Astrocytes are essential regulators of central nervous system (CNS) homeostasis, and their dysfunction can amplify neuroinflammation and neurodegeneration. HIV and methamphetamine (Meth) are known to downregulate β-catenin signaling and induce astrocyte senescence, but whether this senescent state is functionally pathogenic and contributes directly to neuroinflammatory processes remains unclear. Here, we investigated how senescent astrocytes respond across prototypical astrocytic functions.In vitro, human iPSC-derived astrocytes infected with HIV and/or exposed to Meth developed a senescence phenotype (increased p16INK4A) accompanied by reduced expression of the phagocytic receptor MEGF10 and impaired engulfment of apoptotic neurons. HIV/Meth exposure also shifted astrocyte secretomes toward proinflammatory profiles (IL-6, CCL2, CXCL1, and ICAM-1), and astrocyte-conditioned media decreased neuronal PSD95 and NFL and disrupted endothelial adherens and tight junction proteins (VE-cadherin, occludin, claudin-5), resulting in increased monocyte transmigration. Mechanistically, pharmacologic activation or lentiviral expression of active β-catenin protected astrocytes from senescence, preserving MEGF10 expression and phagocytic capacity under HIV/Meth exposure, while MEGF10 overexpression independently restored phagocytosis. In a human-mouse chimera model (NSG mice xenotransplanted with human iPSC-derived astrocytes), HIV infection and/or Meth administration increased p16INK4A and reduced MEGF10 expression in engrafted human astrocytes, recapitulating in vitro findings.Together, these studies demonstrate that HIV and Meth suppress β-catenin signaling to drive a functionally disruptive astrocyte senescence program linked to impaired MEGF10-dependent phagocytosis, diminished neuronal support, and compromised blood-brain barrier integrity. Restoring β-catenin signaling and preserving MEGF10 function emerge as rational strategies to prevent astrocyte-driven neuroinflammation and neuropathogenesis in HIV/Meth co-morbidity.
The nucleolus, a membrane-less organelle within the nucleus, plays a fundamental role in ribosome biogenesis with cellular functions across diverse physiological and pathological states. While its multilayered, liquid-like architecture is formed by complex interactions between nucleolar proteins and nucleic acids, the regulatory mechanism of nucleoplasmic components on nucleolus remains poorly understood, particularly heterochromatin and its associated proteins. In this study, we revealed that Polycomb repressive complex 2.1 (composed of PRC2 core subunits plus Polycomb-like (PCL) homolog), associating with H3K27me3-enriched heterochromatin, accumulates surrounding the nucleolus to form a ring-like structure. Notably, as a key PRC2.1 component, PCL2 undergoes phase separation, while the intrinsically disordered region (IDR) in PCL2 regulates both the fluidity of the condensates and its interaction with nucleolar protein nucleophosmin (NPM1). PCL2 is a key regulator to facilitate NPM1 pentamerization and interaction among nucleolar components, ensuring the efficient progression of rRNA and protein synthesis. Taken together, our findings reveal that PRC2.1(PCL2) plays a crucial role in maintaining nucleolar integrity and rRNA synthesis, highlighting that PRC2.1(PCL2) is required for cell proliferation.
The biological function of the cell membrane is significantly affected by a disrupted lipid bilayer. Maintaining the structural integrity of the lipid bilayer is hence crucial. In this work, a series of molecular dynamics (MD) simulations were carried out by varying ethanol and glucose concentrations to investigate the counteracting effects of glucose concentration on the ethanol-stressed disorganized hydrated model lipid bilayer, 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). Our work highlights that the ethanol's concentration-dependent disruptive role affects bilayer integrity, as evidenced by increased lateral diffusion, reduced bilayer thickness, and enhanced disorder in the structural arrangements of the lipids, including the surface curvature order parameter. We observed that the addition of glucose, to some extent, opposes such disorganization. Our investigation reveals that glucose forms substantial hydrogen bonds with lipid headgroups, thereby reducing ethanol-headgroup interaction and promoting tighter lipid packing. The minimum-distance distribution functions suggest that, while water is excluded from the lipid tail, ethanol is found all the way towards the lipid tails, and the terminal methyl groups interact significantly, causing the lipid tails to bend toward the polar regions of the bilayer. This phenomenon however was observed less in the presence of glucose. An inverse relationship between glucose concentration and translational mobility is consistently observed across all ethanol concentrations studied, while higher ethanol content facilitates glucose mobility, regardless of the glucose concentration. This suggests a complex interplay between glucose and ethanol that modulates the diffusive dynamics of the solvent and solute, affecting lipid diffusion and its structural integrity. The computation of the potential of mean force (PMF) using the umbrella sampling technique highlights that the ethanol penetration process is significantly less favored and energetically hindered in the presence of glucose; as a result, lipid diffusion is slowed down. Our findings provide insights into the molecular mechanisms and underscore the protective role of glucose in stabilizing lipid bilayers under alcohol-induced stress. The study is relevant to various pharmaceutical and biomedical applications, as well as the design of biopreservation strategies for cells and tissues.
The skin is far from a passive shield; it functions as a dynamic "living barrier" whose structural and immunological integrity is paramount in preventing atopic dermatitis (AD) and the subsequent progression of the atopic march toward food allergy (FA). While prophylactic emollient therapy has emerged as a promising strategy for primary prevention, recent large-scale randomized controlled trials have yielded conflicting results. In this review, we examine the complex interplay between emollient formulations, skin barrier biology, and immune responses in the context of AD and FA prevention. We discuss the functional roles of occlusives, humectants, and barrier-replenishing lipids, highlighting how their composition, ratios, and physicochemical properties determine their effects on barrier integrity. We further evaluate the impact of additional formulation components, including food-derived ingredients, haptens, irritants, and pH, as well as external factors such as product handling and application practices, which may inadvertently promote barrier disruption and transcutaneous sensitization. Collectively, current evidence supports a shift from generalized emollient use toward a precision-based, barrier-directed approach. Optimized formulations that restore physiological lipid composition, combined with early intervention and integration of objective biomarkers, may offer a more effective strategy to prevent AD and the atopic march.
Lectins are carbohydrate-binding proteins that recognize specific glycan patterns on cell surfaces and have been increasingly explored as analytical tools in reproductive biology. This study characterized the interaction of a lectin isolated from Abelmoschus esculentus seeds (AEL) with ovine spermatozoa and evaluated its compatibility with sperm functional parameters during short-term refrigeration. Eighteen ejaculates were diluted in a Tris-egg yolk extender supplemented with 0, 1, or 3 µg/mL AEL and stored at 5°C for 24 h. Sperm motility (CASA) and flow cytometry were used to assess plasma membrane integrity and fluidity, mitochondrial membrane potential, and reactive oxygen species production. Confocal microscopy was performed to determine the localization pattern of AEL binding. AEL labeling was predominantly restricted to the sperm midpiece region. Supplementation with AEL at the tested concentrations did not affect motility patterns, membrane integrity, mitochondrial membrane potential, or ROS levels after 24 h of storage (p > 0.05). These findings indicate that AEL exhibits a consistent midpiece-associated binding pattern and does not compromise sperm functional parameters under refrigerated conditions. The results support the use of AEL as a compatible probe for investigating glycan distribution in the midpiece region of ram spermatozoa.
Intestinal barrier dysfunction is a key driver of septic liver injury (SLI). Dachaihu decoction (DCHD), a classic traditional Chinese medicine formula recorded in the Treatise on Cold Damage, is widely used to treat gastrointestinal and hepatic inflammatory conditions. The primary objective of our research was to elucidate the protective effects of DCHD against SLI and the underlying molecular mechanisms. In a murine model of sepsis induced by cecal ligation and puncture (CLP), we evaluated the therapeutic effects of DCHD on SLI by assessing serum liver enzymes, histopathology, oxidative stress, hepatocyte apoptosis, and inflammatory cytokines. Intestinal barrier integrity was examined via transmission electron microscopy, serum biomarkers (D-lactate, DAO, LPS), and tight junction proteins (ZO-1, Occludin, E-cadherin). Gut microbiota composition was analyzed using 16S rRNA sequencing. Chemical profiling of DCHD was performed via UPLC-Q-TOF-MS. Integrated network pharmacology, bioinformatics, and transcriptomic analyses identified the NF-κB/NLRP3/Caspase-1 axis as a potential mechanism, which was validated in vivo and in LPS-stimulated immortalized mouse Kupffer cells (ImKCs). Functional involvement of TLR4 and NLRP3 was further confirmed by genetic silencing of TLR4 with siRNA and pharmacological inhibition using TAK-242 (TLR4 inhibitor) and MCC950 (NLRP3 inhibitor). DCHD treatment attenuated liver injury in CLP-induced septic mice, as evidenced by improved liver function, attenuated histopathology, reduced oxidative stress, suppressed inflammation, and decreased hepatocyte apoptosis. These hepatoprotective effects were associated with reduced intestinal permeability and enhanced barrier integrity, alongside gut microbiota remodeling characterized by enrichment of beneficial bacteria and reduced abundance of gram-negative genera (e.g., Klebsiella, Enterobacter, Proteus), leading to decreased LPS production and translocation to the liver. Integrated network pharmacology and transcriptomics revealed the NF-κB/NLRP3/Caspase-1 axis as a central mechanism, with DCHD downregulating p-p65, p-IκBα, NLRP3, ASC, and Cleaved Caspase-1 in vivo and in LPS-stimulated ImKCs. Functional validation using TLR4 siRNA and the inhibitors TAK-242 and MCC950 confirmed that DCHD might attenuate liver inflammatory injury primarily through the NF-κB/NLRP3/Caspase-1 signaling pathway. DCHD may alleviate SLI by enhancing the intestinal barrier, potentially reducing the translocation of gut-derived LPS to the liver, and subsequently inhibiting the NF-κB/NLRP3/Caspase-1 axis, highlighting its considerable translational potential for SLI therapy.
Tracheal diverticulum is frequently detected incidentally on cross-sectional imaging but may pose a significant intraoperative challenge when anticipated within or adjacent to the thyroid surgical field, particularly in close proximity to the recurrent laryngeal nerve (RLN) and during central compartment (level VI) dissection. Conventionally, surgeons use preoperative computed tomography (CT) to approximate diverticular anatomy and then rely on intraoperative experience and anatomic landmarks for dissection. Recognition is challenging because intraoperative localization relies on CT correlation and anatomic landmarks within the posterior capsular plane; limited exposure may obscure the diverticulum, and a non-aerated sac may collapse and become indistinguishable from surrounding soft tissue, increasing the risk of rupture and subsequent loss of tension with further obscured planes and potential operative-field contamination. However, a standardized intraoperative workflow to functionally localize the diverticulum (and confirm tracheal communication) before definitive posterior dissection, and to verify tracheal integrity after diverticulectomy and repair, is lacking. Here, we describe a stepwise surgical technique for planned thyroidectomy in which a cervical tracheal diverticulum has been identified preoperatively. The workflow integrates preoperative contrast-enhanced CT-based risk stratification with a ventilation-assisted functional visualization maneuver [standardized endotracheal tube (ETT) repositioning within recorded cuff-depth boundaries 'D_cuff, the ETT incisor depth at which the proximal cuff margin is visualized just distal to the vocal cords; D_cuff2, the deeper ETT incisor depth after advancement beyond the diverticular level to keep the cuff distal to the diverticular ostium'] to localize the diverticular sac at the CT-predicted region and confirm tracheal communication before definitive posterior dissection. After localization, posterior dissection proceeds with deliberate RLN identification and protection, followed by controlled diverticulectomy and submerged air-leak testing under gentle manual ventilation to verify tracheal wall integrity.
Breast conservation therapy in the setting of breast cancer has been shown to be a safe method for oncologic treatment in certain patients. However, lumpectomy and radiation have historically been associated with contour deformity, asymmetry, and suboptimal aesthetic outcomes. Oncoplastic breast reconstruction is an approach to breast-conserving surgery that includes reconstruction of partial mastectomy defects in order to minimize the deformity caused by treatment. These techniques serve to optimize breast aesthetics and symmetry in the oncologic setting and have improved patient satisfaction in the setting of breast conservation. There are several key points in the planning and execution of oncoplastic surgery as well as secondary procedures to further improve outcomes. Here, the authors present an overview of patient selection, multidisciplinary planning, and surgical technique for primary and secondary reconstruction, with a focus on specific methods of improving outcomes with autologous fat transfer. This review includes an overview of the literature focusing on patient-reported outcomes (PROs), as these play an important role in a surgeon's postoperative assessment of oncoplastic breast reconstruction. Overall, patient satisfaction is high and the oncologic integrity is preserved. Clinical cases are used to illustrate conceptual framework, pertinent anatomy of various techniques, and preoperative and postoperative comparisons.
Current biodegradable polymers for absorbable tissue ligation clips suffer from not possessing rigidity and toughness simultaneously compared with polyoxymethylene (POM) clips. To overcome the limitations, two series of biodegradable copolymers of poly(ε-caprolactone) and poly(L-lactide) with random and block architectures were designed and synthesized. Compared with random copolymer P(CL-r-LLA), diblock copolymer PCL-b-PLLA exhibited higher crystallinity and superior mechanical strength. The copolymers with 15 mol% CL composition were selected for further study, denoted as RP15 (random) and BP15 (block). The BP15 clips achieved a closure force of 27.9 ± 1.4 N, comparable to commercial POM clips (29.0 ± 2.4 N), while also offering biodegradability. The degradation studies showed that BP15 clips degraded slowly, maintaining structural integrity and sustained closure functionality for up to two weeks, which was essential for safe tissue ligation. Subcutaneous implantation in rats further confirmed that BP15 clips exhibited favorable performances, demonstrating their potential as a promising biodegradable alternative to POM for use as an absorbable tissue ligation clip.
Left transcervical extrapleural esophageal mobilization can avoid pleural entry and one-lung ventilation but is limited by challenging exposure of the right paratracheal compartment and a relevant risk of left recurrent laryngeal nerve (RLN) injury. Prior bilateral transcervical approaches have been constrained by instrument collisions and limited caudal reach. In a fresh-frozen cadaver, a right-first, bilateral transcervical single-port (SP) robotic strategy was performed with sequential right-then-left cervical docking under low-pressure pneumomediastinum. Feasibility endpoints were predefined: (i) reproducible access to the paratracheal and subcarinal planes, (ii) identification and preservation of the RLNs, (iii) maintenance of an extrapleural corridor to the diaphragmatic hiatus, and (iv) collision-free workflow. Right-sided transcervical docking enabled near-circumferential thoracic esophageal mobilization with early identification of the right RLN and systematic mediastinal lymphadenectomy, including reliable exposure of the subcarinal station. The SP configuration allowed stable traction with two working instruments, facilitating controlled dissection without relevant internal or external collisions. The subsequent left-sided phase focused on completion of residual mobilization and targeted lymphadenectomy along the left RLN with minimal traction and limited energy application. All predefined feasibility criteria were met. A threshold-guided (~ 85%) right-first bilateral transcervical SP robotic approach is feasible in a cadaveric model. This strategy improves access to the right paratracheal nodal stations and may reduce traction on the left RLN by shifting traction-intensive steps to the right. Prospective clinical translation is warranted to benchmark RLN outcomes, nodal yield, pleural integrity, and recovery against current transcervical and transthoracic techniques.
The cellular DNA damage response (DDR) is essential for maintaining genome integrity in the face of exogenous assault, as well as errors and breaks introduced during DNA replication. The DDR is a complex network of cellular processes that detects the type and severity of DNA damage and coordinates downstream actions. These include activating specific DNA repair pathways, including cell cycle arrest to allow time for repair, and-if the damage is irreparable-triggering apoptosis to eliminate cells with compromised genomes. DNA viruses have evolved multiple strategies to evade, suppress, or even hijack host DDR factors to promote their own replication. In the absence of such countermeasures, the activation of the host DDR may rapidly detect viral genomes as aberrant DNA, and restrict viral infection. Paradoxically, many DNA viruses depend on many aspects of the host DDR to ensure efficient replication of their genomes. Here, we focus on human cytomegalovirus (HCMV), a beta-herpesvirus with a large complex double-stranded DNA genome, and its relationship with host DDR. Although HCMV infects most of the human population, fundamental gaps remain in understanding how viral replication modulates a robust cellular DDR or recruits it for its own replication. Moreover, the roles of DDR in restricting viral DNA (vDNA) replication, promoting entry into latency, stabilizing latent genomes during cellular proliferation, and promoting vDNA synthesis following infection or reactivation from latency remain poorly understood.
Apolipoprotein M (ApoM), a sphingosine-1-phosphate (S1P) binding protein, is primarily synthesized in the liver and kidney. It is a key component of high-density lipoprotein (HDL) and plays a pivotal role in reverse cholesterol transport, vascular homeostasis and anti-inflammatory responses via the ApoM-S1P axis. Dysregulation of ApoM expression or S1P metabolism is associated with cardiovascular, metabolic and inflammatory disorders, highlighting the importance of understanding its structural and functional dynamics. Despite extensive knowledge of individual transcriptional and post-transcriptional changes in ApoM, the coordinated mechanisms integrating these signals and the impact of naturally occurring non-synonymous single-nucleotide polymorphisms (nsSNPs) remain poorly understood. This study systematically investigated the structural and functional impact of nsSNPs in ApoM using an integrated in silico approach. From 4097 variants, 162 nsSNPs were prioritized, with A51S, F63V and R89S identified as highly deleterious. A51S disrupts a conserved hydrophobic core, resulting in a compact and less dynamic structure with reduced flexibility. F63V induces severe destabilization by altering β-turn integrity and binding pocket architecture, leading to increased solvent exposure and conformational heterogeneity. In contrast, R89S enhances hydrogen bonding and maintains a more stable conformation. These mutation-oriented effects are predicted to influence S1P binding and HDL function. Overall, the findings provide mechanistic insight into ApoM variant-induced dysfunction and establish a framework for prioritizing functionally significant mutations.
Traditional classification of traumatic brain injury (TBI) has relied predominantly on neurological responsiveness, most notably the Glasgow Coma Scale, to stratify injury severity. While this approach has provided a common clinical language for decades, it inadequately captures the biological heterogeneity, anatomical variability, and contextual factors that shape injury trajectories and outcomes. Growing recognition of these limitations has prompted the development of multidimensional classification frameworks, including the clinical, biomarker, imaging, and modifier (CBI-M) model, designed to reflect the complex and dynamic nature of TBI. The objective is to describe a structured, implementation-science-based methodology for transitioning from traditional TBI classification to the CBI-M framework while preserving clinical continuity, data integrity, and system feasibility. We propose a phased transition methodology grounded in implementation science principles, emphasizing backward compatibility, additive integration, reproducibility, scalability, and distributed responsibility. The methodology is organized into three sequential phases: (I) mapping legacy classification elements to CBI-M domains to ensure conceptual continuity; (II) operational integration through workflow-aligned, staged adoption across the trauma care continuum; and (III) system-level embedding via electronic medical records, trauma registries, governance structures, and continuous evaluation mechanisms. Explicit attention is given to data stewardship, accountability, and longitudinal consistency. The proposed methodology provides a practical roadmap for embedding multidimensional TBI classification into routine clinical practice without disrupting existing workflows or registries. By aligning classification domains with established clinical roles and care phases, the approach minimizes documentation burden while enabling incremental adoption on the basis of institutional readiness. Governance and audit mechanisms support fidelity, comparability, and sustainability over time. Transitioning to multidimensional TBI classification requires more than conceptual validity; it demands deliberate implementation strategy. The proposed methodology offers a scalable, system-aware pathway for integrating the CBI-M framework into real-world trauma care, supporting precision-oriented classification while maintaining continuity with legacy systems. This approach provides a foundation for durable adoption, quality improvement, and future research in traumatic brain injury.
Prostate-specific membrane antigen (PSMA) is a validated target for prostate cancer imaging and radiotherapy, with most high-affinity ligands incorporating the lysine-urea-glutamate (KuE) pharmacophore. Despite emerging triphosgene-free approaches, current synthetic strategies still largely rely on hypertoxic triphosgene for asymmetric urea formation. Herein, we report a comparative evaluation of triphosgene-free acyl-transfer reagents for on-resin KuE formation under solid-phase peptide synthesis (SPPS) conditions. Among the reagents examined, 1,1'-carbonyldiimidazole (CDI) emerged as the most effective, enabling near-quantitative urea formation under mild, SPPS-compatible conditions. Application of the optimised CDI-mediated protocol on TentaGel® resin bearing an HMPB linker enabled efficient fully solid-phase assembly of PSMA-617 in >80% crude purity and 31% isolated yield. Purified PSMA-617 was radiolabelled with 68Ga in >99% radiochemical purity, confirming chemical integrity. Additionally, the PSMA-I&T backbone and four PSMA-617 analogues were synthesised in high crude purities, demonstrating rapid on-resin ligand construction for structure-activity relationship (SAR) studies.
Although space travel is becoming more accessible, our understanding of how the space environment and microgravity (μG) affect biology, physiology, and human health remains incomplete. This study examined the effects of μG on synaptic signaling and neuromuscular aging in Caenorhabditis elegans. The D01 cohort, consisting of L4 larvae to young adults raised in μG, exhibited a downregulation of genes linked to synaptic signaling, dopamine response, locomotion, cuticle development, and mitochondrial metabolism. This was accompanied by altered synapse dynamics, reduced motility, and shorter body length. In μG, aged worms showed a reduction in collagen gene expression, increased abnormalities in motor neuron morphology, changes in synaptic vesicle dynamics, and a collapse of mitochondrial morphology in body wall muscles, highlighting exacerbated aging-like phenotypes. The gentle-touch mechanoreceptor MEC-4 was identified as a key mediator of μG-induced body length reduction and changes in extracellular matrix gene expression. mec-4 mutants did not show μG-associated body shortening. The expression of most mechanoreceptor genes, including stretch-activated channels unc-105 and del-1, was downregulated under μG conditions. Notably, the expression of tmc-1 and degt-1 mechanoreceptor genes was downregulated independently of MEC-4. Restoration of physical stimulation using culture medium with small beads in space mitigated many μG-induced neuromuscular defects and expression alterations including those in mechanoreceptor genes. These results highlight the role of mechanical stimuli in maintaining neuromuscular integrity during spaceflight and suggest that restoring tactile input could counter health risks from reduced tactile stimulation during long-term space missions.
Highly crystalline single-walled carbon nanotubes were employed as robust supports for carbon-encapsulated PtPd alloy electrocatalysts synthesized via a rapid, industrially scalable solution plasma method, enhancing long-term durability under frequent start-up/shut-down conditions. Electrochemical evaluation of PtPd@C/SWCNT as a cathode catalyst in a polymer electrolyte fuel cell (PEFC) membrane electrode assembly (MEA) demonstrated superior durability compared to commercial Pt/C and monometallic Pt@C/SWCNT under an accelerated durability test. PtPd@C/SWCNT maintained high performance for 5000 potential cycles and retained over 50% of its electrochemically active surface area (ECSA) after 10 000 cycles under the accelerated durability test of high-potential triangular pulses (1.0-1.5 V) simulating harsh conditions encountered during actual start-up/shut-down operations. Carbon encapsulation effectively inhibited nanoparticle agglomeration and suppressed the oxidation of the SWCNT support in close proximity to the nanoparticles during the durability test of 30 000 cycles. Raman spectroscopy confirmed the excellent corrosion resistance and maintained the crystallinity of the SWCNT support. The negligible thickness change observed in the PtPd@C/SWCNT cathode layer further highlights the benefit of the SWCNT support and carbon encapsulation in maintaining structural integrity under severe operating conditions. XPS analysis indicated a more stable, reduced state of Pt in PtPd@C/SWCNT compared to that of Pt/C. These results highlight the synergistic effects of the SWCNT support and carbon encapsulation in improving both catalyst stability and support durability for prolonged PEFC operation, particularly under demanding heavy-duty vehicle (HDV) conditions.
Diversity, equity, and inclusion (DEI) are foundational values for cultural psychiatry. The current assault on DEI in the United States and other countries represents a direct challenge to the scientific, ethical, and clinical commitments that have guided progress in mental health over recent decades. This paper examines the history, rationale, and successes of DEI frameworks in health care and psychiatry, discusses legitimate critiques, documents the scope and consequences of the current anti-DEI movement in the United States and other jurisdictions, and draws comparisons with Canadian and United Kingdom contexts. We revisit each component of DEI-diversity, equity, and inclusion-to clarify its scientific grounding and moral significance for mental health. We argue that, rather than dismantling DEI, cultural psychiatry and allied disciplines must respond with renewed commitment to the values of DEI as well as those of dignity, empathy, and integrity, as ways to advance social justice and pluralistic civil society.
The pathogenicity of P. aeruginosa is not only mediated by its resistance mechanisms but also by its ability to form persister cells. The persister state is a transient condition in which cells retain a dormant, non- or slow-growing state during exposure to stressors, such as high doses of bactericidal antibiotics, but can resume growth once the stress is removed. In this study, we investigated in vitro the virulence phenotype and stress-induced modulation of persisters in P. aeruginosa PA14 exposed to 30 × the minimum inhibitory concentration (MIC) of imipenem (60 µg/mL) or ciprofloxacin (3.75 µg/mL). Using a biphasic killing assay, we detected persisters with survival levels of 4.8 and 3.4 log10 CFU/mL at 24 h for imipenem and ciprofloxacin, respectively. MIC assays confirmed the absence of acquired resistance. Flow cytometry revealed a time-dependent accumulation of cells with intermediate redox activity and membrane integrity, consistent with the persister phenotype. Both planktonic and biofilm-associated populations harbored persisters, with biofilms exhibiting a greater amount of tolerant cells. Furthermore, persister cells displayed reduced pigment production, biofilm formation, and phagocytosis survival. Gene expression analysis revealed upregulation of higB and pqsA in persisters, along with distinct profiles in recovered cells. Pre-exposure to hydrogen peroxide (H2O2; 0.4%) increased survival by > 1 log10 following antibiotic treatment. Additionally, pretreatment with 5-fluorouracil (5-FU; 256 µg/mL) increased imipenem persistence by twofold. Altogether, our findings show that oxidative and genotoxic stresses promote persistence, and that macrophage interactions modulate persister physiology, offering insight into host-pathogen dynamics and potential therapeutic avenues.
Effective management of IBD necessitates ongoing evaluation of disease activity. Molecular imaging of neutrophil elastase (NE) could address this urgent need as it mechanistically targets the early event of intestinal inflammation. In this study, based on macrocyclic peptide POL6014, [68Ga]Ga-POL6014 was developed for noninvasive assessment of colitis activity and treatment response through targeting of NE. [68Ga]Ga-POL6014 exhibited high specificity and affinity for NE with the KD of 14.81 nM and IC50 of 3.02 nM. In PET imaging, [68Ga]Ga-POL6014 could quickly visualize inflammation sites with colon uptake of 3.15 ± 0.63%ID/g and colon/muscle ratio of 4.92 ± 0.74 at 30 min p.i.. The specificity of [68Ga]Ga-POL6014 was demonstrated by the dose-response blocking studies with POL6014. Furthermore, [68Ga]Ga-POL6014 PET was used to evaluate treatment response to 5-ASA. The treatment significantly inhibited colon uptake of [68Ga]Ga-POL6014 (1.71 ± 0.21 %ID/g), accompanied by a decrease in imaging contrast (2.81 ± 0.26). Immunofluorescence revealed the preservation of mucosal barrier integrity and a remarkable decrease in NE positive cells after 5-ASA treatment. Moreover, there were good correlations between PET quantification with NE expression and DAI score, indicating the reliability of [68Ga]Ga-POL6014. Additionally, [68Ga]Ga-POL6014 was widely distributed and cleared rapidly from nonspecific organs and blood pool, and was excreted through urinary system, suggesting its favorable pharmacokinetics. In conclusion, [68Ga]Ga-POL6014 is a promising tracer that enables in vivo globally mapping of neutrophil-mediated inflammation in the entire gastrointestinal tract for early diagnosis, assessment of disease activity and evaluation of anti-inflammatory treatment response.
Triplet-triplet annihilation upconversion (TTA-UC) liposomes are of emerging interest because of their potential bioapplications in biosensing/imaging and light-driven therapeutic delivery. However, a potential challenge in vivo is their stability, since liposomes are prone to enzymatic degradation. Here, for the first time, we examine the impact of phospholipase hydrolysis on TTA-UC in liposomes. We applied a TTA-UC liposome integrating a BODIPY charge transfer sensitizer and perylene emitter pair within a DOPC membrane, that exhibits intense upconverted blue emission under green excitation. Phospholipase A2 (PLA2) enzyme was applied as the phospholipase as it is ubiquitous in the body and upregulated under a number of conditions. Surprisingly, we observed that PLA2 treatment resulted in only a relatively modest decrease in TTA-UC intensity on exposure of the liposomes to the enzyme. In the presence of imipramine, a competitive inhibitor of PLA2, or absence of Ca2+ on which phospholipase hydrolysis depends, the enzymatic action is inhibited and TTA-UC intensity is indistinguishable from that in enzyme-free solution. Ca2+-dependent enzymatic activity, drug-based inhibition and the impact of hydrolytic products on membrane packing were characterized in pore-suspended lipid bilayers, using confocal-based fluorescence lifetime imaging (FLIM), fluorescence lifetime correlation spectroscopy (FLCS), and electrochemical impedance spectroscopy (EIS). FLIM and FLCS studies show that enzymatic lipid cleavage increases lipid packing and decreases membrane fluidity without significant damage to the bilayer. Thus, we conclude that the decreased TTA-UC output is due to the increased viscosity of the membrane upon hydrolysis. Electrochemcial impedance confirms these observations, where membrane admittance decreases in response to phospholipid hydrolysis indicating tighter lipid packing of the membrane with hydrolytic products. Nanoscale imaging, using atomic force microscopy (AFM) in liquid mode at a mica-supported lipid bilayer, further confirmed that PLA2 causes phospholipid hydrolysis in the presence of Ca2+, resulting in nanoscale pore formation, whereas either in the absence of Ca2+ or with imipramine-treated PLA2, it did not induce lipid hydrolysis. Overall, our findings provide a molecular basis for understanding enzymatic action in general at a liposome bilayer model and show, for the first time, the influence of enzyme hydrolysis on TTA-UC integrity and efficiency in liposomes.