A widespread glutamine-sensing mechanism in the plant kingdom.

Glutamine is the primary metabolite of nitrogen assimilation from inorganic nitrogen sources in microorganisms and plants. The ability to monitor cellular nitrogen status is pivotal for maintaining metabolic homeostasis and sustaining growth. The present study identifies a glutamine-sensing mechanism common in the entire plant kingdom except Brassicaceae. The plastid-localized PII signaling protein controls, in a glutamine-dependent manner, the key enzyme of the ornithine synthesis pathway, N-acetyl-l-glutamate kinase (NAGK), that leads to arginine and polyamine formation. Crystal structures reveal that the plant-specific C-terminal extension of PII, which we term the Q loop, forms a low-affinity glutamine-binding site. Glutamine binding alters PII conformation, promoting interaction and activation of NAGK. The binding motif is highly conserved in plants except Brassicaceae. A functional Q loop restores glutamine sensing in a recombinant Arabidopsis thaliana PII protein, demonstrating the modular concept of the glutamine-sensing mechanism adopted by PII proteins during the evolution of plant chloroplasts.

Nanobead handling on a centrifugal microfluidic LabDisk for automated extraction of cell-free circulating DNA with high recovery rates.

cfDNA is an emerging biomarker with promising uses for the monitoring of cancer or infectious disease diagnostics. This work demonstrates a new concept for an automated cfDNA extraction with nanobeads as the solid phase in a centrifugal microfluidic LabDisk. By using a combination of centrifugal and magnetic forces, we retain the nanobeads in one incubation chamber while sequentially adding, incubating and removing the sample and pre-stored buffers for extraction. As the recovery rate of the typically low concentration of cfDNA is of high importance to attain sufficient amounts for analysis, optimal beadhandling is paramount. The goal is that the cfDNA in the sample adsorbs to the solid phase completely during the binding step, is retained during washing and completely removed during elution. In this work, we improved beadhandling by optimizing the incubation chamber geometry and both frequency and temperature protocols, to maximize recovery rates. For characterization of the extraction performance, synthetic mutant DNA was spiked into human plasma samples. The LabDisk showed better reproducibility in DNA recovery rates with a standard deviation of ±13% compared to a manual approach using spin-columns (±17%) or nanobeads (±26%). The extraction of colorectal cancer samples with both the developed LabDisk and a robotic automation instrument resulted in comparable allele frequencies. Consequently, we present a highly attractive solution for an automated liquid biopsy cfDNA extraction in a small benchtop device.

RespiDisk: a point-of-care platform for fully automated detection of respiratory tract infection pathogens in clinical samples.

We present the RespiDisk enabling the fully automated and multiplex point-of-care (POC) detection of (currently) up to 19 respiratory tract infection (RTI) pathogens from a single sample based on reverse transcriptase polymerase chain reaction (RT-PCR). RespiDisk comprises a RTI-specific implementation of the centrifugal microfluidic LabDisk platform and combines new and existing advanced unit operations for liquid control, thereby automating all assay steps only by a spinning frequency and temperature protocol in combination with the use of a permanent magnet for in situ bead handing. The capabilities of the system were demonstrated with 36 tested quality samples mimicking clinical conditions (clinical and/or cultured material suspended in transport medium or synthetic bronchoalveolar lavage (BAL)) from past external quality assessment (EQA) panels covering 13 of the 19 integrated RTI detection assays. In total, 36 samples × 19 assays/sample resulting in 684 assays were performed with the RespiDisk, and its analytical performance was in full agreement with the routine clinical workflow serving as reference. A strong feature of the platform is its universality since its components allow the simultaneous detection of a broad panel of bacteria and viruses in a single run, thereby enabling the differentiation between antibiotic-treatable diseases. Furthermore, the full integration of all necessary biochemical components enables a reduction of the hands-on time from manual to automated sample-to-answer analysis to about 5 min. The study was performed on an air-heated LabDisk Player instrument with a time-to-result of 200 min.

Population shift of binding pocket size and dynamic correlation analysis shed new light on the anticooperative mechanism of PII protein.

PII protein is one of the largest families of signal transduction proteins in archaea, bacteria, and plants, controlling key processes of nitrogen assimilation. An intriguing characteristic for many PII proteins is that the three ligand binding sites exhibit anticooperative allosteric regulation. In this work, PII protein from Synechococcus elongatus, a model for cyanobacteria and plant PII proteins, is utilized to reveal the anticooperative mechanism upon binding of 2-oxoglutarate (2-OG). To this end, a method is proposed to define the binding pocket size by identifying residues that contribute greatly to the binding of 2-OG. It is found that the anticooperativity is realized through population shift of the binding pocket size in an asymmetric manner. Furthermore, a new algorithm based on the dynamic correlation analysis is developed and utilized to discover residues that mediate the anticooperative process with high probability. It is surprising to find that the T-loop, which is believed to be responsible for mediating the binding of PII with its target proteins, also takes part in the intersubunit signal transduction process. Experimental results of PII variants further confirmed the influence of T-loop on the anticooperative regulation, especially on binding of the third 2-OG. These discoveries extend our understanding of the PII T-loop from being essential in versatile binding of target protein to signal-mediating in the anticooperative allosteric regulation.

Two-stage tuberculosis diagnostics: combining centrifugal microfluidics to detect TB infection and Inh and Rif resistance at the point of care with subsequent antibiotic resistance profiling by targeted NGS.

Globally, tuberculosis (TB) remains the deadliest bacterial infectious disease, and spreading antibiotic resistances is the biggest challenge for combatting the disease. Rapid and comprehensive diagnostics including drug susceptibility testing (DST) would assure early treatment, reduction of morbidity and the interruption of transmission chains. To date, rapid genetic resistance testing addresses only one to four drug groups while complete DST is done phenotypically and takes several weeks. To overcome these limitations, we developed a two-stage workflow for rapid TB diagnostics including DST from a single sputum sample that can be completed within three days. The first stage is qPCR detection of M. tuberculosis complex (MTBC) including antibiotic resistance testing against the first-line antibiotics, isoniazid (Inh) and rifampicin (Rif). The test is automated by centrifugal microfluidics and designed for point of care (PoC). Furthermore, enriched MTBC DNA is provided in a detachable sample tube to enable the second stage: if the PCR detects MTBC and resistance to either Inh or Rif, the MTBC DNA is shipped to specialized facilities and analyzed by targeted next generation sequencing (tNGS) to assess the complete resistance profile. Proof-of-concept testing of the PoC test revealed an analytical sensitivity of 44.2 CFU ml-1, a diagnostic sensitivity of 96%, and a diagnostic specificity of 100% for MTBC detection. Coupled tNGS successfully provided resistance profiles, demonstrated for samples from 17 patients. To the best of our knowledge, the presented combination of PoC qPCR with tNGS allows for the fastest comprehensive TB diagnostics comprising decentralized pathogen detection with subsequent resistance profiling in a facility specialized in tNGS.

Magnetophoresis in Centrifugal Microfluidics at Continuous Rotation for Nucleic Acid Extraction.

Centrifugal microfluidics enables fully automated molecular diagnostics at the point-of-need. However, the integration of solid-phase nucleic acid extraction remains a challenge. Under this scope, we developed the magnetophoresis under continuous rotation for magnetic bead-based nucleic acid extraction. Four stationary permanent magnets are arranged above a cartridge, creating a magnetic field that enables the beads to be transported between the chambers of the extraction module under continuous rotation. The centrifugal force is maintained to avoid uncontrolled spreading of liquids. We concluded that below a frequency of 5 Hz, magnetic beads move radially inwards. In support of magnetophoresis, bead inertia and passive geometrical design features allow to control the azimuthal bead movement between chambers. We then demonstrated ferrimagnetic bead transfer in liquids with broad range of surface tension and density values. Furthermore, we extracted nucleic acids from lysed Anopheles gambiae mosquitoes reaching comparable results of eluate purity (LabDisk: A260/A280 = 1.6 ± 0.04; Reference: 1.8 ± 0.17), and RT-PCR of extracted RNA (LabDisk: Ct = 17.9 ± 1.6; Reference: Ct = 19.3 ± 1.7). Conclusively, magnetophoresis at continuous rotation enables easy cartridge integration and nucleic acid extraction at the point-of-need with high yield and purity.

OralDisk: A Chair-Side Compatible Molecular Platform Using Whole Saliva for Monitoring Oral Health at the Dental Practice.

Periodontitis and dental caries are two major bacterially induced, non-communicable diseases that cause the deterioration of oral health, with implications in patients' general health. Early, precise diagnosis and personalized monitoring are essential for the efficient prevention and management of these diseases. Here, we present a disk-shaped microfluidic platform (OralDisk) compatible with chair-side use that enables analysis of non-invasively collected whole saliva samples and molecular-based detection of ten bacteria: seven periodontitis-associated (Aggregatibacter actinomycetemcomitans, Campylobacter rectus, Fusobacterium nucleatum, Prevotella intermedia, Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola) and three caries-associated (oral Lactobacilli, Streptococcus mutans, Streptococcus sobrinus). Each OralDisk test required 400 µL of homogenized whole saliva. The automated workflow included bacterial DNA extraction, purification and hydrolysis probe real-time PCR detection of the target pathogens. All reagents were pre-stored within the disk and sample-to-answer processing took < 3 h using a compact, customized processing device. A technical feasibility study (25 OralDisks) was conducted using samples from healthy, periodontitis and caries patients. The comparison of the OralDisk with a lab-based reference method revealed a ~90% agreement amongst targets detected as positive and negative. This shows the OralDisk's potential and suitability for inclusion in larger prospective implementation studies in dental care settings.

PII Protein-Derived FRET Sensors for Quantification and Live-Cell Imaging of 2-Oxoglutarate.

The citric acid cycle intermediate 2-oxoglutarate (2-OG, a.k.a. alpha-ketoglutarate) links the carbon and nitrogen metabolic pathways and can provide information on the metabolic status of cells. In recent years, it has become exceedingly clear that 2-OG also acts as a master regulator of diverse biologic processes in all domains of life. Consequently, there is a great demand for time-resolved data on 2-OG fluctuations that can't be adequately addressed using established methods like mass spectrometry-based metabolomics analysis. Therefore, we set out to develop a novel intramolecular 2-OG FRET sensor based on the signal transduction protein PII from Synechococcus elongatus PCC 7942. We created two variants of the sensor, with a dynamic range for 2-OG from 0.1 µM to 0.1 mM or from 10 µM to 10 mM. As proof of concept, we applied the sensors to determine in situ glutamine:2-oxoglutarate aminotransferase (GOGAT) activity in Synechococcus elongatus PCC 7942 cells and measured 2-OG concentrations in cell extracts from Escherichia coli in vitro. Finally, we could show the sensors' functionality in living human cell lines, demonstrating their potential in the context of mechanistic studies and drug screening.

Sensory properties of the PII signalling protein family.

PII signalling proteins constitute one of the largest families of signalling proteins in nature. An even larger superfamily of trimeric sensory proteins with the same architectural principle as PII proteins appears in protein structure databases. Large surface-exposed flexible loops protrude from the intersubunit faces, where effector molecules are bound that tune the conformation of the loops. Via this mechanism, PII proteins control target proteins in response to cellular ATP/ADP levels and the 2-oxoglutarate status, thereby coordinating the cellular carbon/nitrogen balance. The antagonistic (ATP versus ADP) and synergistic (2-oxoglutarate and ATP) mode of effector molecule binding is further affected by PII -receptor interaction, leading to a highly sophisticated signalling network organized by PII . Altogether, it appears that PII is a multitasking information processor that, depending on its interaction environment, differentially transmits information on the energy status and the cellular 2-oxoglutarate level. In addition to the basic mode of PII function, several bacterial PII proteins may transmit a signal of the cellular glutamine status via covalent modification. Remarkably, during the evolution of plant chloroplasts, glutamine signalling by PII proteins was re-established by acquisition of a short sequence extension at the C-terminus. This plant-specific C-terminus makes the interaction of plant PII proteins with one of its targets, the arginine biosynthetic enzyme N-acetyl-glutamate kinase, glutamine-dependent.

Energy Sensing versus 2-Oxoglutarate Dependent ATPase Switch in the Control of Synechococcus PII Interaction with Its Targets NAGK and PipX.

PII proteins constitute a superfamily of highly conserved signaling devices, common in all domains of life. Through binding of the metabolites ATP, ADP and 2-oxoglutarate (2-OG), they undergo conformational changes which allow them to regulate a variety of target proteins including enzymes, transport proteins and transcription factors. But, in reverse, these target proteins also modulate the metabolite sensing properties of PII, as has been recently shown. We used this effect to refine our PII based Förster resonance energy transfer (FRET) sensor and amplify its sensitivity towards ADP. With this enhanced sensor setup we addressed the question whether the PII protein from the model organism Synechococcus elongatus autonomously switches into the ADP conformation through ATPase activity as proposed in a recently published model. The present study disproves ATPase activity as a relevant mechanism for the transition of PII into the ADP state. In the absence of 2-OG, only the ATP/ADP ratio and concentration of ADP directs the competitive interaction of PII with two targets, one of which preferentially binds PII in the ATP-state, the other in the ADP-state.

From PII signaling to metabolite sensing: a novel 2-oxoglutarate sensor that details PII-NAGK complex formation.

The widespread PII signal transduction proteins are known for integrating signals of nitrogen and energy supply and regulating cellular behavior by interacting with a multitude of target proteins. The PII protein of the cyanobacterium Synechococcus elongatus forms complexes with the controlling enzyme of arginine synthesis, N-acetyl-L-glutamate kinase (NAGK) in a 2-oxoglutarate- and ATP/ADP-dependent manner. Fusing NAGK and PII proteins to either CFP or YFP yielded a FRET sensor that specifically responded to 2-oxoglutarate. The impact of the fluorescent tags on PII and NAGK was evaluated by enzyme assays, surface plasmon resonance spectroscopy and isothermal calorimetric experiments. The developed FRET sensor provides real-time data on PII - NAGK interaction and its modulation by the effector molecules ATP, ADP and 2-oxoglutarate in vitro. Additionally to its utility to monitor 2-oxoglutarate levels, the FRET assay provided novel insights into PII - NAGK complex formation: (i) It revealed the formation of an encounter-complex between PII and NAGK, which holds the proteins in proximity even in the presence of inhibitors of complex formation; (ii) It revealed that the PII T-loop residue Ser49 is neither essential for complex formation with NAGK nor for activation of the enzyme but necessary to form a stable complex and efficiently relieve NAGK from arginine inhibition; (iii) It showed that arginine stabilizes the NAGK hexamer and stimulates PII - NAGK interaction.