Homeostatic coordination of cellular phosphate uptake and efflux requires an organelle-based receptor for the inositol pyrophosphate IP8.

Phosphate (Pi) serves countless metabolic pathways and is involved in macromolecule synthesis, energy storage, cellular signaling, and bone maintenance. Herein, we describe the coordination of Pi uptake and efflux pathways to maintain mammalian cell Pi homeostasis. We discover that XPR1, the presumed Pi efflux transporter, separately supervises rates of Pi uptake. This direct, regulatory interplay arises from XPR1 being a binding partner for the Pi uptake transporter PiT1, involving a predicted transmembrane helix/extramembrane loop in XPR1, and its hitherto unknown localization in a subset of intracellular LAMP1-positive puncta (named "XLPVs"). A pharmacological mimic of Pi homeostatic challenge is sensed by the inositol pyrophosphate IP8, which functionalizes XPR1 to respond in a temporally hierarchal manner, initially adjusting the rate of Pi efflux, followed subsequently by independent modulation of PiT1 turnover to reset the rate of Pi uptake. These observations generate a unifying model of mammalian cellular Pi homeostasis, expanding opportunities for therapeutic intervention.

Virulence-associated genes in faecal and clinical Escherichia coli isolates cultured from broiler chickens in Australia.

A healthy chicken's intestinal flora harbours a rich reservoir of Escherichia coli as part of the commensal microbiota. However, some strains, known as avian pathogenic E. coli (APEC), carry specific virulence genes (VGs) that enable them to invade and cause extraintestinal infections such as avian colibacillosis. Although several VG combinations have been identified, the pathogenic mechanisms associated with APEC are ill-defined. The current study screened a subset of 88 E. coli isolates selected from 237 pre-existing isolates obtained from commercial poultry flocks in Australia. The 88 isolates were selected based on their enterobacterial repetitive intergenic consensus (ERIC) and antimicrobial resistance (AMR) profiles and included 29 E. coli isolates cultured from chickens with colibacillosis (referred to as clinical E. coli or CEC) and 59 faecal E. coli (FEC) isolates cultured from clinically healthy chickens. The isolates were screened for the presence of 35 previously reported VGs. Of these, 34 were identified, with iucA not being detected. VGs focG, hlyA and sfa/foc were only detected in FEC isolates. Eight VGs had a prevalence of 90% or above in the CEC isolates. Specifically, astA (100%); feoB (96.6%); iutA, iss, ompT, iroN and hlyF (all 93.1%); and vat (89.7%). The prevalence of these were significantly lower in FEC isolates (astA 79.7%, feoB 77.9%, iutA 52.5%, iss 45.8%, ompT 50.9%, iroN 37.3%, hlyF 50.9% and vat 42.4%). The odds ratios that each of these eight VGs were more likely to be associated with CEC than FEC ranged from 7.8 to 21.9. These eight VGs may be used to better define APEC and diagnostically detect APEC in Australia. Further investigations are needed to identify the roles of these VGs in pathogenicity.

Intracellular delivery and deep tissue penetration of nucleoside triphosphates using photocleavable covalently bound dendritic polycations.

Nucleoside triphosphates (NTPs) are essential in various biological processes. Cellular or even organismal controlled delivery of NTPs would be highly desirable, yet in cellulo and in vivo applications are hampered owing to their negative charge leading to cell impermeability. NTP transporters or NTP prodrugs have been developed, but a spatial and temporal control of the release of the investigated molecules remains challenging with these strategies. Herein, we describe a general approach to enable intracellular delivery of NTPs using covalently bound dendritic polycations, which are derived from PAMAM dendrons and their guanidinium derivatives. By design, these modifications are fully removable through attachment on a photocage, ready to deliver the native NTP upon irradiation enabling spatiotemporal control over nucleotide release. We study the intracellular distribution of the compounds depending on the linker and dendron generation as well as side chain modifications. Importantly, as the polycation is bound covalently, these molecules can also penetrate deeply into the tissue of living organisms, such as zebrafish.

Structures of 3-acetylpyridine adenine dinucleotide and ADP-ribose bound to the electron input module of respiratory complex I.

Energy-converting NADH:ubiquinone oxidoreductase, respiratory complex I, is a major enzyme of energy metabolism that couples NADH oxidation and ubiquinone reduction with proton translocation. The NADH oxidation site features different enzymatic activities with various nucleotides. While the kinetics of these reactions are well described, only binding of NAD+ and NADH have been structurally characterized. Here, we report the structures of the electron input module of Aquifex aeolicus complex I with bound ADP-ribose and 3-acetylpyridine adenine dinucleotides at resolutions better than 2.0 Å. ADP-ribose acts as inhibitor by blocking the "ADP-handle" motif essential for nucleotide binding. The pyridine group of APADH is minimally offset from flavin, which could contribute to its poorer suitability as substrate. A comparison with other nucleotide co-structures surprisingly shows that the adenine ribose and the pyrophosphate moiety contribute most to nucleotide binding, thus all adenine dinucleotides share core binding modes to the unique Rossmann-fold in complex I.

Primary abandonment of the sac in the management of scrotal hernias: a dual-institution experience of short-term outcomes.

PURPOSE: Management of scrotal hernias presents as a common challenge, with operative interventions to address these hernias associated with higher rates of morbidity compared to those of less-complex pathology. Surgeons have advocated for the use of techniques such as primary abandonment of the distal sac as a potential means to reduce complications for operative intervention, with preliminary findings demonstrating feasibility. We sought to assess outcomes related to primary sac abandonment among patients undergoing minimally invasive (MIS) repair of scrotal hernias. METHODS: A review of prospectively maintained databases among two academic hernia centers was conducted to identify patients who underwent MIS inguinal hernia repairs with primary sac abandonment. Patient demographics, hernia risk factors, intraoperative factors, and postoperative outcomes were evaluated. Short-term outcomes related to patient-reported experiences and surgical-site occurrences requiring procedural intervention were queried. RESULTS: Sixty-seven male patients [median age: 51.6 years; interquartile range (IQR): 45-65 years] underwent inguinal hernia repair with primary sac abandonment. Anatomic polypropylene mesh was used in 98.5% cases. Rates of postoperative complications were low and included postoperative urinary retention (6%), clinically identified or patient-reported seromas/hematomas within a 30-day follow-up period (23.9%), deep venous thrombosis (1.5%), and pelvic hematoma (1.5%). No seromas or hematomas necessitated procedural interventions, with resolution of symptoms within three months of their operation date. CONCLUSION: We report a multi-center experience of patients managed with primary abandonment of the sac technique during repair of inguinoscrotal hernias. Utilization of this technique appears to be safe and reproducible with a low burden of short-term complications.

Understanding barriers to reducing antimicrobials on Australian dairy farms: A qualitative analysis.

INTRODUCTION: Reducing antibiotic use in production animal systems is one strategy which may help to limit the development of antimicrobial resistance. To reduce antimicrobial use in food-producing animals, it is important to first understand how antibiotics are used on farm and what barriers exist to decreasing their use. In dairy production systems, mastitis is one of the most common reasons for administering antimicrobials. Therefore, it is important to understand the motivations and behaviours of dairy farmers in relation to the diagnosis, treatment and prevention of mastitis. MATERIALS AND METHODS: In this study, we interviewed a sample of dairy farmers and dairy industry professionals from the major dairying regions of eastern Australia regarding their current practices used to diagnose, treat, and control subclinical and clinical mastitis. Inductive thematic analysis was used to code interview transcripts and identify the recurrent themes. RESULTS: Four overarching themes were identified: (1) the challenges associated with the detection and diagnosis of clinical mastitis, including with laboratory culture, (2) the motivations behind treatment decisions for different cases, (3) decisions around dry cow therapy and the role of herd recording, and (4) concerns regarding the development of antimicrobial resistance. DISCUSSION: This study identifies several challenges which may limit the ability of Australian dairy farmers to reduce antimicrobial use on farm, such as the need for rapid and reliable diagnostic tests capable of identifying the pathogenic causes of mastitis and the difficulties associated with conducting herd recording for the implementation of selective dry cow therapy. Industry professionals were concerned that farmers were not using individual cow records to aid in treatment decisions, which could result in unnecessary antimicrobial use. The results of this study can act as the basis for future research aimed at assessing these issues across the broader Australian dairy industry.

The Ip6k1 and Ip6k2 Kinases Are Critical for Normal Renal Tubular Function.

SIGNIFICANCE STATEMENT: Kidneys are gatekeepers of systemic inorganic phosphate balance because they control urinary phosphate excretion. In yeast and plants, inositol hexakisphosphate kinases (IP6Ks) are central to regulate phosphate metabolism, whereas their role in mammalian phosphate homeostasis is mostly unknown. We demonstrate in a renal cell line and in mice that Ip6k1 and Ip6k2 are critical for normal expression and function of the major renal Na + /Pi transporters NaPi-IIa and NaPi-IIc. Moreover, Ip6k1/2-/- mice also show symptoms of more generalized kidney dysfunction. Thus, our results suggest that IP6Ks are essential for phosphate metabolism and proper kidney function in mammals. BACKGROUND: Inorganic phosphate is an essential mineral, and its plasma levels are tightly regulated. In mammals, kidneys are critical for maintaining phosphate homeostasis through mechanisms that ultimately regulate the expression of the Na + /Pi cotransporters NaPi-IIa and NaPi-IIc in proximal tubules. Inositol pyrophosphate 5-IP 7 , generated by IP6Ks, is a main regulator of phosphate metabolism in yeast and plants. IP6Ks are conserved in mammals, but their role in phosphate metabolism in vivo remains unexplored. METHODS: We used in vitro (opossum kidney cells) and in vivo (renal tubular-specific Ip6k1/2-/- mice) models to analyze the role of IP6K1/2 in phosphate homeostasis in mammals. RESULTS: In both systems, Ip6k1 and Ip6k2 are responsible for synthesis of 5-IP 7 . Depletion of Ip6k1/2 in vitro reduced phosphate transport and mRNA expression of Na + /Pi cotransporters, and it blunts phosphate transport adaptation to changes in ambient phosphate. Renal ablation of both kinases in mice also downregulates the expression of NaPi-IIa and NaPi-IIc and lowered the uptake of phosphate into proximal renal brush border membranes. In addition, the absence of Ip6k1 and Ip6k2 reduced the plasma concentration of fibroblast growth factor 23 and increased bone resorption, despite of which homozygous males develop hypophosphatemia. Ip6k1/2-/- mice also show increased diuresis, albuminuria, and hypercalciuria, although the morphology of glomeruli and proximal brush border membrane seemed unaffected. CONCLUSIONS: Depletion of renal Ip6k1/2 in mice not only altered phosphate homeostasis but also dysregulated other kidney functions.

Hierarchical Mechanical Transduction of Precision-Engineered DNA Hydrogels with Sacrificial Bonds.

Engineering the response to external signals in mechanically switchable hydrogels is important to promote smart materials applications. However, comparably little attention has focused on embedded precision mechanisms for autonomous nonlinear response in mechanical profiles in hydrogels, and we lack understanding of how the behavior from the molecular scale transduces to the macroscale. Here, we design a nonlinear stress-strain response into hydrogels by engineering sacrificial DNA hairpin loops into model network hydrogels formed from star-shaped building blocks. We characterize the force-extension response of single DNA hairpins and are able to describe how the specific topology influences the nonlinear mechanical behavior at different length scales. For this purpose, we utilize force spectroscopy as well as microscopic and macroscopic deformation tests. This study contributes to a better understanding of designing nonlinear strain-adaptive features into hydrogel materials.

Spontaneous and Selective Peptide Elongation in Water Driven by Aminoacyl Phosphate Esters and Phase Changes.

Nature chose phosphates to activate amino acids, where reactive intermediates and complex machinery drive the construction of polyamides. Outside of biology, the pathways and mechanisms that allow spontaneous and selective peptide elongation in aqueous abiotic systems remain unclear. Herein we work to uncover those pathways by following the systems chemistry of aminoacyl phosphate esters, synthetic counterparts of aminoacyl adenylates. The phosphate esters act as solubility tags, making hydrophobic amino acids and their oligomers soluble in water and enabling selective elongation and different pathways to emerge. Thus, oligomers up to dodecamers were synthesized in one flask and on the minute time scale, where consecutive additions activated autonomous phase changes. Depending on the pathway, the resulting phases initially carry nonpolar peptides and amphiphilic oligomers containing phosphate esters. During elongation and phosphate release, shorter oligomers dominate in solution, while the aggregated phase favors the presence of longer oligomers due to their self-assembly propensity. Furthermore we demonstrated that the solution phases can be isolated and act as a new environment for continuous elongation, by adding various phosphate esters. These findings suggest that the systems chemistry of aminoacyl phosphate esters can activate a selection mechanism for peptide bond formation by merging aqueous synthesis and self-assembly.

Inositol pyrophosphate dynamics reveals control of the yeast phosphate starvation program through 1,5-IP8 and the SPX domain of Pho81.

Eukaryotic cells control inorganic phosphate to balance its role as essential macronutrient with its negative bioenergetic impact on reactions liberating phosphate. Phosphate homeostasis depends on the conserved INPHORS signaling pathway that utilizes inositol pyrophosphates and SPX receptor domains. Since cells synthesize various inositol pyrophosphates and SPX domains bind them promiscuously, it is unclear whether a specific inositol pyrophosphate regulates SPX domains in vivo, or whether multiple inositol pyrophosphates act as a pool. In contrast to previous models, which postulated that phosphate starvation is signaled by increased production of the inositol pyrophosphate 1-IP7, we now show that the levels of all detectable inositol pyrophosphates of yeast, 1-IP7, 5-IP7, and 1,5-IP8, strongly decline upon phosphate starvation. Among these, specifically the decline of 1,5-IP8 triggers the transcriptional phosphate starvation response, the PHO pathway. 1,5-IP8 inactivates the cyclin-dependent kinase inhibitor Pho81 through its SPX domain. This stimulates the cyclin-dependent kinase Pho85-Pho80 to phosphorylate the transcription factor Pho4 and repress the PHO pathway. Combining our results with observations from other systems, we propose a unified model where 1,5-IP8 signals cytosolic phosphate abundance to SPX proteins in fungi, plants, and mammals. Its absence triggers starvation responses.

Sangivamycin is preferentially incorporated into viral RNA by the SARS-CoV-2 polymerase.

Sangivamycin (S) is an adenosine (A) nucleoside analog with low nanomolar antiviral activity against SARS-CoV-2 in vitro. Previously, low nanomolar antiviral efficacy was revealed when tested against multiple viral variants in several cell types. SARS-CoV-2 RNA isolated from live virus infected cells and the virions released from these cells was analyzed by mass spectrometry (MS) for S incorporation. Dose-dependent incorporation occurred up to 1.8 S per 1,000 nucleotides (49 S per genome) throughout the viral genomes isolated from both infected cells and viral particles, but this incorporation did not change the viral mutation rate. In contrast, host mRNA, affinity purified from the same infected and treated cells, contained little or no S. Sangivamycin triphosphate (STP) was synthesized to evaluate its incorporation into RNA by recombinant SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) under defined in vitro conditions. SARS-CoV-2 RdRp showed that S was not a chain terminator and S containing oligonucleotides templated as A. Though the antiviral mechanism remains to be determined, the data suggests that SARS-CoV-2 RdRp incorporates STP into SARS-CoV-2 RNA, which does not significantly impair viral RNA synthesis or the mutation rate.

Activities, substrate specificity, and genetic interactions of fission yeast Siw14, a cysteinyl-phosphatase-type inositol pyrophosphatase.

IMPORTANCE: The inositol pyrophosphate signaling molecule 1,5-IP8 modulates fission yeast phosphate homeostasis via its action as an agonist of RNA 3'-processing and transcription termination. Cellular 1,5-IP8 levels are determined by a balance between the activities of the inositol polyphosphate kinase Asp1 and several inositol pyrophosphatase enzymes. Here, we characterize Schizosaccharomyces pombe Siw14 (SpSiw14) as a cysteinyl-phosphatase-family pyrophosphatase enzyme capable of hydrolyzing the phosphoanhydride substrates inorganic pyrophosphate, inorganic polyphosphate, and inositol pyrophosphates 5-IP7, 1-IP7, and 1,5-IP8. Genetic analyses implicate SpSiw14 in 1,5-IP8 catabolism in vivo, insofar as: loss of SpSiw14 activity is lethal in the absence of the Nudix-type inositol pyrophosphatase enzyme Aps1; and siw14∆ aps1∆ lethality depends on synthesis of 1,5-IP8 by the Asp1 kinase. Suppression of siw14∆ aps1∆ lethality by loss-of-function mutations of 3'-processing/termination factors points to precocious transcription termination as the cause of 1,5-IP8 toxicosis.

Non-Hydrolysable Analogues of Cyclic and Branched Condensed Phosphates: Chemistry and Chemical Proteomics.

Studies into the biology of condensed phosphates almost exclusively cover linear polyphosphates. However, there is evidence for the presence of cyclic polyphosphates (metaphosphates) in organisms and for enzymatic digestion of branched phosphates (ultraphosphates) with alkaline phosphatase. Further research of non-linear condensed phosphates in biology would profit from interactome data of such molecules, however, their stability in biological media is limited. Here we present syntheses of modified, non-hydrolysable analogues of cyclic and branched condensed phosphates, called meta- and ultraphosphonates, and their application in a chemical proteomics approach using yeast cell extracts. We identify putative interactors with overlapping hits for structurally related capture compounds underlining the quality of our results. The datasets serve as starting point to study the biological relevance and functions of meta- and ultraphosphates. In addition, we examine the reactivity of meta- and ultraphosphonates with implications for their "hydrolysable" analogues: Efforts to increase the ring-sizes of meta- or cyclic ultraphosphonates revealed a strong preference to form trimetaphosphate-analogue structures by cyclization and/or ring-contraction. Using carbodiimides for condensation, the so far inaccessible dianhydro product of ultraphosphonate, corresponding to P4 O11 2- , was selectively obtained and then ring-opened by different nucleophiles yielding modified cyclic ultraphosphonates.

ß-lapachone regulates mammalian inositol pyrophosphate levels in an NQO1- and oxygen-dependent manner.

Inositol pyrophosphates (PP-InsPs) are energetic signaling molecules with important functions in mammals. As their biosynthesis depends on ATP concentration, PP-InsPs are tightly connected to cellular energy homeostasis. Consequently, an increasing number of studies involve PP-InsPs in metabolic disorders, such as type 2 diabetes, aspects of tumorigenesis, and hyperphosphatemia. Research conducted in yeast suggests that the PP-InsP pathway is activated in response to reactive oxygen species (ROS). However, the precise modulation of PP-InsPs during cellular ROS signaling is unknown. Here, we report how mammalian PP-InsP levels are changing during exposure to exogenous (H2O2) and endogenous ROS. Using capillary electrophoresis electrospray ionization mass spectrometry (CE-ESI-MS), we found that PP-InsP levels decrease upon exposure to oxidative stressors in HCT116 cells. Application of quinone drugs, particularly ß-lapachone (ß-lap), under normoxic and hypoxic conditions enabled us to produce ROS in cellulo and to show that ß-lap treatment caused PP-InsP changes that are oxygen-dependent. Experiments in MDA-MB-231 breast cancer cells deficient of NAD(P)H:quinone oxidoreductase-1 (NQO1) demonstrated that ß-lap requires NQO1 bioactivation to regulate the cellular metabolism of PP-InsPs. Critically, significant reductions in cellular ATP concentrations were not directly mirrored in reduced PP-InsP levels as shown in NQO1-deficient MDA-MB-231 cells treated with ß-lap. The data presented here unveil unique aspects of ß-lap pharmacology and its impact on PP-InsP levels. The identification of different quinone drugs as modulators of PP-InsP synthesis will allow the overall impact on cellular function of such drugs to be better appreciated.

Assigning the Absolute Configuration of Inositol Poly- and Pyrophosphates by NMR Using a Single Chiral Solvating Agent.

Inositol phosphates constitute a family of highly charged messenger molecules that play diverse roles in cellular processes. The various phosphorylation patterns they exhibit give rise to a vast array of different compounds. To fully comprehend the biological interconnections, the precise molecular identification of each compound is crucial. Since the myo-inositol scaffold possesses an internal mirror plane, enantiomeric pairs can be formed. Most commonly employed methods for analyzing InsPs have been geared towards resolving regioisomers, but they have not been capable of resolving enantiomers. In this study, we present a general approach for enantiomer assignment using NMR measurements. To achieve this goal, we used 31P-NMR in the presence of L-arginine amide as a chiral solvating agent, which enables the differentiation of enantiomers. Using chemically synthesized standard compounds allows for an unambiguous assignment of the enantiomers. This method was applied to highly phosphorylated inositol pyrophosphates, as well as to lowly phosphorylated inositol phosphates and bisphosphonate analogs. Our method will facilitate the assignment of biologically relevant isomers when isolating naturally occurring compounds from biological specimens.

Bacterial Pathogen Infection Triggers Magic Spot Nucleotide Signaling in Arabidopsis thaliana Chloroplasts through Specific RelA/SpoT Homologues.

Magic spot nucleotides (p)ppGpp are important signaling molecules in bacteria and plants. In the latter, RelA-SpoT homologue (RSH) enzymes are responsible for (p)ppGpp turnover. Profiling of (p)ppGpp is more difficult in plants than in bacteria due to lower concentrations and more severe matrix effects. Here, we report that capillary electrophoresis mass spectrometry (CE-MS) can be deployed to study (p)ppGpp abundance and identity in Arabidopsis thaliana. This goal is achieved by combining a titanium dioxide extraction protocol and pre-spiking with chemically synthesized stable isotope-labeled internal reference compounds. The high sensitivity and separation efficiency of CE-MS enables monitoring of changes in (p)ppGpp levels in A. thaliana upon infection with the pathogen Pseudomonas syringae pv. tomato (PstDC3000). We observed a significant increase of ppGpp post infection that is also stimulated by the flagellin peptide flg22 only. This increase depends on functional flg22 receptor FLS2 and its interacting kinase BAK1 indicating that pathogen-associated molecular pattern (PAMP) receptor-mediated signaling controls ppGpp levels. Transcript analyses showed an upregulation of RSH2 upon flg22 treatment and both RSH2 and RSH3 after PstDC3000 infection. Arabidopsis mutants deficient in RSH2 and RSH3 activity display no ppGpp accumulation upon infection and flg22 treatment, supporting the involvement of these synthases in PAMP-triggered innate immune responses to pathogens within the chloroplast.

In Vivo Metabolic Imaging of [1-13 C]Pyruvate-d3 Hyperpolarized By Reversible Exchange With Parahydrogen.

Metabolic magnetic resonance imaging (MRI) using hyperpolarized (HP) pyruvate is becoming a non-invasive technique for diagnosing, staging, and monitoring response to treatment in cancer and other diseases. The clinically established method for producing HP pyruvate, dissolution dynamic nuclear polarization, however, is rather complex and slow. Signal Amplification By Reversible Exchange (SABRE) is an ultra-fast and low-cost method based on fast chemical exchange. Here, for the first time, we demonstrate not only in vivo utility, but also metabolic MRI with SABRE. We present a novel routine to produce aqueous HP [1-13 C]pyruvate-d3 for injection in 6 minutes. The injected solution was sterile, non-toxic, pH neutral and contained ≈30 mM [1-13 C]pyruvate-d3 polarized to ≈11 % (residual 250 mM methanol and 20 µM catalyst). It was obtained by rapid solvent evaporation and metal filtering, which we detail in this manuscript. This achievement makes HP pyruvate MRI available to a wide biomedical community for fast metabolic imaging of living organisms.

Non-canonical nucleosides: Biomimetic triphosphorylation, incorporation into mRNA and effects on translation and structure.

Recent advances in mRNA therapeutics demand efficient toolkits for the incorporation of nucleoside analogues into mRNA suitable for downstream applications. Herein, we report the application of a versatile enzyme cascade for the triphosphorylation of a broad range of nucleoside analogues, including unprotected nucleobases containing chemically labile moieties. Our biomimetic system was suitable for the preparation of nucleoside triphosphates containing adenosine, cytidine, guanosine, uridine and non-canonical core structures, as determined by capillary electrophoresis coupled to mass spectrometry. This enabled us to establish an efficient workflow for transcribing and purifying functional mRNA containing these nucleoside analogues, combined with mass spectrometric verification of analogue incorporation. Our combined methodology allows for analyses of how incorporation of nucleoside analogues that are commercially unavailable as triphosphates affect mRNA properties: The translational fidelity of the produced mRNA was demonstrated in analyses of how incorporated adenosine analogues impact translational recoding. For the SARS-CoV-2 frameshifting site, analyses of the mRNA pseudoknot structure using circular dichroism spectroscopy allowed insight into how the pharmacologically active 7-deazaadenosine destabilises RNA secondary structure, consistent with observed changes in recoding efficiency.