Nat Protoc. 2025 Jun 9. doi: 10.1038/s41596-025-01223-y. Online ahead of print.

NO ABSTRACT

PMID:40490534 | DOI:10.1038/s41596-025-01223-y

Free Radic Biol Med. 2025 Jun 7:S0891-5849(25)00757-9. doi: 10.1016/j.freeradbiomed.2025.06.008. Online ahead of print.

ABSTRACT

Glioblastoma multiforme (GBM) is the most aggressive and lethal malignant brain tumor, with limited treatment options and poor patient prognosis. Novel therapeutic strategies are urgently needed to extend survival and improve quality of life for GBM patients. Our transcriptomic analysis revealed that GBM cells exhibit substantial upregulation of DNA damage response (DDR) pathways, identifying DDR as a promising therapeutic target. To leverage this vulnerability, we investigated pharmacological ascorbate (P-AscH-; millimolar concentrations of vitamin C) as a potential treatment for GBM. Our findings demonstrated that P-AscH- induces ROS-mediated cytotoxicity in both low- and high-grade glioma cells, primarily via extracellular H2O2 production. This generated H2O2 triggers oxidative DNA damage, PARP1 hyperactivation, and impairment of DDR signaling by targeting key regulators, including Chk1 and RPA2. Concurrently, extracellular H2O2 following P-AscH- exposure also disrupts the mTOR signaling pathway, a critical regulator of cell survival and DDR. Mechanistic studies showed that H2O2 impairs both mTORC1 and mTORC2 pathways, as evidenced by decreased phosphorylation of mTORC1 substrates (S6K, S6 and 4EBP1) and the mTORC2 substrate Akt, along with reduced total protein levels of these key components. Furthermore, we demonstrated that P-AscH- augments the cytotoxic effects of standard-of-care temozolomide and synergistically enhances the anticancer effects of mTOR inhibitors. These improved therapeutic responses were validated in a 3D-GBM spheroid model. Collectively, our findings suggest that P-AscH- disrupts both DDR and mTOR signaling pathways, potentially sensitizing GBM cells to both existing therapies and investigational drugs. These results underscore the promise of P-AscH- as an adjunctive treatment for GBM and other malignancies.

PMID:40490203 | DOI:10.1016/j.freeradbiomed.2025.06.008

ACS Synth Biol. 2025 Jun 9. doi: 10.1021/acssynbio.4c00809. Online ahead of print.

ABSTRACT

Chemogenetic regulators of transgene activity, such as the tetracycline-inducible system derived from the tetracycline resistance operon of the bacterial transposon Tn10, are critical and widely used systems in cellular engineering. The tetracycline-inducible system is prized for its selectivity, high affinity, inducibility, reversibility, and differential control of gene transcription. However, its optimization for binary on/off expression limits its application in systems biology and the modeling and construction of complex regulatory systems with intricate input/output paradigms. To overcome this limitation, we developed a high-throughput reporter system to investigate a saturated mutagenesis library of tetracycline resistance operator variants. Using this system, we mapped the functional interactions of Tet repressor DNA binding protein at single-nucleotide resolution in mammalian cells. Our comprehensive screen revealed a spectrum of variant effects, ranging from a nearly complete loss of repression to levels indistinguishable from the natural operator, validated through orthogonal assays. This comprehensive characterization of the sequence-specificity of a tetracycline resistance operator facilitates the construction of variably suppressive, inducible systems for dynamic and modular control over gene expression in mammalian cell culture.

PMID:40489706 | DOI:10.1021/acssynbio.4c00809

Annu Rev Chem Biomol Eng. 2025 Jun;16(1):409-432. doi: 10.1146/annurev-chembioeng-100722-104442.

ABSTRACT

The search for what differentiates inanimate matter from living things began in antiquity as a search for a fundamental life force embedded deep within living things-a special material unit owned only by life-later transforming to a more circumspect search for unique gains in function that transform nonliving matter to that which can reproduce, adapt, and survive. Aristotelian thinking about the matter/life distinction and Vitalistic philosophy's vital force persisted well into the Scientific Revolution, only to be debunked by Pasteur and Brown in the nineteenth century. Acceptance of the atomic reality and understanding of the uniqueness of life's heredity, evolution, and reproduction led to formation of the Central Dogma. With startling speed, technological development then gave rise to structural biology, systems biology, and synthetic biology-and a search to replicate and synthesize that gain in function that transforms matter to life. Yet one still cannot build a living cell de novo from its atomic and molecular constituents, and "what I cannot create, I do not understand," in the words of Richard Feynman. In the last two decades, new recognition of old ideas-spatial organization and compartmentalization-has renewed focus on Brownian and flow physics. In this article, we explore how experimental and computational advances in the last decade have embraced the deep coupling between physics and cellular biochemistry to shed light on the matter/life nexus. Whole-cell modeling and synthesis are offering promising new insights that may shed light on this nexus in the cell's watery, crowded milieu.

PMID:40489303 | DOI:10.1146/annurev-chembioeng-100722-104442

J Mol Evol. 2025 Jun 9. doi: 10.1007/s00239-025-10255-7. Online ahead of print.

ABSTRACT

Drd2 dopamine receptor mRNAs are alternatively spliced in rodents and primates by skipping exon 6 to produce the D2S protein, or including exon 6 to produce the D2L protein. These protein isoforms have differing roles in pre- vs. post-synaptic signaling, cytoplasmic vesicle processing, and calcium-mediated desensitization. Genetic alteration in the D2S/D2L ratio affects human behavior and cognition at multiple levels, including working memory. Here we show that exon 6 originated early in vertebrate evolution, after the duplication and divergence of D2 and D4 dopamine receptor genes, but before the duplication and divergence of D2 and D3 dopamine receptor genes. Exon 6 encodes a relatively conserved sequence in the third cytoplasmic loop of the D2-D3 receptor. Its amino acid sequence is relatively short (24-33 amino acids), and is not strictly necessary for dopamine signal transduction. Exon skipping of Drd2 exon 6 was not detectable in the brains of cyclostomes, sharks, fish, relatively primitive amphibians (Xenopus, Notophthalmus), relatively primitive reptiles (turtles), relatively primitive birds (ostrich), or relatively primitive mammals (monotremes and marsupials). However, exon skipping of Drd2 exon 6 did occur at significant levels in the brains of more derived amphibians, reptiles, birds and mammals. Thus, skipping of Drd2 exon 6 arose convergently and specifically in the more derived tetrapod lineages, none of which deleted this exon. In contrast, exon 6 was convergently deleted during Drd3 evolution in an apparently random subset of the species of sharks, fish, amphibians, reptiles, birds, and mammals.

PMID:40488893 | DOI:10.1007/s00239-025-10255-7

Biointerphases. 2025 May 1;20(3):038601. doi: 10.1116/6.0004726.

NO ABSTRACT

PMID:40488549 | DOI:10.1116/6.0004726

ISME Commun. 2025 May 20;5(1):ycaf084. doi: 10.1093/ismeco/ycaf084. eCollection 2025 Jan.

ABSTRACT

Soil water repellency (SWR), the reduced affinity of soil for water, is a phenomenon that affects soils globally. With worsening climate change, SWR is expected to increase emphasizing the need to understand the mechanisms driving SWR development and persistence. The importance of the soil microbes in SWR has been postulated for decades, but limited research has been conducted into whole-community interactions and the role of community metabolic activity. To address this gap in knowledge, we investigated the direct effect of microbial community composition, activity, and diversity, as well as their associated metabolites on the development and persistence of SWR by inoculating microcosms containing model soils with 15 different microbial communities and quantified respiration and SWR over time. Six communities that consistently produced either a hydrophobic or hydrophilic phenotype were characterized using metagenomics and metabolomics to determine the impact of microbial and metabolite composition and diversity on SWR. We identified several bacterial genera with significant changes in abundance between SWR phenotypes including Nocardiopsis and Kocuria in hydrophilic and Streptomyces and Cutibacterium in hydrophobic. We discovered that hydrophilic communities were more positively connected when compared to hydrophobic communities, which could be due to an increase in defense mechanism genes. Additionally, we identified specific metabolites associated with hydrophilic and hydrophobic phenotypes including an increase in the osmolyte ectoine in hydrophilic and an increase in plant-derived decomposition products in hydrophobic communities. Finally, our research suggests that fungi, previously thought to cause hydrophobicity, may actually contribute to hydrophilicity through their preferential consumption of hydrophobic compounds.

PMID:40487918 | PMC:PMC12143472 | DOI:10.1093/ismeco/ycaf084

Front Cell Infect Microbiol. 2025 May 23;15:1597009. doi: 10.3389/fcimb.2025.1597009. eCollection 2025.

ABSTRACT

Pseudomonas aeruginosa is an opportunistic pathogen causing severe infections of the lung, burn wounds and eyes. Due to its intrinsic high antibiotic resistance the bacterium is difficult to eradicate. A promising therapeutic option is the use of P. aeruginosa-specific bacteriophages. Thus, the implementation of a phage therapy requires their selection, production and systematic administration using multiple strains of the bacterial target. Here, we used 25 phages and tested their susceptibility on 141 different P. aeruginosa strains isolated from patients with different types of infection. Comparative host spectrum analyses were carried out using double agar overlay plaque assay (DPA) and planktonic killing assay (PKA), which resulted in 70% of the cases in the same host range. All phages were assigned to known phage genera, but some of the phages are new species. Isolated members of the genera Pakpunavirus, Pbunavirus (myoviruses), Pawinskivirus, Elvirus (myoviruses, jumbo phages), Litunavirus and Bruynoghevirus (podoviruses) demonstrated great therapeutic potential due to strong lysis behavior on diverse strains. Seven phages were excluded for therapeutic purposes due to genetic determinants that confer lysogenicity. Due to automation with lower time expenditure in execution and analysis, PKA has the higher potential for implementation in diagnostics. Finally, different combinations of phages were tested in silico with various P. aeruginosa strains. Highly efficient phage combinations eradicating multiple P. aeruginosa strains were found. Thus, a solid basis for the development of a broad host range phage therapy was laid.

PMID:40487314 | PMC:PMC12141865 | DOI:10.3389/fcimb.2025.1597009

Chronic Dis Transl Med. 2025 Apr 2;11(2):140-147. doi: 10.1002/cdt3.70001. eCollection 2025 Jun.

ABSTRACT

BACKGROUND: Computer-aided detection (CAD) software has been introduced to automatically interpret digital chest X-rays. This study aimed to evaluate the performance of CAD software (JF CXR-1 v3.0, which was developed by a domestic Hi-tech enterprise) in tuberculosis (TB) case finding in China.

METHODS: In 2019, we conducted an internal evaluation of the performance of JF CXR-1 v3.0 by reading standard images annotated by a panel of experts. In 2020, using the reading results of chest X-rays by a panel of experts as the reference standard, we conducted an on-site prospective study to evaluate the performance of JF CXR-1 v3.0 and local radiologists in TB case finding in 13 township health centers in Zhongmu County, Henan Province.

RESULTS: Internal assessment results based on 277 standard images showed that JF CXR-1 v3.0 had a sensitivity of 85.94% (95% confidence interval [CI]: 77.42%, 94.45%) and a specificity of 74.65% (95% CI: 68.81%, 80.49%) to distinguish active TB from other imaging conditions. In the on-site evaluation phase, images from 3705 outpatients who underwent chest X-ray detection were read by JF CXR-1 v3.0 and local radiologists in parallel. The imaging diagnosis of local radiologists for active TB had a sensitivity of 32.89% (95% CI: 22.33%, 43.46%) and a specificity of 99.28% (95% CI: 99.01%, 99.56%), while JF CXR-1 v3.0 showed a significantly higher sensitivity of 92.11% (95% CI: 86.04%, 98.17%) (p < 0.05) and maintained high specificity at 94.54% (95% CI: 93.81%, 95.28%).

CONCLUSIONS: CAD software could play a positive role in improving the TB case finding capability of township health centers.

PMID:40486951 | PMC:PMC12142701 | DOI:10.1002/cdt3.70001

J Toxicol Environ Health A. 2025 Jun 9:1-15. doi: 10.1080/15287394.2025.2516815. Online ahead of print.

ABSTRACT

During the tobacco crop sorting process, workers come in contact with various substances found in dried leaves, including tobacco-specific nitrosamines: N'-nitrosonornicotine (NNN), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), and nicotine (NIC). The objective of this study was to assess the cytotoxic and genotoxic effects initiated by exposure to aqueous extract of dry tobacco leaves (TLE) and by compounds NNN, NNK, and NIC, both alone and in combination, using the human hepatocarcinoma cell line (HepG2) as a model. The relationship of exposure with mechanisms of oxidative damage was also assessed. To determine cytotoxicity, the MTT assay was employed, and to assess genotoxicity, the alkaline comet assay and its modified version with the enzyme FPG were utilized, along with the measurement of telomere length. Further systems biology tools were employed to generate the protein network for the combination of NIC, NNN, and NNK. No marked cytotoxicity was observed through the MTT assay. Genotoxicity was detected at the highest concentrations of TLE (1.25 and 5 mg/ml), for NIC, NNN, NNK, and in combinations of these samples. The modified comet assay with formamidopyrimidine DNA glycosylase (FPG) enzyme found that genotoxicity was related to oxidative damage. No significant difference in telomere length was observed. Through in silico analysis, data demonstrated that the predominant proteins associated with the combined agents NNN, NNK, and NIC exhibited connections with signaling pathways related to oxidative damage mechanisms and carcinogenic processes.

PMID:40485624 | DOI:10.1080/15287394.2025.2516815

Adv Sci (Weinh). 2025 Jun 9:e15820. doi: 10.1002/advs.202415820. Online ahead of print.

ABSTRACT

A Physical Unclonable Function (PUF) is a security primitive that exploits inherent variations in manufacturing protocols to generate unique, random-like identifiers. These identifiers are used for authentication and encryption purposes in hardware security applications in the semiconductor industry. Inspired by the success of silicon PUFs, herein it is leverage Terminal deoxynucleotidyl Transferase (TdT), a template-independent polymerase belonging to the X-family of DNA polymerases, to augment the intrinsic entropy generated during DNA lesion repair and rapidly produce genetic PUFs that satisfy the following properties: robustness (i.e., they repeatedly produce the same output), uniqueness (i.e., they do not coincide with any other identically produced PUF), and unclonability (i.e., they are virtually impossible to replicate). Furthermore, a post-sequencing feature selection methodology based on logistic regression to facilitate PUF classification is developed. This experimental and computational pipeline drastically reduces production time and cost compared to conventional genetic barcoding without compromising the stringent PUF criteria of uniqueness and unclonability. This results provide novel insights into the function of TdT and represent a major step toward utilization of PUFs as a biosecurity primitive for cell line authentication and provenance attestation.

PMID:40485605 | DOI:10.1002/advs.202415820

Physiol Plant. 2025 May-Jun;177(3):e70322. doi: 10.1111/ppl.70322.

NO ABSTRACT

PMID:40485589 | DOI:10.1111/ppl.70322

BMC Med. 2025 Jun 9;23(1):340. doi: 10.1186/s12916-025-04172-1.

ABSTRACT

BACKGROUND: In the ongoing opioid epidemic, the mortality risk of opioid initiation in patients with dementia or mild cognitive impairment (MCI) remains understudied despite their vulnerability. This study evaluates mortality risks associated with opioid exposure in patients diagnosed with dementia or MCI by comparing outcomes between the initiation and continuation groups.

METHODS: We conducted a retrospective cohort study using data from a Northern California academic healthcare system (Stanford Health Care Alliance; 2015/01/01-2024/07/31), including 27,757 patients aged 50-100 with dementia or MCI. Of these, 14,105 received opioids after diagnosis and were classified as initiation (opioid-naïve; n=9443) or continuation (non-naïve; n=4662) groups. Cox regression assessed 14-day mortality risk. Aalen's additive model examined time-varying impact up to 180 days. Potential causes of death were extracted from clinical notes using GPT-3.5-Turbo. We also analyzed an independent community healthcare system cohort (Providence Health & Service; n=208,306) from western US states (2015/01/01-2023/05/31) as a replication cohort.

RESULTS: In the primary cohort, 4.1% (572/14,105) of patients died within 14 days of opioid exposure. The initiation group had a significantly higher 14-day mortality risk than the continuation group (adjusted hazard ratio (aHR), 2.00 (1.59-2.52); P<0.0001). The replication cohort had a 14-day mortality rate of 6.2% (7022/113,343) with a smaller difference between the initiation (n=77,168) and continuation (n=36,175) groups (aHR 1.22 (1.16-1.30); P<0.0001). In both cohorts, elevated risk stabilized after day 30. In the primary cohort, respiratory conditions (62% vs. 48%, P<0.1), particularly pneumonia (38% vs. 19%, P<0.05), were more prevalent among the initiation group who died early.

CONCLUSIONS: Starting opioids in patients with dementia or MCI is associated with elevated short-term mortality risks, with the initiation group having twice the 14-day mortality risk in academic settings and a smaller but significant increase in community healthcare systems. The first 30 days after initiation represent a critical risk window, likely due to a lack of tolerance to opioid adverse effects. These findings underscore the need for cautious initiation, tailored follow-up protocols accounting for healthcare setting characteristics, and close monitoring during the first month in this vulnerable population.

PMID:40484923 | DOI:10.1186/s12916-025-04172-1

Cell Rep. 2025 Jun 6;44(6):115792. doi: 10.1016/j.celrep.2025.115792. Online ahead of print.

ABSTRACT

Castration-resistant prostate cancer (CRPC) remains an incurable disease in need of improved treatments. CAMKK2 is an emerging therapeutic target whose oncogenic effects in prostate cancer have, to date, been largely attributed to its activation of AMP-activated protein kinase (AMPK). Here, we demonstrate that CAMKK2 promotes prostate cancer growth through an alternative downstream pathway involving CAMKI and CREB. Unbiased transcriptomics identify CREB-mediated transcription as a CAMKK2-regulated process, findings that we validate using diverse molecular, genetic, and pharmacological approaches in vitro and in vivo. CAMKK2 promotes CREB phosphorylation/activation through CAMKIα independently of AMPK, CAMKIV, or other CAMKI isoforms. Functionally, the CREB family members CREB1 and ATF1 exhibit close redundancy, necessitating co-targeting for optimal anti-tumor efficacy. An inhibitor of CREB1/ATF1 blocks CRPC with minimal side effects. Mechanistically, CAMKK2 and CREB increase CRPC growth through augmenting cholesterol metabolism. Together, these findings identify an oncogenic pathway that could be exploited for the treatment of CRPC.

PMID:40483692 | DOI:10.1016/j.celrep.2025.115792

Neurochem Res. 2025 Jun 8;50(3):190. doi: 10.1007/s11064-025-04440-9.

ABSTRACT

Asparagus racemosus Willd, an Ayurvedic medicine, is known for its antioxidant, antiviral, immune-boosting, and neuro-nutraceutical benefits, particularly in female health. However, its metabolites, mechanisms of action, and target proteins are yet to be fully understood. The present study aimed to identify the metabolite constitution and metabolite-associated proteins in neuroprotective mechanisms in neurodegenerative disease. Mass spectrometry-based untargeted metabolomics and network pharmacology approaches were used to identify metabolites in A. racemosus root extract. In vitro studies, including oxidative stress regulation, neuronal apoptosis, and western blot analysis, were conducted to assess the plant's impact on Alzheimer's disease (AD). We identified 44,014 spectra in positive and negative modes, corresponding to 31,931 non-redundant metabolites at the MS1 level and 5,608 at the MS2 level, from A. racemosus root extract, which include metabolites belonging to phenols, lipids, flavonoids, isoprenoids, and fatty acyls. Novel and known compounds were identified, such as asparagine, sitosterol, arginine, muzanzagenin, pinene, flavone, and kaempferol. Network pharmacology predicted 44 potential human protein targets linked to Alzheimer's disease from these metabolites. These proteins belong to neuromodulator classes, including BACE1, CHRM3, APP, MAP2K1, GSK3B, and TNF, and some of the metabolites of A. racemosus including muzanzagenin interact with BACE1 protein. In vitro validation showed that A. racemosus regulates ROS levels, apoptosis pathways, and BACE1 expression in Alzheimer's disease (AD), highlighting its therapeutic potential. This study integrates network pharmacology and metabolomics, paving the way for clinical research into the neuropharmacological effects of A. racemosus on neurological disorders.

PMID:40483633 | DOI:10.1007/s11064-025-04440-9

Plant Biotechnol J. 2025 Jun 8. doi: 10.1111/pbi.70149. Online ahead of print.

ABSTRACT

The Anaphase-Promoting Complex/Cyclosome (APC/C) is an E3 ubiquitin ligase that plays a crucial role in ubiquitin-dependent proteolysis of key cell cycle regulators, which is completed by the 26S proteasome. Previously, SAMBA, a plant-specific regulator of the APC/C, was identified in Arabidopsis as a critical factor controlling organ size through the regulation of cell proliferation. Here, by assessing its role in the crop tomato (Solanum lycopersicum), we confirm that SAMBA is a conserved APC/C regulator in plants and shows additional roles, including the modulation of fruit shape and changes in sugar metabolism. Two slsamba genome-edited lines were produced and characterized, and showed delayed growth, reduced plant size, and altered fruit morphology, which were linked to changes in cell division and expansion. Notably, untargeted metabolomics revealed altered flavonoid profiles, along with elevated Brix values in the fruits, indicating a sweeter taste. Accordingly, transcriptomics uncovered a change in temporal gene expression gradients during early fruit development, correlating with the alterations in sugar metabolism and revealing changes in cell wall biosynthesis genes. This study provides the first evidence of SAMBA's role in regulating fruit development, metabolic content, and ultimately, quality. These important findings offer potential applications for improving the nutritional quality and overall performance of tomatoes.

PMID:40483582 | DOI:10.1111/pbi.70149

J Physiol. 2025 Jun 7. doi: 10.1113/JP287970. Online ahead of print.

ABSTRACT

Central terminals of primary afferents and dorsal horn neurons usually exhibit spontaneous activity, the two phenomena being interrelated. Spontaneous activity may constitute a system for adjusting the level of excitation of spinal circuits and the processing of somatosensory information. Superficial dorsal horn neurons fire action potentials in a coordinated form, giving rise to population events. These population events are altered by peripheral inflammation, suggesting their implication in central sensitisation. In this work, we aimed to define the role of primary afferents in the occurrence of this coordinated activity. Channelrhodopsin-2, archaerhodopsin-3 or the hM4Di-DREADD receptor were expressed in primary afferents by Cre-recombination under control of the advillin promoter. Dorsal roots and superficial dorsal horn neurons were simultaneously recorded using in vitro spinal cord slices from neonatal mice. Depolarisation of primary afferents by activation of channelrhodopsin-2 inhibited dorsal root activity and the coordinated firing of dorsal horn neurons. DREADD activation reduced the activity in the afferents and depressed coordinated activity in dorsal horn neurons. In contrast, hyperpolarisation of afferents by archaerhodopsin-3 augmented dorsal root responses and increased the coordinated activity of spinal neurons. The present results demonstrate a direct implication of primary afferents in the generation of coordinated spontaneous firing in superficial dorsal horn neurons. KEY POINTS: The input of somatosensory information through primary afferents is a process subjected to regulation at the level of the spinal cord, even before it reaches second-order neurons. Primary afferent and spinal cord neurons exhibit spontaneous activity, which is altered in pathological models of pain. This study demonstrates the role of primary afferents as a fundamental coordinating element for the spontaneous activity of dorsal horn neurons. These results show that modulating the activity of the central terminals of primary afferents may have profound implications in both the excitability of spinal cord circuits and the processing of somatosensory information.

PMID:40483563 | DOI:10.1113/JP287970

Curr Opin Neurobiol. 2025 Jun 6;93:103061. doi: 10.1016/j.conb.2025.103061. Online ahead of print.

ABSTRACT

Generating neuronal diversity from a limited number of neural stem cells is fundamental for the proper functioning of the brain. However, the mechanisms that govern neural fate determination have long been elusive due to the intricate interplay of multiple independent factors that influence a cell's commitment to specific fates. While classical genetics and labeling tools have laid the groundwork for identifying cell types and understanding neural complexity, recent breakthroughs in single-cell transcriptomics and whole-brain connectomics represent a significant advancement in enabling a comprehensive characterization of brain cell types and the underlying mechanisms that encode these neuronal identities. This review focuses on recent developments in our understanding of neural cell fate determination in Drosophila, emphasizing three key mechanisms: spatial patterning, temporal patterning, and neuron-type specific terminal selector transcription factors.

PMID:40482397 | DOI:10.1016/j.conb.2025.103061

J Physiol. 2025 Jun 7. doi: 10.1113/JP286760. Online ahead of print.

ABSTRACT

Ca2+-dependent exocytosis initiates with the formation of fusion pores comprising the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex. Although cellular signalling typically occurs in transient oscillations on the order of tens of seconds, it remains unclear how such rapid SNARE phosphorylation influences fusion pore kinetics, analogous to transient regulation observed in ion channels. Here we demonstrate that protein kinase A (PKA)-mediated phosphorylation of SN25b (the neuronal isoform of synaptosome-associated protein of 25 kD) modulates secretion rate and fusion pore kinetics in PC12 cells (rat pheochromocytoma derivatives). Upon acute application of KCl and forskolin, cells overexpressing SN25b exhibited a reduced secretion rate compared to the control. This reduction was occluded by overexpressing a PKA-phosphodeficient mutant, SN25b-T138A, rather than a PKA-phosphomimetic mutant, SN25b-T138E. Notably, SN25b, SN25b-T138A or SN25b-T138E did not alter the fraction of incomplete fusion events or quantal size compared to the control. Further kinetic analysis indicated that SN25b-T138A destabilized initial fusion pores by promoting the closure and dilatation of fusion pores. Mechanistically, in situ proximity ligation assays showed that SN25b-T138A reduced its interaction with the other t-SNARE syntaxin-1 compared to the control and SN25b, correlating with destabilized fusion pores. Moreover, compared to SN25b-T138E, SN25b-T138A decreased whole-cell Ca2+ currents and weakened its interaction with synaptobrevin-2 and L-type Ca2+ channel subunits. These changes in interaction were associated with increased secretion and full-fusion rate, implying efficient disassembly after dilatation. Together, PKA-mediated phosphorylation of SN25b rapidly modulates fusion pore kinetics in response to transient signalling oscillations, thereby fine-tuning exocytotic efficiency in real time. KEY POINTS: Protein kinase A (PKA)-mediated SNAP-25 phosphorylation rapidly reduces the rate of secretion. PKA-phosphodeficiency of SNAP-25 destabilizes the kinetics of initial fusion pores, correlating with its decreased interaction with syntaxin-1. PKA-phosphodeficiency of SNAP-25 decreases the interaction with synaptobrevin-2 and the L-type calcium channel subunit, leading to efficient priming. PKA-mediated SNAP-25 phosphorylation rapidly regulates fusion pore kinetics and shapes exocytotic kinetics on the order of tens of seconds.

PMID:40481809 | DOI:10.1113/JP286760

Braz J Microbiol. 2025 Jun 6. doi: 10.1007/s42770-025-01707-9. Online ahead of print.

ABSTRACT

Phage therapy is a promising therapeutic weapon in the fight against multidrug-resistant Klebsiella pneumoniae infections. A Klebsiella phage, KpTRp1, was isolated from wastewater sample collected in Tunisia. It exhibited specific lytic activity against a reference strain of K. pneumoniae serotype K2. Phage isolation and enrichment were performed using the double-layer agar method. It was characterized by transmission electron microscopy, physiological investigation and genomic sequence analysis. Transmission electron micrographs of KpTRp1 showed a myovirus morphology. The optimal multiplicity of infection by KpTRp1 was 0.0001 with a latent period lasted for 30 min. Its rise phase showed a burst period of almost 150 min and released a small burst size of about six plaque-forming units per infected cell. KpTRp1 was stable over a broad pH and temperature range. The phage genome sequencing revealed a linear double-stranded DNA genome of 48,003 base pairs with a G + C content of 48.79%, defining a new species of Sircambvirus genus of Jameshumphriesvirinae subfamily. Among the 68 open reading frames identified, about half were hypothetical proteins with unknown functions, the others being involved in DNA replication and metabolism, phage structure, host recognition and lysis. Remarkably, lytic activity of KpTRp1 against K. pneumoniae serotype K2 was confirmed by genomic analysis of life cycle, making it suitable candidate for phage therapy, especially since no antibiotic resistance, or lysogenic genes were detected in the phage genome.

PMID:40481305 | DOI:10.1007/s42770-025-01707-9