die Häufigkeiten verschiedener nahverwandter Subpopulationen der sublineage II der Gat-tung Nitrospira basierend auf ihren Biomasseanteilen untersucht. Um diese zu erheben wurde die Methode Fluoreszenz in situ Hybridisierung (FISH) in Kombination mit Konfokalmikrosko-pie sowie der Quantifizierungssoftware daime eingesetzt. Die durchgeführten Erhebungen zeig-ten, dass sublineage II-angehörige Populationen der Gattung Nitrospira in dieser Kläranlage höchstabundant waren. Innerhalb dieser sublineage II konnte weiters eine stabile Koexistenz von zwei Subpopulationen über den gesamten Beprobungszeitraum nachgewiesen werden. Die-se stabile Koexistenz könnte auf verschiedenen physiologischen Anforderungen oder Fähigkei-ten dieser Subpopulationen basieren. Zusammenfassend konnten neue Einblicke in die Ökophysiologie und die Gemeinschaftstruktur von Nitritoxidierenden Bakterien gewonnen werden, welche wiederum Grundlage für weitere Experimente darstellen. Diese Informationen wiederum sind von enormer Bedeutung, um den Prozess der Nitrifikation in Kläranlagen besser verstehen zu können.The biogeochemical nitrogen cycle is of paramount importance for the biosphere since nitrogen is essential to all organisms. Therefore, microbes participating in this cycle have been investigated for more than one century. But still, much is unknown. Especially the nitrification process - the transformation of ammonia/ammonium to nitrate - is of particular interest since many new aspects have been revealed in the last decade. This study focussed on the further investigation of the genus Nitrospira - a genus of nitrite-oxidizing bacteria (NOB) performing the oxidation of nitrite to nitrate - the second step in the nitrification process. This bacterial genus was investigated concerning its ecophysiology on the one hand and its distribution in the wastewater treatment plant of the University of Veterinary Medicine Vienna on the other hand. The key enzyme for the oxidation of nitrite to nitrate is the membrane-associated nitrite-oxidoreductase (Nxr) which in ”Candidatus Nitrospira defluvii” - a member of sublineage I of the genus Nitrospira - is present in two copies in the genome. The alpha-subunits of the encoded enzymes differ from each other indicating possible differences of their substrate-affinities. Therefore, the first part of this study focussed on the expression of these alpha-subunits with different nitrite concentrations to reveal a possible substrate-dependent regulation. For this purpose, qPCR assays specific for these genes were developed. First experiments supported the hypothesis that one of the Nxr’s is constitutively expressed, whereas the second copy is upregulated at a nitrite-concentration of 3 mM - the optimal concentration for growth of Ca. N. defluvii. Furthermore, this enzyme was shown to be capable of performing the reverse step - the reduction of nitrate to nitrite - in other organisms. Therefore, the developed qPCR assays were also used to check for the expression of the two encoded Nxr enzymes under denitrifying conditions. First results, however, did not support this hypothesis. Another part of this thesis tackled to a possible mixotrophic lifestyle of this organism by examining the expression of the enzyme citrate synthase. This enzyme is essential in the oxidative tricarboxylic acid cycle (oTCA), which is yet unknown to be functional in Ca. N. defluvii. To analyze its expression, a qPCR assay specific for the gltA gene encoding the citrate synthase was developed. This assay allows the investigation of Ca. N. defluvii with regard to the use of organic compounds with the oTCA cycle. Future experiments with cultures of Ca. N. defluvii should test the use of different organic compounds after incubating the cultures under oxic conditions. Such data could explain the distribution of this sublineage as well as its dominance in some wastewater treatment plants (WWTPs). Another part of this study dealt with the distribution of Nitrospira populations in the WWTP of the University of Veterinary Medicine Vienna. Different subpopulations of sublineage II of the genus Nitrospira were examined concerning their abundance in samples taken over six years. For this purpose, fluorescence in situ hybridization (FISH) in combination with confocal microscopy and the image analysis software daime was applied. This study showed that members of sublineage II of the genus Nitrospira are highly dominant in this WWTP. Furthermore, stable coexistence of different subpopulations over the whole sampling period was revealed. This likely is caused by differences in the ecophysiology of the subpopulations which may represent different ecotypes. Due to different physiological demands or capabilities they could inhabit different niches in WWTP. Taken together, new insights into the ecophysiology and community structure of nitrite-oxidizing bacteria have been revealed and provide material for further investigations. Such information about the genus Nitrospira is important to better understand the nitrification process in WWTPs

PERANCANGAN KOMIK BERTEMA PANDEMI SEBAGAI SARANA HIBURAN KHALAYAK DEWASA MUDA

Pandemi Covid-19 yang sedang dialami dunia membuat berbagai sektor terpuruk. Berbagai macam cara dilakukan untuk menekan penyebaran Covid-19 salah satunya yaitu pemberlakuan lockdown. Menurut data yang didapatkan melalui kursioner menunjukan bahwa 80% responden mengalami rasa bosan. Perancangan ini bertujuan untuk mengatasi rasa bosan bagi dewasa muda yang sedang mengalami dan menjalankan lockdown. Melalui perancangan Komik ini diharapkan dapat terancangnya komik yang dapat mengatasi rasa bosan selama pandemi atau lockdown. Kata kunci : Komik, Pandemi, Bosa

Association of NAFLD and Insulin Resistance with Non Metastatic Bladder Cancer Patients: A Cross-Sectional Retrospective Study

Among risk factors (apart from smoking) likely involved in bladder cancer (BCa), metabolic syndrome (MS), obesity and type 2 diabetes mellitus (T2DM) have been explored with contrasting results. In spite of these studies, there is little data on the association between nonalcoholic fatty liver disease (NAFLD), its main driver, i.e., insulin resistance (IR), and BCa. Implanting a cross-sectional retrospective study we tried to investigate both NAFLD and IR prevalence in a hospital based population of BCa patients. We studied laboratory data from 204 patients with histologically confirmed non metastatic BCa and 50 subjects with no BCa, but with bladder diseases (no Ca BD). We evaluated the presence of NAFLD by the triglycerides/glucose Index (TyG Index), using a cut-off of 0.59 and by the Aspartate Aminotransferase/Alanine Aminotransferase AST/ALT ratio. IR was assessed by the same TyG Index (cut-off 4.68) and the triglycerides/High-Density Lipoprotein HDL ratio (cut-off 2.197). The diagnosis of impaired fasting glucose (IFG), condition of prediabetes, as well as that of T2DM was assessed according to canonical guidelines. The TyG Index predicted NAFLD presence in both groups (p = 0.000), but the BCa group showed a major percentage of NAFLD cases with respect to no Ca BD group (59% versus 40%). A greater proportion of IR (47%) in BCa group than in no Ca BD one (37%) was evidenced by the TyG Index with its median value significantly different (p = 0.0092). This high rate of IR in the BCa group was confirmed by the triglycerides/HDL ratio (p = 0.02). Prediabetes and T2DM were more prevalent in the BCa group than no Ca BD group (p = 0.024). In this study a consistent NAFLD presence was found in BCa patients. This is an important comorbidity factor that deserves further consideration in prospective studies. The higher prevalence of NAFLD, IR, prediabetes and T2DM in the BCa group evidences the need that these disorders should be reckoned as adjunct factors that could impact on this cancerous disease

8-Hydroxy-2-Deoxyguanosine and 8-Iso-Prostaglandin F2α: Putative Biomarkers to assess Oxidative Stress Damage Following Robot-Assisted Radical Prostatectomy (RARP)

Objective: Prostate cancer (PCa) is the most common type of cancer. Biomarkers help researchers to understand the mechanisms of disease and refine diagnostic panels. We measured urinary 8-hydroxy-2-deoxyguanosine (8-OHdG) and 8-iso-prostaglandin F2α (8-IsoF2α) to assess oxidative stress damage in PCa patients undergoing robot-assisted radical prostatectomy (RARP). Methods: Forty PCa patients were enrolled in the study. Urine was collected before (T0) and 3 months after the RARP procedure (T1). 8-OHdG and 8-IsoF2α were measured through liquid chromatography-tandem mass spectrometry. Sex- and age-matched healthy subjects served as controls (CTRL). Results: At T0, patients exhibited significantly higher levels of 8-OHdG than CTRL (p = 0.026). At T1, 23/40 patients who completed the 3-month follow-up showed levels of 8-OHdG that were significantly lower than at T0 (p = 0.042), and comparable to those of the CTRL subjects (p = 0.683). At T0, 8-Iso-PGF2α levels were significantly higher in PCa patients than in CTRL subjects (p = 0.0002). At T1, 8-Iso-PGF2α levels were significantly lower than at T0 (p < 0.001) and were comparable to those of CTRL patients (p = 0.087). Conclusions: A liquid chromatography-tandem mass spectrometry method reveals enhanced OHdG and 8-Iso-PGF2α in the urine of PCa patients. RARP normalizes such indices of oxidative stress. Large-sized sample studies and long-term follow-ups are now needed to validate these urinary biomarkers for use in the early prevention and successful treatment of PCa

Metabolomic Approaches for Detection and Identification of Biomarkers and Altered Pathways in Bladder Cancer

Metabolomic analysis has proven to be a useful tool in biomarker discovery and the molecular classification of cancers. In order to find new biomarkers, and to better understand its pathological behavior, bladder cancer also has been studied using a metabolomics approach. In this article, we review the literature on metabolomic studies of bladder cancer, focusing on the different available samples (urine, blood, tissue samples) used to perform the studies and their relative findings. Moreover, the multi-omic approach in bladder cancer research has found novel insights into its metabolic behavior, providing excellent start-points for new diagnostic and therapeutic strategies. Metabolomics data analysis can lead to the discovery of a “signature pathway” associated with the progression of bladder cancer; this aspect could be potentially valuable in predictions of clinical outcomes and the introduction of new treatments. However, further studies are needed to give stronger evidence and to make these tools feasible for use in clinical practice

Renal Cell Carcinoma as a Metabolic Disease: An Update on Main Pathways, Potential Biomarkers, and Therapeutic Targets

: Clear cell renal cell carcinoma (ccRCC) is the most frequent histological kidney cancer subtype. Over the last decade, significant progress has been made in identifying the genetic and metabolic alterations driving ccRCC development. In particular, an integrated approach using transcriptomics, metabolomics, and lipidomics has led to a better understanding of ccRCC as a metabolic disease. The metabolic profiling of this cancer could help define and predict its behavior in terms of aggressiveness, prognosis, and therapeutic responsiveness, and would be an innovative strategy for choosing the optimal therapy for a specific patient. This review article describes the current state-of-the-art in research on ccRCC metabolic pathways and potential therapeutic applications. In addition, the clinical implication of pharmacometabolomic intervention is analyzed, which represents a new field for novel stage-related and patient-tailored strategies according to the specific susceptibility to new classes of drugs

Novel Insights into Autophagy and Prostate Cancer: A Comprehensive Review

Autophagy is a complex process involved in several cell activities, including tissue growth, differentiation, metabolic modulation, and cancer development. In prostate cancer, autophagy has a pivotal role in the regulation of apoptosis and disease progression. Several molecular pathways are involved, including PI3K/AKT/mTOR. However, depending on the cellular context, autophagy may play either a detrimental or a protective role in prostate cancer. For this purpose, current evidence has investigated how autophagy interacts within these complex interactions. In this article, we discuss novel findings about autophagic machinery in order to better understand the therapeutic response and the chemotherapy resistance of prostate cancer. Autophagic-modulation drugs have been employed in clinical trials to regulate autophagy, aiming to improve the response to chemotherapy or to anti-cancer treatments. Furthermore, the genetic signature of autophagy has been found to have a potential means to stratify prostate cancer aggressiveness. Unfortunately, stronger evidence is needed to better understand this field, and the application of these findings in clinical practice still remains poorly feasible

Spring Water of an Alpine Karst Aquifer Is Dominated by a Taxonomically Stable but Discharge-Responsive Bacterial Community

Alpine karst aquifers are important groundwater resources for the provision of drinking water all around the world. Yet, due to difficult accessibility and long-standing methodological limitations, the microbiology of these systems has long been understudied. The aim of the present study was to investigate the structure and dynamics of bacterial communities in spring water of an alpine limestone karst aquifer (LKAS2) under different hydrological conditions (base vs. event flow). The study was based on high-throughput 16S rRNA gene amplicon sequencing, study design and sample selection were guided by hydrology and pollution microbiology data. Spanning more than 27 months, our analyses revealed a taxonomically highly stable bacterial community, comprising high proportions of yet uncultivated bacteria in the suspended bacterial community fraction. Only the three candidate phyla Parcubacteria (OD1), Gracilibacteria (GN02), Doudnabacteria (SM2F11) together with Proteobacteria and Bacteroidetes contributed between 70.0 and 88.4% of total reads throughout the investigation period. A core-community of 300 OTUs consistently contributed between 37.6 and 56.3% of total reads, further supporting the hypothesis of a high temporal stability in the bacterial community in the spring water. Nonetheless, a detectable response in the bacterial community structure of the spring water was discernible during a high-discharge event. Sequence reads affiliated to the class Flavobacteriia clearly increased from a mean proportion of 2.3% during baseflow to a maximum of 12.7% during the early phase of the studied high-discharge event, suggesting direct impacts from changing hydrological conditions on the bacterial community structure in the spring water. This was further supported by an increase in species richness (Chao1) at higher discharge. The combination of these observations allowed the identification and characterization of three different discharge classes (Q1–Q3). In conclusion, we found a taxonomically stable bacterial community prevailing in spring waters from an alpine karst aquifer over the entire study period of more than 2 years. Clear response to changing discharge conditions could be detected for particular bacterial groups, whereas the most responsive group – bacteria affiliated to the class of Flavobacteriia – might harbor potential as a valuable natural indicator of “system disturbances” in karst aquifers

Spatial patterns of enzymatic activity in large water bodies: Ship-borne measurements of beta-D-glucuronidase activity as a rapid indicator of microbial water quality

This study used automated enzymatic activity measurements conducted from a mobile research vessel to detect the spatial variability of beta‑d‑glucuronidase (GLUC) activity in large freshwater bodies. The ship-borne observations provided the first high-resolution spatial data of GLUC activity in large water bodies as rapid indication of fecal pollution and were used to identify associations with hydrological conditions and land use. The utility of this novel approach for water quality screening was evaluated by surveys of the Columbia River, the Mississippi River and the Yahara Lakes, covering up to a 500 km river course and 50 km2 lake area. The ship-borne measurements of GLUC activity correlated with standard E. coli analyses (R2 = 0.71) and revealed the effects of (1) precipitation events and urban run-off on GLUC activity in surface waters, (2) localized point inlets of potential fecal pollution and (3) increasing GLUC signals along gradients of urbanization. We propose that this ship-borne water quality screening to be integrated into future water inventory programs as an initial or complementary tool (besides established fecal indicator parameters), due to its ability to provide near real-time spatial information on potential fecal contamination of large surface water resources and therefore being helpful to greatly reduce potential human health risks.Austrian Science Fund (FWF)Vienna University of TechnologyNorth Temperate Lakes–Long Term Ecological Researc

Global Distribution of Human-Associated Fecal Genetic Markers in Reference Samples from Six Continents

Numerous bacterial genetic markers are available for the molecular detection of human sources of fecal pollution in environmental waters. However, widespread application is hindered by a lack of knowledge regarding geographical stability, limiting implementation to a small number of well-characterized regions. This study investigates the geographic distribution of five human-associated genetic markers (HF183/BFDrev, HF183/BacR287, BacHum-UCD, BacH, and Lachno2) in municipal wastewaters (raw and treated) from 29 urban and rural wastewater treatment plants (750-4»400»000 population equivalents) from 13 countries spanning six continents. In addition, genetic markers were tested against 280 human and nonhuman fecal samples from domesticated, agricultural and wild animal sources. Findings revealed that all genetic markers are present in consistently high concentrations in raw (median log10 7.2-8.0 marker equivalents (ME) 100 mL-1) and biologically treated wastewater samples (median log10 4.6-6.0 ME 100 mL-1) regardless of location and population. The false positive rates of the various markers in nonhuman fecal samples ranged from 5% to 47%. Results suggest that several genetic markers have considerable potential for measuring human-associated contamination in polluted environmental waters. This will be helpful in water quality monitoring, pollution modeling and health risk assessment (as demonstrated by QMRAcatch) to guide target-oriented water safety management across the globe.Fil: Mayer, René E.. Vienna University of Technology; Austria. Interuniversity Cooperation Centre for Water and Health; AustriaFil: Reischer, Georg. Vienna University of Technology; AustriaFil: Ixenmaier, Simone K.. Vienna University of Technology; Austria. Interuniversity Cooperation Centre for Water and Health; AustriaFil: Derx, Julia. Vienna University of Technology; AustriaFil: Blaschke, Alfred Paul. Vienna University of Technology; AustriaFil: Ebdon, James E.. University of Brighton; Reino UnidoFil: Linke, Rita. Vienna University of Technology; Austria. Interuniversity Cooperation Centre Water And Health; AustriaFil: Egle, Lukas. Vienna University of Technology; AustriaFil: Ahmed, Warish. Csiro Land And Water; AustraliaFil: Blanch, Anicet R.. Universidad de Barcelona; EspañaFil: Byamukama, Denis. Makerere University; UgandaFil: Savill, Marion. Affordable Water Limited;Fil: Mushi, Douglas. Sokoine University Of Agriculture; TanzaniaFil: Cristobal, Hector Antonio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Investigaciones para la Industria Química. Universidad Nacional de Salta. Facultad de Ingeniería. Instituto de Investigaciones para la Industria Química; ArgentinaFil: Edge, Thomas A.. Canada Centre for Inland Waters. Environment and Climate Change Canada; CanadáFil: Schade, Margit A.. Bavarian Environment Agency; AlemaniaFil: Aslan, Asli. Georgia Southern University; Estados UnidosFil: Brooks, Yolanda M.. Michigan State University; Estados UnidosFil: Sommer, Regina. Interuniversity Cooperation Centre Water And Health; Austria. Medizinische Universitat Wien; AustriaFil: Masago, Yoshifumi. Tohoku University; JapónFil: Sato, Maria I.. Cia. Ambiental do Estado de Sao Paulo. Departamento de Análises Ambientais; BrasilFil: Taylor, Huw D.. University of Brighton; Reino UnidoFil: Rose, Joan B.. Michigan State University; Estados UnidosFil: Wuertz, Stefan. Nanyang Technological University. Singapore Centre for Environmental Life Sciences Engineering and School of Civil and Environmental Engineering; SingapurFil: Shanks, Orin. U.S. Environmental Protection Agency; Estados UnidosFil: Piringer, Harald. Vrvis Research Center; AustriaFil: Mach, Robert L.. Vienna University of Technology; AustriaFil: Savio, Domenico. Karl Landsteiner University of Health Sciences; AustriaFil: Zessner, Matthias. Vienna University of Technology; AustriaFil: Farnleitner, Andreas. Vienna University of Technology; Austria. Interuniversity Cooperation Centre Water And Health; Austria. Karl Landsteiner University of Health Sciences; Austri