Polyphosphate affects cytoplasmic and chromosomal dynamics in nitrogen-starved Pseudomonas aeruginosa.

Polyphosphate (polyP) synthesis is a ubiquitous stress and starvation response in bacteria. In diverse species, mutants unable to make polyP have a wide variety of physiological defects, but the mechanisms by which this simple polyanion exerts its effects remain unclear. One possibility is that polyP's many functions stem from global effects on the biophysical properties of the cell. We characterize the effect of polyphosphate on cytoplasmic mobility under nitrogen-starvation conditions in the opportunistic pathogen Pseudomonas aeruginosa. Using fluorescence microscopy and particle tracking, we quantify the motion of chromosomal loci and cytoplasmic tracer particles. In the absence of polyP and upon starvation, we observe a 2- to 10-fold increase in mean cytoplasmic diffusivity. Tracer particles reveal that polyP also modulates the partitioning between a "more mobile" and a "less mobile" population: Small particles in cells unable to make polyP are more likely to be "mobile" and explore more of the cytoplasm, particularly during starvation. Concomitant with this larger freedom of motion in polyP-deficient cells, we observe decompaction of the nucleoid and an increase in the steady-state concentration of ATP. The dramatic polyP-dependent effects we observe on cytoplasmic transport properties occur under nitrogen starvation, but not carbon starvation, suggesting that polyP may have distinct functions under different types of starvation.

Endothelin-1-mediated Brainstem Glial Activation Produces Asthmatic Airway Vagal Hypertonia Via Enhanced ATP-P2X4 Receptor Signaling in Sprague-Dawley Rats.

The occurrence of major asthma symptoms is largely attributed to airway vagal hypertonia, of which the central mechanisms remain unclear. This study tests the hypotheses that endothelin-1-mediated brainstem glial activation produces asthmatic airway vagal hypertonia via enhanced action of adenosine 5'-triphosphate on neuronal purinergic P2X4 receptors. A rat model of asthma was prepared using ovalbumin. Airway vagal tone was evaluated by the recurrent laryngeal discharge and plethysmographic measurement of pulmonary function. The changes in the brainstem were examined using ELISA, Western blot, luciferin-luciferase, quantitative reverse transcription-polymerase chain reaction, enzyme activity assay and immunofluorescent staining, respectively. The results showed that in the medulla of rats, endothelin receptor type B and P2X4 receptors were primarily expressed in astrocytes and neurons, respectively, and both of which, along with endothelin-1 content, were significantly increased after ovalbumin sensitization. Ovalbumin sensitization significantly increased recurrent laryngeal discharge, which was blocked by acute intracisternal injection of P2X4 receptor antagonist 5-BDBD, knockdown of brainstem P2X4 receptors, and chronic intraperitoneal injection of endothelin receptor type B antagonist BQ788, respectively. Ovalbumin sensitization activated microglia and astrocytes and significantly decreased ecto-5'-nucleotidase activity in the medulla, and all of which, together with the increase of medullary P2X4 receptor expression and decrease of pulmonary function, were reversed by chronic BQ788 treatment. These results demonstrated that in rats, allergic airway challenge activates both microglia and astrocytes in the medulla via enhanced endothelin-1/endothelin receptor type B signaling, which subsequently causes airway vagal hypertonia via augmented adenosine 5'-triphosphate/P2X4 receptor signaling in central neurons of airway vagal reflex.

Advancing Genetic Alphabet Expansion: Synthesis of 7-(2-Thienyl)-Imidazo[4,5-b]pyridine (Ds) and 4-(4-Pentyne-1,2-diol)-1-Propynyl-2-Nitropyrrole (Diol-Px) for Use in Replicable Unnatural Base Pairs for PCR Applications.

Expanding the genetic alphabet enhances DNA recombinant technologies by introducing unnatural base pairs (UBPs) beyond the standard A-T and G-C pairs, leading to biomaterials with novel and increased functionalities. Recent developments include UBPs that effectively function as a third base pair in replication, transcription, and/or translation processes. One such UBP, Ds-Px, demonstrates extremely high specificity in replication. Chemically synthesized DNA fragments containing Ds bases are amplified by PCR with the 5'-triphosphates of Ds and Px deoxyribonucleosides (dDsTP and dPxTP). The Ds-Px pair system has applications in enhanced DNA data storage, generation of high-affinity DNA aptamers, and incorporation of functional elements into RNA through transcription. This protocol describes the synthesis of the amidite derivative of Ds (dDs amidite), the triphosphate dDsTP, and the diol-modified dPxTP (Diol-dPxTP) for PCR amplifications involving the Ds-Px pair. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of Ds deoxyribonucleoside (dDs) Basic Protocol 2: Synthesis of dDs amidite Basic Protocol 3: Synthesis of dDs triphosphate (dDsTP) Basic Protocol 4: Synthesis of Pn deoxyribonucleoside (4-iodo-dPn) Basic Protocol 5: Synthesis of acetyl-protected diol-modified Px deoxyribonucleoside (Diol-dPx) Basic Protocol 6: Synthesis of Diol-dPx triphosphate (Diol-dPxTP) Basic Protocol 7: Purification of triphosphates Support Protocol 1: Synthesis of Hoffer's chlorosugar Support Protocol 2: Preparation of 0.5 M pyrophosphate in DMF Support Protocol 3: Preparation of 2 M TEAB buffer.

Heat flows enrich prebiotic building blocks and enhance their reactivity.

The emergence of biopolymer building blocks is a crucial step during the origins of life1-6. However, all known formation pathways rely on rare pure feedstocks and demand successive purification and mixing steps to suppress unwanted side reactions and enable high product yields. Here we show that heat flows through thin, crack-like geo-compartments could have provided a widely available yet selective mechanism that separates more than 50 prebiotically relevant building blocks from complex mixtures of amino acids, nucleobases, nucleotides, polyphosphates and 2-aminoazoles. Using measured thermophoretic properties7,8, we numerically model and experimentally prove the advantageous effect of geological networks of interconnected cracks9,10 that purify the previously mixed compounds, boosting their concentration ratios by up to three orders of magnitude. The importance for prebiotic chemistry is shown by the dimerization of glycine11,12, in which the selective purification of trimetaphosphate (TMP)13,14 increased reaction yields by five orders of magnitude. The observed effect is robust under various crack sizes, pH values, solvents and temperatures. Our results demonstrate how geologically driven non-equilibria could have explored highly parallelized reaction conditions to foster prebiotic chemistry.

Enhancement of skin rejuvenation and hair growth through novel near-infrared light emitting diode (nNIR) lighting: in vitro and in vivo study.

The study aimed to explore the impact of a novel near-infrared LED (nNIR) with an extended spectrum on skin enhancement and hair growth. Various LED sources, including White and nNIRs, were compared across multiple parameters: cytotoxicity, adenosine triphosphate (ATP) synthesis, reactive oxygen species (ROS) reduction, skin thickness, collagen synthesis, collagenase expression, and hair follicle growth. Experiments were conducted on human skin cells and animal models. Cytotoxicity, ATP synthesis, and ROS reduction were evaluated in human skin cells exposed to nNIRs and Whites. LED irradiation effects were also studied on a UV-induced photoaging mouse model, analyzing skin thickness, collagen synthesis, and collagenase expression. Hair growth promotion was examined as well. Results revealed both White and nNIR were non-cytotoxic to human skin cells. nNIR enhanced ATP and collagen synthesis while reducing ROS levels, outperforming the commonly used 2chip LEDs. In the UV-induced photoaging mouse model, nNIR irradiation led to reduced skin thickness, increased collagen synthesis, and lowered collagenase expression. Additionally, nNIR irradiation stimulated hair growth, augmented skin thickness, and increased hair follicle count. In conclusion, the study highlighted positive effects of White and nNIR irradiation on skin and hair growth. However, nNIR exhibited superior outcomes compared to White. Its advancements in ATP content, collagen synthesis, collagenase inhibition, and hair growth promotion imply increased ATP synthesis activity. These findings underscore nNIR therapy's potential as an innovative and effective approach for enhancing skin and promoting hair growth.

Structure properties of Canna edulis RS3 (double enzyme hydrolysis) and RS4 (OS-starch and cross-linked starch): Influence on fermentation products and human gut microbiota.

This study investigated the in vitro fermentation characteristics of different structural types of Canna edulis resistant starch (RS). RS3 was prepared through a double enzyme hydrolysis method, and RS4 (OS-starch and cross-linked starch) was prepared using octenyl succinic anhydride and sodium trimetaphosphate/sodium tripolyphosphate, respectively. The RS3 and RS4 samples were structurally analyzed using scanning electron microscopy, Fourier-transform infrared spectroscopy, differential scanning calorimetry, and X-ray diffraction analysis. This was followed by in vitro fermentation experiments. The results revealed microstructure differences in the two groups of starch samples. Compared to native starch, RS3 and RS4 exhibited a lower degree of order and endothermic energy, with lower crystallinity (RS3: 29.59 ± 1.11 %; RS4 [OS-starch]: 28.01 ± 1.32 %; RS4 [cross-linked starch]: 30.44 ± 1.73 %) than that in native starch (36.29 ± 0.89 %). The RS content was higher in RS3 (63.40 ± 2.85 %) and RS4 (OS-starch: 71.21 ± 1.28 %; cross-linked starch: 74.33 ± 0.643 %) than in native starch (57.71 ± 2.95 %). RS3 and RS4 exhibited slow fermentation rates, promoting the production of short-chain fatty acids. RS3 and cross-linked starch significantly increased the production of acetate and butyrate. Moreover, RS3 significantly promoted the abundance of Lactobacillus, while OS-starch and cross-linked starch significantly enhanced the abundance of Dorea and Coprococcus, respectively. Hence, the morphological structure and RS content of the samples greatly influenced the fermentation rate. Moreover, the different varieties of RS induced specific gut microbial regulation. Hence, they show potential applications in functional foods for tailored gut microbiota management.

Polyphosphate kinase regulates LPS structure and polymyxin resistance during starvation in E. coli.

Polyphosphates (polyP) are chains of inorganic phosphates that can reach over 1,000 residues in length. In Escherichia coli, polyP is produced by the polyP kinase (PPK) and is thought to play a protective role during the response to cellular stress. However, the molecular pathways impacted by PPK activity and polyP accumulation remain poorly characterized. In this work, we used label-free mass spectrometry to study the response of bacteria that cannot produce polyP (Δppk) during starvation to identify novel pathways regulated by PPK. In response to starvation, we found 92 proteins significantly differentially expressed between wild-type and Δppk mutant cells. Wild-type cells were enriched for proteins related to amino acid biosynthesis and transport, while Δppk mutants were enriched for proteins related to translation and ribosome biogenesis, suggesting that without PPK, cells remain inappropriately primed for growth even in the absence of the required building blocks. From our data set, we were particularly interested in Arn and EptA proteins, which were down-regulated in Δppk mutants compared to wild-type controls, because they play a role in lipid A modifications linked to polymyxin resistance. Using western blotting, we confirm differential expression of these and related proteins in K-12 strains and a uropathogenic isolate, and provide evidence that this mis-regulation in Δppk cells stems from a failure to induce the BasRS two-component system during starvation. We also show that Δppk mutants unable to up-regulate Arn and EptA expression lack the respective L-Ara4N and pEtN modifications on lipid A. In line with this observation, loss of ppk restores polymyxin sensitivity in resistant strains carrying a constitutively active basR allele. Overall, we show a new role for PPK in lipid A modification during starvation and provide a rationale for targeting PPK to sensitize bacteria towards polymyxin treatment. We further anticipate that our proteomics work will provide an important resource for researchers interested in the diverse pathways impacted by PPK.

A microfluidic chip-based capillary zone electrophoresis-mass spectrometry method for measuring adenosine 5'-Triphosphate and its similar nucleotide analogues.

BACKGROUND: Extracellular ATP is involved in disorders that cause inflammation of the airways and cough, thus limiting its release has therapeutic benefits. Standard luminescence-based ATP assays measure levels indirectly through enzyme degradation and do not provide a simultaneous readout for other nucleotide analogues. Conversely, mass spectrometry can provide direct ATP measurements, however, common RPLC and HILIC methods face issues because these molecules are unstable, metal-sensitive analytes which are often poorly retained. These difficulties have traditionally been overcome using passivation or ion-pairing chromatography, but these approaches can be problematic for LC systems. As a result, more effective analytical methods are needed. RESULTS: Here, we introduce a new application that uses microfluidic chip-based capillary zone electrophoresis-mass spectrometry (µCZE-MS) to measure ATP and its analogues simultaneously in biofluids. The commercially available ZipChip Interface and a High-Resolution Bare-glass microchip (ZipChip, HRB, 908 Devices Inc.) coupled to a Thermo Scientific Tribrid Orbitrap, were successfully used to separate and detect various nucleotide standards, as well as ATP, ADP, AMP, and adenosine in plasma and BALF obtained from naïve Brown Norway rats. The findings demonstrate that this approach can rapidly and directly detect ATP and its related nucleotide analogues, while also highlighting the need to preserve these molecules in biofluids with chelators like EDTA. In addition, we demonstrate that this µCZE-MS method is also suitable for detecting a variety of metabolites, revealing additional potential future applications. SIGNIFICANCE: This innovative µCZE-MS approach provides a robust new tool to directly measure ATP and other nucleotide analogues in biofluids. This can enable the study of eATP in human disease and potentially contribute to the creation of ATP-targeting therapies for airway illnesses.

Mechanisms of cadmium mitigation in tomato plants under orthophosphate and polyphosphate fertilization regimes.

Cadmium (Cd) is one of the most toxic elements in soil, affecting morphological, physiological, and biochemical processes in plants. Mineral plant nutrition was tested as an effective approach to mitigate Cd stress in several crop species. In this regard, the present study aimed to elucidate how different phosphorus (P) fertilization regimes can improve some bio-physiological processes in tomato plants exposed to Cd stress. In a hydroponic experiment, the impact of two phosphorus fertilizer forms (Polyphosphate (poly-P): condensed P-form with 100% polymerization rate and orthophosphate (ortho-P): from orthophosphoric acid) on the photosynthetic activity, plant growth, and nutrient uptake was assessed under three levels of Cd stress (0, 12, and 25 µM of CdCl2). The obtained results confirmed the negative effects of Cd stress on the chlorophyll content and the efficiency of the photosynthesis machinery. The application of poly-P fertilizer significantly improved the chlorophyll stability index (82%) under medium Cd stress (Cd12), as compared to the ortho-P form (55%). The analysis of the chlorophyll α fluorescence transient curve revealed that the amplitude of Cd effect on the different steps of electron transfer between PSII and PSI was significantly reduced under the poly-P fertilization regime compared to ortho-P, especially under Cd12. The evaluation of the RE0/RC parameter showed that the electron flux reducing end electron acceptors at the PSI acceptor side per reaction center was significantly improved in the poly-P treatment by 42% under Cd12 compared to the ortho-P treatment. Moreover, the use of poly-P fertilizer enhanced iron uptake and its stoichiometric homeostasis in the shoot tissue which maintained an adequate absorption of iron under Cd stress conditions. Findings from this study revealed for the first time that inorganic polyphosphate fertilizers can reduce Cd toxicity in tomato plants by enhancing photosynthesis activity, nutrient uptake, plant growth, and biomass accumulation despite the high level of cadmium accumulation in shoot tissues.

Combining recombinase polymerase amplification with tyrosine modified 2'-deoxyuridine-5'-triphosphate for direct voltammetric detection of double-stranded DNA: Application to potato pathogen Dickeya solani.

The approach based on a combination of isothermal recombinase polymerase amplification (RPA), 2'-deoxyuridine-5'-triphosphate modified with tyrosine aromatic group (dUTP-Y1), and direct voltammetric detection of RPA product carrying electroactive labels was successfully applied to the potato pathogen Dickeya solani. The artificial nucleotide dUTP-Y1 demonstrated a good compatibility with RPA, enabling by targeting a section of D. solani genome with a unique sequence to produce the full-size modified products at high levels of substitution of dTTP by dUTP-Y1 (up to 80-90 %) in the reaction mixture. The optimized procedure of square wave voltammetry allowed to reliably detect the product generated by RPA at 80 % substitution of dTTP by dUTP-Y1 (dsDNA-Y1) in microliter sample volumes on the surface of disposable carbon screen printed electrodes at the potential of about 0.6 V. The calibration curve for the amplicon detection was linear in coordinates 'Ip, A vs. Log (c, M)' within the 0.05-1 µM concentration range. The limit of detection for dsDNA-Y1 was estimated as 8 nM. The sensitivity of the established electrochemical approach allowed to detect amplicons generated in a single standard 50 µL RPA reaction after their purification with silica-coated magnetic beads. The overall detectability of D. solani with the suggested combination of RPA and voltammetric registration of dsDNA-Y1 can be as low as a few copies of bacterial genome per standard reaction. In total, amplification, purification, and electrochemical detection take about 120-150 min. Considering the potential of direct electrochemical analysis for miniaturization, as well as compliance with low-cost and low-power requirements, the findings provide grounds for future development of microfluidic devices integrating isothermal amplification, amplicon purification and detection based on the tyrosine modified nucleotide for the purpose of 'on-site' detection of various pathogens.

Phosphatase-degradable nanoparticles providing sustained drug release.

AIM: The study aimed to develop enzyme-degradable nanoparticles comprising polyphosphates and metal cations providing sustained release of the antibacterial drug ethacridine (ETH). METHODS: Calcium polyphosphate (Ca-PP), zinc polyphosphate (Zn-PP) and iron polyphosphate nanoparticles (Fe-PP NPs) were prepared by co-precipitation of sodium polyphosphate with cations and ETH. Developed nanocarriers were characterized regarding particle size, PDI, zeta potential, encapsulation efficiency and drug loading. Toxicological profile of nanocarriers was assessed via hemolysis assay and cell viability on human blood erythrocytes and HEK-293 cells, respectively. The enzymatic degradation of NPs was evaluated in presence of alkaline phosphatase (ALP) monitoring the release of monophosphate, shift in zeta potential and particle size as well as drug release. The antibacterial efficacy against Escherichia coli was determined via microdilution assay. RESULTS: NPs were obtained in a size range between 300 - 480 nm displaying negative zeta potential values. Encapsulation efficiency was in the range of 83.73 %- 95.99 %. Hemolysis assay underlined sufficient compatibility of NPs with blood cells, whereas drug and NPs showed a concentration dependent effect on HEK-293 cells viability. Ca- and Zn-PP NPs exhibited remarkable changes in zeta potential, particle size, monophosphate and drug release upon incubation with ALP, compared to Fe-PP NPs showing only minor differences. The released ETH from Ca- and Zn-PP nanocarriers retained the antibacterial activity against E. coli, whereas no antibacterial effect was observed with Fe-PP NPs. CONCLUSION: Polyphosphate nanoparticles cross-linked with divalent cations and ETH hold promise for sustained drug delivery triggered by ALP for parental administration.

Utilization of spent coffee grounds as charring agent to prepare flame retardant poly(lactic acid) composites with improved toughness.

The objective was to utilize spent coffee grounds (SCG) as charring agent to combine with ammonium polyphosphate (APP) to prepare flame retardant poly(lactic acid) (PLA) composites with improved toughness. PLA/APP-SCG and PLA/APP-SCG/KH560 composites were prepared, and silane coupling agent KH560 was applied to improve particle-matrix interfacial compatibility. The particle-matrix interface, char formation, flame retardancy, mechanical properties and fracture morphology of PLA composites were studied. Results showed that PLA/APP-SCG5% and PLA/APP-SCG20% passed UL-94 V-0 rating, and increase in charred residues was favorable for improving flame retardancy. Improved toughness was also obtained compared to PLA, attributed to debonding of APP from matrix under external force as well as plasticization effect of coffee oil contained in SCG. PLA/APP-SCG5%/KH560 and PLA/APP-SCG20%/KH560 showed smaller elongation at break and impact strength compared to PLA/APP-SCG5% and PLA/APP-SCG20%, respectively. The improved interfacial compatibility was unfavorable for debonding of APP from matrix, and both APP and SCG played the role of enhancing strength, thus decreasing toughness. PLA/APP-SCG/KH560 counterparts were actually set as parallel samples to prove that PLA/APP-SCG composites showed improved toughness with weak interfacial compatibility. This study has provided a practical approach to utilize bio-derived wastes as charring agent to prepare flame retardant PLA composites with enhanced toughness.

Repair of a rat calvaria defect with injectable strontium (Sr)-doped polyphosphate dicalcium phosphate dehydrate (P-DCPD) ceramic bone grafts.

The trace element strontium (Sr) enhances new bone formation. However, delivering Sr, like other materials, in a sustained manner from a ceramic bone graft substitute (BGS) is difficult. We developed a novel ceramic BGS, polyphosphate dicalcium phosphate dehydrate (P-DCPD), which delivers embedded drugs in a sustained pattern. This study assessed the in vitro and in vivo performance of Sr-doped P-DCPD. In vitro P-DCPD and 10%Sr-P-DCPD were nontoxic and eluents from 10%Sr-P-DCPD significantly enhanced osteoblastic MC3T3 cell differentiation. A sustained, zero-order Sr release was observed from 10%Sr-P-DCPD for up to 70 days. When using this BGS in a rat calvaria defect model, both P-DCPD and 10% Sr-P-DCPD were found to be biocompatible and biodegradable. Histologic data from decalcified and undecalcified tissue showed that 10%Sr-P-DCPD had more extensive new bone formation compared with P-DCPD 12-weeks after surgery and the 10%Sr-P-DCPD had more organized new bone and much less fibrous tissue at the defect margins. The new bone was formed on the surface of the degraded ceramic debris within the bone defect area. P-DCPD represented a promising drug-eluting BGS for repair of critical bone defects.

The Physiological Inorganic Polymers Biosilica and Polyphosphate as Key Drivers for Biomedical Materials in Regenerative Nanomedicine.

There is a need for novel nanomaterials with properties not yet exploited in regenerative nanomedicine. Based on lessons learned from the oldest metazoan phylum, sponges, it has been recognized that two previously ignored or insufficiently recognized principles play an essential role in tissue regeneration, including biomineral formation/repair and wound healing. Firstly, the dependence on enzymes as a driving force and secondly, the availability of metabolic energy. The discovery of enzymatic synthesis and regenerative activity of amorphous biosilica that builds the mineral skeleton of siliceous sponges formed the basis for the development of successful strategies for the treatment of osteochondral impairments in humans. In addition, the elucidation of the functional significance of a second regeneratively active inorganic material, namely inorganic polyphosphate (polyP) and its amorphous nanoparticles, present from sponges to humans, has pushed forward the development of innovative materials for both soft (skin, cartilage) and hard tissue (bone) repair. This energy-rich molecule exhibits a property not shown by any other biopolymer: the delivery of metabolic energy, even extracellularly, necessary for the ATP-dependent tissue regeneration. This review summarizes the latest developments in nanobiomaterials based on these two evolutionarily old, regeneratively active materials, amorphous silica and amorphous polyP, highlighting their specific, partly unique properties and mode of action, and discussing their possible applications in human therapy. The results of initial proof-of-concept studies on patients demonstrating complete healing of chronic wounds are outlined.

Synergistic effect of stereo-complexation and interfacial compatibility in ammonium polyphosphate grafted polylactic acid fibers for simultaneously improved toughness and flame retardancy.

Flammability and poor toughness of unmodified PLA limit its applications in various fields. Though ammonium polyphosphate (APP) is a green and effective flame retardant, it has poor compatibility with the matrix, leading to a decrease in mechanical properties. Stereo-complexation greatly improves the strength and heat resistance of traditional PLA. However, the effect of flame retardants on the formation of stereo-complexed crystals and the impact of stereo-complexation on flame retardancy have not been studied previously. In this research, PDLA chains were first in-situ reacted with APP particles for improved interfacial compatibility. By utilizing the characteristic of PLA enantiomers that can form stereo-complexed crystals, near-complete stereo-complexed PLA fibers with flame retardancy were produced via clean and continuous melt spinning. The compatibility between PDLA-g-APP and PLLA matrix was studied by SEM, rheological analyses and DSC. Strength and flexibility of the fibers were simultaneously enhanced compared to traditional PLA due to the synergistic effect of interfacial compatibility and stereo-complexation. Compared to traditional PLA, the peak heat release rate and total heat release in microcalorimetry test were reduced by 33 % and 22 %, respectively. The flame-retardant fibers achieved a V-0 rating in the UL-94 test, and an increase in LOI value from 19.4 % to 28.2 %.

The molecular language of RNA 5' ends: guardians of RNA identity and immunity.

RNA caps are deposited at the 5' end of RNA polymerase II transcripts. This modification regulates several steps of gene expression, in addition to marking transcripts as self to enable the innate immune system to distinguish them from uncapped foreign RNAs, including those derived from viruses. Specialized immune sensors, such as RIG-I and IFITs, trigger antiviral responses upon recognition of uncapped cytoplasmic transcripts. Interestingly, uncapped transcripts can also be produced by mammalian hosts. For instance, 5'-triphosphate RNAs are generated by RNA polymerase III transcription, including tRNAs, Alu RNAs, or vault RNAs. These RNAs have emerged as key players of innate immunity, as they can be recognized by the antiviral sensors. Mechanisms that regulate the presence of 5'-triphosphates, such as 5'-end dephosphorylation or RNA editing, prevent immune recognition of endogenous RNAs and excessive inflammation. Here, we provide a comprehensive overview of the complexity of RNA cap structures and 5'-triphosphate RNAs, highlighting their roles in transcript identity, immune surveillance, and disease.

Cytosolic 5'-Nucleotidase III and Nucleoside Triphosphate Diphosphohydrolase 1 Dephosphorylate the Pharmacologically Active Metabolites of Gemcitabine and Emtricitabine.

Gemcitabine (dFdC) and emtricitabine (FTC) are first-line drugs that are used for the treatment of pancreatic cancer and human immunodeficiency virus, respectively. The above drugs must undergo sequential phosphorylation to become pharmacologically active. Interindividual variability associated with the responses of the above drugs has been reported. The molecular mechanisms underlying the observed variability are yet to be elucidated. Although this could be multifactorial, nucleotidases may be involved in the dephosphorylation of drug metabolites due to their structural similarity to endogenous nucleosides. With these in mind, we performed in vitro assays using recombinant nucleotidases to assess their enzymatic activities toward the metabolites of dFdC and FTC. From the above in vitro experiments, we noticed the dephosphorylation of dFdC-monophosphate in the presence of two 5'-nucleotidases (5'-NTs), cytosolic 5'-nucleotidase IA (NT5C1A) and cytosolic 5'-nucleotidase III (NT5C3), individually. Interestingly, FTC monophosphate was dephosphorylated only in the presence of NT5C3 enzyme. Additionally, nucleoside triphosphate diphosphohydrolase 1 (NTPDase 1) exhibited enzymatic activity toward both triphosphate metabolites of dFdC and FTC. Enzyme kinetic analysis further revealed Michaelis-Menten kinetics for both NT5C3-mediated dephosphorylation of monophosphate metabolites, as well as NTPDase 1-mediated dephosphorylation of triphosphate metabolites. Immunoblotting results confirmed the presence of NT5C3 and NTPDase 1 in both pancreatic and colorectal tissue that are target sites for dFdC and FTC treatment, respectively. Furthermore, sex-specific expression patterns of NT5C3 and NTPDase 1 were determined using mass spectrometry-based proteomics approach. Based on the above results, NT5C3 and NTPDase 1 may function in the control of the levels of dFdC and FTC metabolites. SIGNIFICANCE STATEMENT: Emtricitabine and gemcitabine are commonly used drugs for the treatment of human immunodeficiency virus and pancreatic cancer. To become pharmacologically active, both the above drugs must be phosphorylated. The variability in the responses of the above drugs can lead to poor clinical outcomes. Although the sources of drug metabolite concentration variability are multifactorial, it is vital to understand the role of nucleotidases in the tissue disposition of the above drug metabolites due to their structural similarities to endogenous nucleosides.

Development of a chitosan and whey protein-based, biodegradable, colorimetric/fluorescent dual-channel monitoring label for real-time sensing of shrimp freshness.

To address the growing and urgent need for quick and accurate food spoilage detection systems as well as to reduce food resource wastage, recent research has focused on intelligent bio-labels using pH indicators. Accordingly, we developed a dual-channel intelligent label with colorimetric and fluorescent capabilities using black lycium anthocyanin (BLA) and 9,10-bis(2,2-dipyridylvinyl) anthracene (DSA4P) as colorimetric and fluorescent indicators within a composite film consisting of chitosan (Cs), whey protein (Wp), and sodium tripolyphosphate (STPP). The addition of STPP as a cross-linking agent significantly improved the hydrophobicity, mechanical properties, and thermal stability of the Cs/Wp composite films under low pH conditions. After the incorporation of BLA and DSA4P, the resulting dual-channel intelligent label (Cs/Wp/STPP/BLA/DSA4P) exhibited superior hydrophobicity, as indicated by a water contact angle of 78.03°. Additionally, it displayed enhanced mechanical properties, with a tensile strength (TS) of 3.04 MPa and an elongation at break (EAB) of 81.07 %, while maintaining a low transmittance of 28.48 % at 600 nm. After 25 days of burial in soil, the label was significantly degraded, which showcases its eco-friendly nature. Moreover, the label could visually detect color changes indicating volatile ammonia concentrations (25-25,000 ppm). The color of the label in daylight gradually shifted from brick-red to light-red, brownish-yellow, and finally light-green as the ammonia concentration increased. Correspondingly, its fluorescence transitioned from no fluorescence to green fluorescence with increasing ammonia concentration, gradually intensifying under 365-nm UV light. Furthermore, the label effectively monitored the freshness of shrimp stored at temperatures of 4 °C, 25 °C, and - 18 °C. Thus, the label developed in this study exhibits significant potential for enhancing food safety monitoring.

Probing the legitimate initiation of RNA synthesis by Qß replicase with oligonucleotide primers.

Oligoribonucleotides complementary to the template 3' terminus were tested for their ability to initiate RNA synthesis on legitimate templates capable of exponential amplification by Qß replicase. Oligonucleotides shorter than the distance to the nearest predicted template hairpin proved able to serve as primers, with the optimal length varying for different templates, suggesting that during initiation the template retains its native fold incorporating the 3' terminus. The priming activity of an oligonucleotide is greatly enhanced by its 5'-triphosphate group, the effect being strongly dependent on Mg2+ ions. This indicates that, unlike other studied RNA polymerases, Qß replicase binds the 5'-triphosphate of the initiating nucleotide GTP, and this binding is needed for the replication of legitimate templates.

Inorganic polyphosphate: from basic research to diagnostic and therapeutic opportunities in ALS/FTD.

Inorganic polyphosphate (polyP) is a simple, negatively charged biopolymer with chain lengths ranging from just a few to over a thousand ortho-phosphate (Pi) residues. polyP is detected in every cell type across all organisms in nature thus far analyzed. Despite its structural simplicity, polyP has been shown to play important roles in a remarkably broad spectrum of biological processes, including blood coagulation, bone mineralization and inflammation. Furthermore, polyP has been implicated in brain function and the neurodegenerative diseases amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), Alzheimer's disease and Parkinson's disease. In this review, we first address the challenges associated with identifying mammalian polyP metabolizing enzymes, such as Nudt3, and quantifying polyP levels in brain tissue, cultured neural cells and cerebrospinal fluid. Subsequently, we focus on recent studies that unveil how the excessive release of polyP by human and mouse ALS/FTD astrocytes contributes to these devastating diseases by inducing hyperexcitability, leading to motoneuron death. Potential implications of elevated polyP levels in ALS/FTD patients for innovative diagnostic and therapeutic approaches are explored. It is emphasized, however, that caution is required in targeting polyP in the brain due to its diverse physiological functions, serving as an energy source, a chelator for divalent cations and a scaffold for amyloidogenic proteins. Reducing polyP levels, especially in neurons, might thus have adverse effects in brain functioning. Finally, we discuss how activated mast cells and platelets also can significantly contribute to ALS progression, as they can massively release polyP.