FLASH radiotherapy, characterized by ultra-high dose rates (> 40 Gy/s), potentially spares normal tissues while maintaining antitumor efficacy (the FLASH effect). While electron and proton FLASH are explored, pulsed X-ray sources like plasma focus devices offer unique possibilities. Previous work has reported hyper-radiosensitivity in colorectal cancer cells exposed to ultra-high-dose-rate pulsed X-rays from a kilojoule plasma focus (PF) device, without significant effects on non-cancerous cells. This study further investigates the biological effects of ultra-high-dose-rate (~ 10⁷ Gy/min), low-total-dose pulsed X-rays generated by a PF-2 kJ device on colorectal cancer cell lines, focusing on DNA damage, cell cycle progression, and gene expression. Low-total-dose (~ 0.25 Gy), ultra-high-dose-rate pulsed X-rays (0.025 Gy/pulse, a total of 10 pulses, pulses temporally separated by 15–20 s) generated by a PF-2 kJ device induced a significant increase in the SubG1 population in HCT116 and DLD1 cells over 72 h, an effect indicative of apoptosis, which was not observed with conventional X-rays at similar total doses. In addition, pulsed X-rays induced apoptosis in radioresistant MCF-7 breast cancer cells. Whereas conventional X-rays did not cause a significant increase in double-strand breaks (DSBs), surrogate marker γ-H2AX and phosphor-P53(Ser15) signal were detected 30 min following pulsed X-ray exposure and persisted for up to 24 h, and no evidence of G2/M cell cycle arrest was detected in exposed cells. Gene expression analysis and preliminary transcriptomic data further suggest a DNA damage response leading to cell death and global change in general biological processes related to regulation of gene expression. Low-total-dose (~ 0.25 Gy), ultra-high-dose-rate pulsed X-rays generated by a PF-2 kJ device induce significant and sustained DNA damage (DSBs) leading to increased apoptosis in colorectal (HCT-116, DLD-1) and breast (MCF-7) cancer cells, compared to conventional X-rays. These effects, coupled with distinct changes in gene expression, suggest that ultra-high-dose-rate pulsed X-rays may overcomeradio-resistancee without eliciting a conventional DNA damage repair or cell cycle checkpoint response. These findings support the potential of PF-generated pulsed X-rays as a novel sourcof e radiotherapy modality and warrant further investigation, particularly in in vivo models, to assess clinical applicability and safety.

Cortical layer 1 (L1) is a key site for integrating top-down and bottom-up information and is populated by inhibitory interneurons, including vasoactive intestinal peptide (VIP)-expressing cells. These interneurons regulate information flow across the cortical column by disinhibiting pyramidal neurons, yet the subthreshold ionic mechanisms that shape their excitability in the medial prefrontal cortex (mPFC) remain poorly understood. Here, we characterize the electrophysiological properties of L1b VIP interneurons in mouse mPFC, focusing on the role of the hyperpolarization-activated cation current (Ih) and inward-rectifying potassium current (Kir). Using whole-cell recordings in acute slices, we find that L1b VIP interneurons exhibit constitutively active Ih and Kir conductances. Inhibition of Ih with ZD-7288 hyperpolarized the resting membrane potential (RMP), increased input resistance (Rin), and prolonged action potential (AP) duration without altering rheobase or firing frequency, while increasing EPSP-spike coupling probability. Blocking Kir with BaCl2 depolarized the RMP, increased Rin and membrane time constant, reduced rheobase, increased firing frequency, and similarly enhanced EPSP-spike probability and AP duration. Voltage-clamp experiments confirmed the presence of ZD-7288-sensitive Ih and BaCl2-sensitive Kir currents of small amplitude but operating on a high-resistance membrane, consistent with a strong impact on excitability. In contrast, neither Ih nor Kir inhibition affected the amplitude or frequency of spontaneous or miniature EPSCs, indicating that these currents do not measurably alter basal excitatory synaptic transmission. Immunofluorescence revealed weak somatic HCN1 and no detectable HCN2 expression in L1b VIP interneurons. Notably, Kir inhibition unmasked an Ih-dependent voltage sag during hyperpolarizing steps, suggesting that constitutive Kir activity normally masks Ih recruitment at subthreshold potentials. Together, these results indicate that Ih and Kir are active near RMP in L1b VIP interneurons and jointly regulate their passive properties, intrinsic excitability, and EPSP-spike coupling, therefore shaping how L1 VIP cells filter incoming signals and influence information flow within the prefrontal cortical column.

Mitral regurgitation is highly prevalent and elevates the risk of heart failure. Regurgitant flow induced shear stress disrupts Ca2+ homeostasis in atrial cardiomyocytes. Overloaded Ca2+ is a crucial regulator of endoplasmic reticulum stress-mediated apoptosis, although the regulatory mechanisms remain unclear. We aimed to explore the relationship between shear stress and Ca2+ homeostasis. Through employing an in vitro model replicating atrial cardiomyocytes during mitral regurgitation, we observed that shear stress increased Ca2+ oscillation and the amplitude of Ca2+ waves through extracellular Ca2+ influx. Suppression of mechanosensitive Ca2+ channels or store-operated calcium entry resulted in decreased intracellular Ca2+ concentration and oscillation. Conversely, the inhibition of voltage-gated Ca2+ channels did not significantly impact the Ca2+ concentration. Remarkably, shear stress elevated the expression of endoplasmic reticulum stress and apoptosis-related proteins in HL-1 cells in a time-dependent manner. Also, in a novel rodent mitral regurgitation model, shear stress increased apoptosis and endoplasmic reticulum stress expression compared to sham rats. Our findings underscore that shear stress induces apoptosis through mechanosensitive and store-operated Ca2+ channels, leading to Ca2+ overload and endoplasmic reticulum stress. Understanding the mechanisms behind shear stress-disturbed Ca2+ homeostasis may enhance the treatment of mitral regurgitation-induced heart failure.

Translation is an extremely fine-tuned process. Both speed and fidelity are required to sustain translational demand and protein homeostasis in cells, traits that depend on several elements, including transfer RNA (tRNA) modifications. Additionally, it has been demonstrated that proliferative and differentiated tissues display differential susceptibilities to deficiencies in certain tRNA modifications. By using the Gal4/UAS system we manipulated the expression levels of the Drosophila melanogaster orthologue of TrmO (dTrmO), a tRNA-methyltransferase that methylates t6A in position 37 of tRNAs decoding ACY codons, during development. Our results show that this protein is necessary for proper translation of ACT codons, and its function is differentially required in proliferative and non-proliferative tissues. Furthermore, we observed that reductions of dTrmO are detrimental for cell growth and proliferation, and muscle function due to impaired translation of Msp300, a nuclear membrane protein specific of muscle fibres. Strikingly, the translation-related phenotypes produced caused by decreased levels of dTrmO in the wing and eye-antenna imaginal disc, fat body, striated muscles, as well as overall organismal growth defects, can be rescued by overexpression of tRNA-Thr-AGT. Results point to the importance of dTrmO in translation of ACT codons and consequently in cell proliferation and growth of tissues, thus providing further insight about the active role of tRNA modifications in the context of eumetazoan development.

To summarize the role of endoplasmic reticulum stress (ERS) in the pathogenesis of diabetic retinopathy (DR) and evaluate potential ERS-targeted interventions. This review analyzes recent preclinical and clinical studies focusing on the molecular mechanisms of ERS and its impact on retinal inflammation, oxidative stress, and angiogenesis in DR. ERS, triggered by hyperglycemia-induced oxidative stress and glucotoxicity, activates the unfolded protein response (UPR) via inositol-requiring enzyme 1 (IRE1), PKR-like endoplasmic reticulum kinase (PERK), and activating transcription factor 6 (ATF6) pathways. While initially protective, prolonged ERS leads to apoptosis, chronic inflammation, and neovascularization. Key downstream mediators include C/EBP homologous protein (CHOP), X-box binding protein 1 (XBP1), and activating transcription factor 4 (ATF4). ERS inhibitors such as 4-phenylbutyric acid and tauroursodeoxycholic acid, along with selective modulators of UPR signaling, have shown neuroprotective and anti-inflammatory effects in DR models. Combination therapies integrating antioxidants and anti-inflammatory agents demonstrate synergistic efficacy. However, clinical translation remains limited by delivery barriers and incomplete understanding of UPR-specific actions in the human retina. Targeting ERS presents a promising therapeutic strategy for DR, with the potential to preserve vision and improve outcomes for diabetic patients. Future research should focus on elucidating the precise molecular pathways and developing targeted, personalized ERS-modulating therapies.

The complex interaction between the immune system and metabolic homeostasis is becoming recognized, as immune sensors affect key metabolic tissues, including the liver and adipose tissue. The cGAS-cGAMP-STING pathway, previously recognized as a cytosolic DNA-sensing pathway, is currently associated with lipid metabolism in addition to its inflammatory function. Although STING is acknowledged for its connection to cholesterol, the metabolic functions of its upstream component molecules-the DNA sensor cGAS and the resulting product cGAMP-are largely unexplored. We propose that cGAS and cGAMP serve as crucial, previously unidentified regulators of systemic lipid homeostasis throughout the lifetime. We investigated the long-term metabolic consequences of intrinsic cGAS deficiency, leading to the absence of cGAMP, in male mice fed on a standard chow diet. cGAS knockout (cGASKO) mice demonstrated a consistent increase in body weight across their lifespan, primarily attributed to adipocyte hypertrophy and increased adipose tissue mass. Increased weight correlated with elevated adiposity. This condition was associated with reduced weight-bearing strength, despite unchanged general locomotor activity and food intake during young age. Liver histology revealed modest cellular infiltration and absent steatosis, suggesting potential low-grade inflammation. Circulating triglyceride and glucose levels exhibited transient, age-dependent variations-decreased glucose and increased triglycerides in young age, which stabilized in adult and old ages, reflecting a possible compensatory metabolic adaptation with time. Conversely, total cholesterol levels were consistently and significantly elevated across all age groups, underscoring the importance of the cGAS-cGAMP axis in cholesterol homeostasis throughout life. Our study identified the cGAS-cGAMP axis as an interesting regulator of cholesterol homeostasis and fat accumulation in aging, independent of STING activation. The persistent deficiency of cGAS and cGAMP leads to lifelong hypercholesterolemia and adipose hypertrophy. This research highlights an unexpected metabolic function of the cGAS-cGAMP pathway and indicates the necessity of evaluating this axis in relation to physiological aging and metabolic disorders.

BackgroundCarbapenemase-mediated resistance to carbapenems is a significant public health concern due to its potential for widespread dissemination. The KPC family of carbapenemases, encoded by the blaKPC gene and often associated with Tn4401-like transposons, is particularly important for its ability to be transferred through diverse plasmid types. In Chile, KPC-producing Gram-negative bacteria have been detected in clinical settings; however, their occurrence in wastewater (WW) remains unknown. This study addresses this gap by characterizing carbapenem-resistant Enterobacterales isolates from a wastewater treatment plant (WWTP) in the Gran Concepción Metropolitan Area, Chile.ResultsThis study identifies three carbapenem-resistant Enterobacterales isolates, namely Klebsiella pasteurii M2/A/C/34, Klebsiella pneumoniae subsp. pneumoniae M3/A/M/3, and Citrobacter freundii sensu stricto. M4/A/C/32, all exhibiting multidrug-resistant profiles and carrying the blaKPC-2 gene encoding KPC-like carbapenemases. These isolates also possessed genes for extended-spectrum β-lactamases (ESBLs) and aminoglycoside-modifying enzymes (AMEs). Sequence typing revealed that M2/A/C/34, M3/A/M/3, and M4/A/C/32 belonged to novel sequence types, specifically ST470, ST273, and ST214, respectively. All isolates carried plasmids belonging to groups commonly associated with ARGs, including IncF, IncP, and IncA. Both Klebsiella isolates (M2/A/C/34 and M3/A/M/3) carried the class 1 integron (intl1) gene. Phylogenomic analysis reveals that M2/A/C/34 is related to strains from China and Pakistan, while M3/A/M/3 shares similarities with a strain from Germany, indicating their potential dissemination.ConclusionsThis study represents the first detection of carbapenem-resistant Enterobacterales carrying blaKPC-2 in Chilean WW, including the novel identification of K. pasteurii. These findings emphasize the critical role of genomic surveillance in WW under the One Health framework, enabling the monitoring of carbapenemase-producing bacteria and associated ARGs. Sustained surveillance efforts are essential to comprehend the dynamics of antibiotic resistance in environmental reservoirs and to develop strategies for its containment and mitigation.