Altered post-lanosterol cholesterol biosynthesis causes a heterogeneous group of rare inherited metabolic disorders, including Smith-Lemli-Opitz syndrome, desmosterolosis, lathosterolosis, and congenital hemidysplasia with ichthyosiform nevus and limb defects syndrome. These conditions are characterized by impaired cholesterol synthesis together with the accumulation of disease-specific sterol intermediates. Current experimental and clinical evidence suggests that pathogenesis reflects both cholesterol insufficiency and sterol-mediated toxicity, including oxidative stress, perturbed developmental signaling, membrane dysfunction, and impaired neurodevelopment. Experimental models have played a central role in elucidating these mechanisms and in evaluating emerging therapeutic strategies. This review provides a comprehensive overview of currently available experimental models used to investigate inherited cholesterol biosynthesis disorders, including genetically engineered animal models, patient-derived fibroblasts, immortalized and CRISPR/Cas9-edited cell lines, and induced pluripotent stem cell-based systems. Particular emphasis is placed on Smith-Lemli-Opitz syndrome, the most extensively studied disorder within this group, while recent advances in modeling desmosterolosis, lathosterolosis, and congenital hemidysplasia with ichthyosiform nevus and limb defects syndrome are also critically discussed. We compare the strengths and limitations of each experimental platform, highlighting their contributions to understanding sterol metabolism, developmental abnormalities, and cell-type-specific disease mechanisms. Finally, we discuss current challenges and future perspectives, including the development of patient-specific induced pluripotent stem cell models, genome editing approaches, and next-generation multicellular systems. Collectively, this review provides an updated framework for selecting appropriate experimental models to investigate cholesterol biosynthesis disorders and accelerate the development of mechanism-based therapeutic strategies.

Stress urinary incontinence (SUI) is a highly prevalent condition associated with pelvic floor damage, fibroblast dysfunction, and impaired extracellular matrix (ECM) remodeling. This study aimed to investigate the regenerative potential and underlying molecular mechanisms of decidua-derived mesenchymal stromal cells (DMSCs) in a rat model of vaginal distension (VD) and in human suburethral fibroblasts from SUI patients. Adult female rats were subjected to VD and treated with periurethral DMSC injections, followed by functional and transcriptomic analyses, one and six weeks after VD. In parallel, an in vitro co-culture system was used to evaluate the paracrine effects of DMSCs on SUI fibroblasts. DMSC treatment significantly increased leak point pressure (LPP) one week after VD, approaching values observed in control animals (19.8 ± 0.45 vs. 22.3 ± 2.08 mmHg, p = 0.09), while promoting transcriptional changes consistent with tissue repair. Gene expression analyses revealed transient increases in proliferative and inflammatory markers, followed by earlier normalization compared to untreated animals. In vitro, DMSCs reduced p16 expression, increased p21 and Klotho levels, and rebalanced ECM remodeling by decreasing MMP-1 and increasing MMP-2. These findings indicate that DMSCs promote a regenerative microenvironment by modulating senescence and ECM dynamics. Overall, DMSCs may represent a promising disease-modifying strategy for SUI by enhancing tissue repair and functional recovery.

The adeno-associated virus (AAV) has become the vector of choice for gene therapy and experimental gene delivery, owing to its non-pathogenic nature and ability to achieve persistent gene expression across diverse tissues. AAV has emerged as a key platform in cellular reprogramming and regenerative medicine, with applications spanning transcription factor delivery for in vivo lineage conversion and tissue repair across the CNS, heart, and musculoskeletal systems. However, significant limitations remain, particularly in the context of induced pluripotent stem cell (iPSC) engineering. We assess barriers to efficient iPSC transduction including receptor-dependent entry deficits and activation of p53-dependent DNA damage responses. Although AAV is widely described as non-integrating, evidence indicates that integration events occur in rapidly proliferating and actively reprogramming cells. Critically, we synthesize evidence that cell-type-specific promoters lose fidelity when paired with neurogenic transgene payloads, a cross-tissue problem not addressed in existing AAV reviews, and that published in vivo reprogramming efficiencies may be substantially confounded by promoter leakage in the absence of formal lineage tracing. These aspects, underrepresented in recent platform-level reviews, are specifically emphasized here as a resource for researchers designing rigorous AAV-based reprogramming and gene therapy strategies.

Obstructive sleep apnea (OSA) syndrome is characterized by repetitive nocturnal airway obstruction and is associated with intermittent hypoxia (IH). The leading cause of death among OSA patients is cardiovascular morbidity, which is greatly enhanced by IH. Despite the existence of standard treatment, cardiovascular morbidity remains unaddressed. Given the central role of IH in OSA-related cardiac damage, the present study aimed to elucidate the mechanisms underlying IH-induced cardiac injury in order to better understand and potentially improve upon current therapeutic approaches. Using human embryonic stem cell-derived cardiomyocytes (hESC-CMs) as a novel in vitro model, IH was successfully induced, and its effects on key signaling pathways were investigated. Following IH exposure, significant activation of ERK1/2, ERK5, and Erbin was demonstrated. Notably, the concurrent increase in both ERK1/2 activation and Erbin expression following IH suggests a more complex regulatory relationship between these molecules than previously appreciated. Furthermore, pathway-specific inhibition of ERK1/2 and ERK5 attenuated the IH-induced decline in beating rate, with significant restoration, following normoxic recovery. This study provides an innovative approach for in vitro investigation of OSA-associated cardiovascular morbidity and supports the search for novel pharmacological agents and molecular targets to improve the diagnosis and treatment of affected patients.

People with diabetes often have a high rate of oral implant failure and unstable long-term treatment effects. It is urgent to find the potential mechanisms and effective cure methods to address the increasing demand for dental restoration in recent years. This paper aims to find an influential factor, and a probable treatment resulting from it, to improve poor osseointegration and achieve favorable bone regeneration. To find out the influential factor of poor diabetic osseointegration, we compared marginal bone loss among patients taking different hypoglycemic drugs. To verify our pre-clinical findings, we performed in vitro experiments as well as micro-CT, fluorescence intensity, and histopathological analysis methods after establishing a diabetic rat model controlled by different systemic hypoglycemic drugs. To enhance implant osseointegration and find out the underlying mechanism, we proposed the GelMA-alogliptin hydrogel system and tested the scratch wound healing, cell adhesion, CCK-8, live-dead cell staining, osteogenesis, qPCR, and Western blot methods based on diabetic rat-derived BMSCs, HUVECs and BMMS, as well as the in vivo experiments in diabetic rats. People with diabetes under DPP4i (alogliptin) exhibit the lowest marginal bone loss. The systemic alogliptin and locally delivered peri-implant GelMA-alogliptin hydrogel treatment promoted bone regeneration around diabetic implants, enhanced diabetic-derived BMSC activity and migration through GLP1R/GSK3β/β-catenin and induced osteogenic differentiation, and promoted vascular regeneration and inflammation control. Alogliptin has positive effects on the oral implant health of people with diabetes. A locally delivered GelMA-alogliptin hydrogel system exhibits competitive ability to improve diabetic bone microenvironment and enhance implant osseointegration.

Extracellular stimuli, including hormones, growth factors and nutrients in the milieu of cells often initiate intracellular changes via signaling pathways, of which the cyclic 5', 3'-adnosine monophosphate (cAMP)-dependent protein kinase (PKA) signaling pathway is prototypical. Research in the past decades has demonstrated that PKA plays versatile roles during cell proliferation and differentiation, mainly through phosphorylating a plethora of protein substrates by its protein kinase activity. Studies using model systems including yeast, neurons and mammalian germ cells indicate that PKA functionality is regulated by not only the cell-type-specific expression of its regulatory and catalytic subunits, but also the spatiotemporal distribution of its binding proteins and secondary messengers. How PKA elicits its functional specificity in a spatiotemporal manner constitutes fundamental mechanisms that regulate development, aging and regeneration. In this review, we first summarize basic aspects that drive the functional diversity of PKA and then focus on the less studied regulatory roles of PKA during synthesis of cellular proteins, the functional units of the cell. Direct links between PKA signaling and protein synthesis machinery are yet to be fully characterized. We anticipate that research in this area, combining model systems and newly developed methodologies, will continue to deepen our understanding of animal development and the etiology of human diseases.

Gastrointestinal acute radiation syndrome (GI-ARS) is a deadly consequence of radiation exposure. We hypothesized that the peptide ghrelin is enteroprotective after radiation injury, and that ghrelin promotes intestinal stem cell regeneration via the vagus nerve. We subjected mice to 12-Gy partial body irradiation (PBI) with 5% bone marrow sparing. Some mice were vagotomized prior to PBI. We then injected the mice with human ghrelin (6 nmol/mouse) or vehicle at 24, 48, and 72 h post-irradiation, and collected blood and tissues at 96 h. PBI caused an 80% reduction in plasma citrulline, 32% shorter villi, 59% fewer crypts, a 14-fold increase in TUNEL+ cells, a 9-fold increase in intestinal permeability (FD4), and 4- to 30-fold increases in bacterial translocation (16S rRNA) to the liver and mesentery. Ghrelin significantly improved all these parameters. Remarkably, vagotomy attenuated ghrelin's protective effects by 22-58%. Mechanistically, ghrelin increased proliferating crypt cells by 2.3-fold, Lgr5+ active stem cells by 2.6-fold, Clu+ revival stem cells by 3.3-fold (immunofluorescence), and Clu mRNA by 1.6-fold compared to PBI alone, and all these effects were significantly diminished by vagotomy. Thus, ghrelin mitigates GI-ARS through vagus nerve-dependent activation of Clu+ revival stem cells and Lgr5+ stem cells, identifying a novel vagal-dependent neuroenteric pathway that regulates intestinal crypt regeneration after radiation injury.

Bone regeneration remains a significant clinical challenge, particularly for large or critical-sized defects caused by trauma, disease, or congenital abnormalities. Mesenchymal stem cells (MSCs) have emerged as a promising cell source for bone tissue engineering, with their osteogenic differentiation playing a crucial role in bone repair. Biomaterials serve as scaffolds that facilitate MSC-mediated bone regeneration by providing structural support and mimicking the extracellular matrix (ECM). This review explores recent advancements in biomaterials designed to promote MSC osteogenesis through two primary approaches: biochemical and physicomechanical stimuli. Therapeutic agent-loaded scaffolds, incorporating growth factors, small molecules, gene materials, peptides, proteins, and extracellular vesicles (EVs), have been extensively studied for their ability to enhance osteogenic differentiation. However, concerns regarding toxicity, off-target effects, and regulatory limitations have led to increasing interest in biomaterials that utilize physicomechanical cues such as stiffness, viscoelasticity, topography, porosity, and dynamic forces (shear stress, compression, vibration) as alternative or complementary strategies. Furthermore, the synergistic effects of multiple physicomechanical cues are being explored to regulate MSC behavior for promoting bone regeneration. This review discusses current challenges, emerging trends, and future directions in the development of next-generation biomaterials that integrate biochemical and physicomechanical approaches for clinical applications in bone repair and regeneration.

Cortical immature neurons (cINs) represent a unique population of prenatally generated, non-dividing neurons that maintain an immature phenotype, characterized by doublecortin (DCX) and polysialylated neural cell adhesion molecule (PSA-NCAM) expression, into adulthood. Unlike canonical adult neurogenesis involving continuous neuron generation from stem cell niches, cINs constitute a distinct form of structural plasticity termed "neurogenesis without division". This review comprehensively examines the molecular markers, morphological diversity, developmental origins, and maturation trajectories of cINs across species. We highlight the striking inverse interspecies relationship between cIN abundance and canonical adult neurogenesis, reflecting distinct biophysical and structural shifts in neural plasticity mechanisms across mammalian lineages. Furthermore, we discuss factors modulating cIN phenotype, including neurotransmitter systems, stress, sensory experience, and aging. Clinical evidence implicating cIN alterations in temporal lobe epilepsy, traumatic brain injury, and stroke is evaluated, revealing potential roles in both pathological circuit remodeling and endogenous repair. Critical gaps remain regarding the molecular programs maintaining immaturity, differentiation triggers, and the functional consequences of circuit integration. Understanding cIN biology offers new perspectives on cortical plasticity and may inform therapeutic strategies targeting endogenous cellular reserves for brain repair.

Guided bone regeneration (GBR) heavily relies on barrier membranes, with collagen being the clinical standard material. Human amniotic epithelial cells (hAECs) represent a promising, non-controversial stem cell source with substantial osteogenic and immunomodulatory potential. This study aimed to comparatively evaluate the structural characteristics of three commercial collagen membranes (Biocollagen®, Derma®, and VantyColl®) and their influence on the biological behavior, viability, and osteogenic differentiation of hAECs and hFOB 1.19 human fetal osteoblasts. The microarchitecture was assessed via scanning electron microscopy (SEM). Biological response was evaluated over 14 days, using MTT assays, calcium and phosphorus quantification, alkaline phosphatase (ALP) activity, and quantitative real-time PCR (qRT-PCR) for osteogenic markers (Runx2, Osterix, ALP, and OPN). SEM revealed a dense lamellar structure for Biocollagen®, a fibrillar and oriented architecture for Derma®, and a highly porous network for VantyColl®. Both cell types adhered to and proliferated on all membranes. Derma® provided the best long-term proliferative support for both lineages. Conversely, VantyColl® induced robust early osteoblastic differentiation, marked by exceptional upregulation of Osterix (24.93-fold) and Runx2 (2.64-fold), though it exhibited diminished long-term hAEC viability. Ultimately, collagen membrane microarchitecture dictates cell fate; dense fibrillar networks (Derma®) favor sustained growth and late matrix maturation (OPN), whereas high-porosity scaffolds (VantyColl®) amplify early osteoinductive cascades.

Maternal obesity and a Western diet high in fat and sugars are increasing worldwide and may contribute to rising neurodevelopmental and neurobehavioral disorders in offspring. Both human and animal studies link these factors to adverse outcomes. However, the cellular mechanisms driving altered early-life neurogenesis, particularly in the hippocampus, remain unclear. To assess fructose effects on neural stem cell (NSC) differentiation and neuronal morphology, hippocampal NSCs from E12.5 C57BL/6 mouse embryos were cultured and treated with fructose (8.75 or 12.5 mM) for 7 days. Neuronal and astrocyte populations, along with neuronal morphology, were analyzed by immunofluorescence, and protein expression was assessed by Western blot. Fructose treatment significantly reduced neuronal and increased astrocyte counts, leading to a decreased neuron-to-astrocyte ratio compared to controls. These findings were supported by decreased MAP2 (neuronal) and increased GFAP (astrocyte) protein expression. Furthermore, fructose also significantly reduced neurite lengths without affecting neurite number. Morphological analysis revealed decreased soma size, reduced area/perimeter, and altered soma area-to-perimeter ratios, indicating impaired structural integrity. Thus, fructose exposure shifts NSC differentiation toward an astroglial lineage while suppressing neuronal development and impairs neuronal growth and structural complexity. Future studies are necessary to determine the influence of these cellular changes on synaptic plasticity and learning, as well as memory.

Cancer stem cell (CSC) persistence drives recurrence and therapy resistance in oral squamous cell carcinoma (OSCC), but what keeps cells locked in this stem-like state is poorly understood. In this narrative review, we propose that CSC state is sustained not by any single pathway but by joint dysregulation of three interacting cell-biological systems: membrane potential (Vmem), communication between neighbouring cells through gap junctional intercellular communication (GJIC), and the Hippo-YAP pathway. We argue that these systems act together on one common point-the YAP protein, retained in the nucleus-which switches on a SOX2-centred stemness gene programme and stabilises a self-reinforcing CSC state. Drawing on evidence from cancer genomics, developmental bioelectricity, connexin biology, and OSCC-specific studies, we reconstruct how membrane depolarisation, loss of gap junction coupling, FAT1 mutation, and Hippo pathway inactivation could converge on persistent nuclear YAP, and how betel quid-the principal risk factor across South and Southeast Asia-may engage all three systems at once. Because the model holds that each input reinforces the others, it predicts that targeting several together should displace CSC state more durably than targeting any one alone. We set out the testable predictions this framework generates.

Small-molecule kinase inhibitors offer a compelling therapeutic strategy for glioblastoma, yet their clinical efficacy remains severely limited by blood-brain barrier penetration and active efflux transporter extrusion. This review evaluates current medicinal chemistry approaches and translational paradigms to overcome these drug delivery and biological constraints. A critical analysis of the literature reveals that direct structural optimization faces a multidimensional balancing act; next-generation design must prioritize macrocyclization, structural rigidification, and bioisosteric capping to lower polar surface area and evade P-glycoprotein and BCRP efflux. Furthermore, carrier-mediated prodrugs targeting the LAT1 transporter provide a viable rescue strategy for highly potent scaffolds. Reviewing recent clinical failures, such as paxalisib and osimertinib, underscores that single-node monotherapies fail due to compensatory pathway hyperactivation and clonal heterogeneity, whereas multi-targeted agents or rational dual-node combinations prevent rapid tumor adaptation. Additionally, combining kinase inhibitors with DNA damage repair inhibitors, immune checkpoint modulation, or MR-guided focused ultrasound could provide powerful synergistic networks. Finally, bridging the translational gap requires complementing conventional serum-cultured cell lines with patient-derived glioma stem cells and orthotopic xenografts to better recapitulate the cellular architecture of the disease. Ultimately, overcoming the therapeutic challenges in glioblastoma demands a fundamental pivot toward rigorous neuro-pharmacological design and multi-lineage network oncology.

The automaticity of human-induced Pluripotent Stem Cell-derived cardiomyocytes (hiPSC-CMs) is governed by coupled Ca2+ and membrane clocks, coordinated through local Ca2+ releases (LCRs) and cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) signaling. We investigated the role of PKA in hiPSC-CM energetics by measuring its dynamics in the cytosol and mitochondria, and its crosstalk with Ca2+. We tested three hypotheses: (i) Ca2+-activated PKA signaling regulates energy balance; (ii) adenylyl cyclase activity correlates with spontaneous beating; and (iii) PKA compartmentalization is Ca2+-dependent. We also compared hiPSC-CMs with rabbit sinoatrial node cells (SANCs). The key findings are: (i) PKA inhibition (H-89), Ca2+ chelation (BAPTA), or mitochondrial Ca2+ blockade (Ru360) led to energy imbalance; (ii) H-89 induced compartmentalized PKA activity in the cytosol, mitochondrial matrix, and outer mitochondrial membrane; (iii) Ca2+ chelation with BAPTA reduced PKA activity globally; and (iv) PKA dynamics and Ca2+-dependent regulation were similar in hiPSC-CMs and rabbit SANCs. In conclusion, intracellular Ca2+-mediated PKA compartmentalization is present in hiPSC-CMs and rabbit SANCs.

Stromal vascular fraction (SVF), an uncultured adipose-derived cell population, exerts regenerative effects primarily through paracrine mechanisms. While local intratesticular injection of SVF alleviates busulfan-induced testicular injury, the intravenous route remains unexplored. This study investigated whether intravenous SVF administration could mitigate busulfan-induced spermatogenic impairment. Adult male Sprague-Dawley rats received busulfan (15 mg/kg, intraperitoneal) or vehicle; the busulfan + SVF group received 2 × 106 allogeneic SVF cells intravenously immediately after busulfan injection. Two weeks later, we evaluated sperm parameters, testicular histopathology (Johnsen's score, seminiferous tubule diameter, germinal epithelial thickness), and immunohistochemical expression of SOX9 (Sertoli cells), CYP11A1 (Leydig cells), and DDX4 (germ cells). Busulfan alone induced severe spermatogenic defects, including reduced sperm count and motility, increased abnormal morphology, marked histopathological damage, and decreased expression of SOX9, CYP11A1, and DDX4. In contrast, intravenous SVF treatment significantly improved sperm count, motility, and testicular architecture, and restored the expression of SOX9, CYP11A1, and DDX4 within two weeks. This is the first demonstration that intravenous SVF attenuates early busulfan-induced testicular injury, providing preliminary evidence for a rapid, culture-free approach as a less invasive alternative to local injection. However, longer follow-up and fertility studies are needed to confirm sustained spermatogenic restoration.

Cardiovascular diseases (CVDs) remain the foremost cause of death globally, responsible for 19.2 million deaths and 437 million disability-adjusted life years in 2023, with prevalent cases having more than doubled since 1990. No approved therapy restores myocardium lost to infarction. The adult heart replaces cardiomyocytes at approximately 1% per year in young adults, declining to about 0.45% per year with ageing, far below what is needed to recover the more than one billion cells destroyed by a large myocardial infarction. Cell-based regenerative strategies have been investigated for more than two decades, encompassing bone marrow mononuclear cells (BM-MNCs), mesenchymal stromal cells (MSCs), cardiac progenitor cells, cardiosphere-derived cells (CDCs), skeletal myoblasts, and induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). Safety has been consistent. Efficacy has been modest and variable: the CADUCEUS trial demonstrated scar mass reduction with CDCs without proportionate ejection fraction improvement; the Phase 1/2 POSEIDON trial confirmed MSC safety in 30 patients; and the Phase 3 DREAM-HF trial, enrolling 537 patients, failed its primary endpoint (HR 1.2, p = 0.406). Mechanistic work has established that transplanted cells engraft poorly and exert their benefit principally through paracrine signalling mediated by secreted extracellular vesicles and exosomes carrying microRNAs, trophic factors, and immunomodulatory proteins. For iPSC-CMs, electrophysiological immaturity and arrhythmogenic risk in primate models remain unresolved barriers. Emerging strategies include CRISPR-engineered hypoimmune iPSC lines, bioengineered cardiac patches, injectable hydrogel scaffolds, and engineered exosome platforms. This review provides a comprehensive synthesis of preclinical and clinical evidence, examines translational barriers, and identifies the scientific and regulatory priorities required before these therapies can enter routine clinical practice.

Local poultry breeds represent an essential component of agricultural biodiversity and play an important role in ensuring the resilience of livestock production systems under changing environmental conditions. The Slovak national chicken breed Oravka is a dual-purpose genotype adapted to mountain and sub-mountain production environments and classified as an endangered genetic resource. The conservation of this breed therefore requires integration of traditional breeding programmes with modern reproductive biotechnologies. The present literature review summarises the origin, breeding characteristics and adaptive potential of the Oravka breed and provides a comprehensive overview of current ex situ conservation strategies based on cryopreservation of reproductive and stem cells. Special attention is devoted to the cryopreservation of primordial germ cells, mesenchymal stem cells and rooster spermatozoa, which represent key biological material stored within national gene bank infrastructure. Differences in cryosurvival between blastodermal cells and germline stem cells are discussed together with methodological limitations of avian embryo cell preservation. The review highlights the importance of combining in situ and ex situ conservation approaches and emphasises the role of poultry gene banks as strategic tools for safeguarding genetic diversity under climate change conditions. The Oravka breed represents a valuable model for integrated conservation strategies linking biodiversity protection, reproductive biotechnology and sustainable poultry production in Europe.

Mesenchymal stromal cells (MSCs) derived from perinatal tissues represent a promising resource for regenerative medicine due to their proliferative, immunomodulatory, and differentiation properties. Among these tissues, the umbilical cord is considered an accessible and ethically acceptable source of MSCs. This study evaluated the feasibility of whole umbilical cord cryopreservation in canine and equine species and assessed the biological properties of MSCs isolated from cryopreserved tissues compared with fresh samples. Equine Wharton's jelly and canine umbilical cord tissues were processed either immediately or after cryopreservation using two different freezing media: 10% DMSO + 10% FBS (DF) and 0.05 M glucose + 0.05 M sucrose + 1.5 M ethylene glycol (GSE). Isolated MSCs were characterized through proliferation, clonogenicity, adhesion and migration assays, trilineage differentiation, and molecular marker analysis. MSCs were successfully isolated from all experimental groups and maintained the typical morphology, plastic adherence, proliferative capacity, multilineage differentiation potential, and mesenchymal marker expression associated with MSC populations. Overall, cryopreservation did not significantly affect the main biological and functional properties of the cells. These findings support the use of cryopreserved umbilical cord tissues for veterinary biobanking and regenerative medicine applications.

Water limitation during soybean (Glycine max (L.) Merr.) establishment restricts leaf expansion and root-shoot development, whereas microbial biostimulants may modify these responses only within particular moisture contexts. We tested whether a mixed-species suspension of Bacillus subtilis and Bacillus amyloliquefaciens produced water-regime-dependent dose responses in a controlled-environment tray-cell experiment. The seedlings received a single rhizosphere-directed application of 0, 0.05, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.75 or 2.0 mL of undiluted inoculum per cell before exposure for 15 days to full irrigation (W100), 50% or 25% of the W100 replacement volume (W50 and W25), or no irrigation (W0). Each of the 44 treatment combinations comprised six independent biological replicates (264 cells). Two-way ANOVA detected significant water-regime, dose and interaction effects for most thermal, growth, biomass and allocation traits, with water regime as the dominant source of variation. Averaged across doses, relative to W100, W0 increased the leaf-surface temperature by 8.8% and reduced the root length by 44.3%, shoot length by 25.4%, stem diameter by 38.2%, leaf area by 72.3%, total dry mass by 20.1% and specific leaf area by 70.4% on average. The concurrent 17.3% increase in the leaf mass fraction indicated the retention of proportional leaf dry-matter investment despite strongly restricted surface expansion. The bacterial response was non-linear and water-regime-specific. Within W0, 0.05 mL increased root dry mass by 116.7% and total dry mass by 39.3% relative to the untreated W0 control. The highest integrated mean z-score occurred at 0.05 mL under W0, 0.2 mL under W25, 1.2 mL under W50 and 0.2 mL under W100. Correlation analysis identified coordinated trait covariation, whereas hierarchical clustering and principal component analysis separated a warm, low-expansion W0 phenotype from a cool, high-vigour W100 phenotype. These findings provide a quantitative experimental basis for selecting candidate pre-drought doses for seedling-stage screening. Validation across soils, application timings, formulation persistence and reproductive-stage yield are required before commercial recommendation.

Esophageal squamous cell carcinoma (ESCC) is an aggressive malignancy with a poor prognosis, highlighting the urgent need to elucidate its molecular mechanisms to develop targeted therapies. Long non-coding RNAs (lncRNAs) play a critical role in cancer progression. However, the majority of lncRNAs involved in ESCC progression remain to be elucidated.