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Natural killer (NK) cells are promising platforms for off-the-shelf immunotherapy, yet nonviral precision engineering remains limited by poor HDR efficiency, DNA toxicity, and manufacturing challenges. The aim of this study was to establish a high-yield, nonviral knock-in platform. Through extensive in-depth rational screens, we achieved ∼90% HDR insertion of therapeutic payloads while maintaining 100% postediting recovery. By hijacking endogenous transcriptional programs, we installed genetic circuits into defined genomic loci to tune transgene expression. To enable context-dependent therapeutic responses, we integrated a synthetic positive feedback circuit at the CISH locus, which enhanced NK cell persistence and drove strong expression of anti-CD22/19 dual CAR. A hypoxia-responsive IL-12 circuit gated by the PFKFB4 promoter restored cytotoxicity under environmental stress. Finally, we showed this platform is compatible with GMP manufacturing and supports clinical-scale expansion. These findings provide a scalable framework for programmable, nonviral editing of NK cell effector functions for therapeutic and research applications.

Sugarcane molasses serves as a vital non-food feedstock for ethanol fermentation, and its efficient utilisation depends on breeding of excellent microbial strains. Therefore, identification of industrial strains for ethanol fermentation is central to improving fermentation efficiency. To identify high-efficiency ethanol-fermenting strains, we developed a method for preparing high-throughput polymerase chain reaction (PCR) templates within 3 min using seven industrial Saccharomyces cerevisiae strains, integrating single-cell morphology and phenotypic variations in ethanol fermentation. Method specificity was confirmed by amplifying and sequencing the internal transcribed spacer region, and the efficiency was validated by extracting genomic DNA using cetyltrimethylammonium bromide (CTAB). Subsequent genetic diversity analysis using random amplified polymorphic DNA (RAPD) with eight effective primers screened from twenty-two random primers revealed genetic similarity coefficients ranging between 0.47 and 0.96. The high-yield strains 1015-04-01 and 1002-03-03 showed the highest similarity (0.96), whereas strain 1015-04-01 and the low-yield strain 1415 exhibited the lowest similarity (0.47). Unweighted pair group method with arithmetic mean (UPGMA) cluster analysis demonstrated that the high-yield strain 1016-02-04, and strains 1415 and 1313 formed a major cluster, and the low-yield strains 1415 and 1313 formed a distinct subcluster; four other high-yield strains constituted a separate cluster, indicating certain genetic differences between high- and low-yield strains, together with partial genetic overlap. The RAPD results exhibited a certain correlation with differences in final ethanol concentration and single-cell morphology, providing preliminary genetic marker references for the rapid identification and screening of high-performance strains used in ethanol fermentation from sugarcane molasses.

Cholangiocarcinoma (CCA) is an aggressive malignant tumor with extremely poor prognosis, and its global incidence is increasing, posing a growing public health burden. Behavioral factors, such as diet, alcohol consumption, smoking, physical activity, and psychological status, are closely associated with the development of CCA. This narrative review delves into the multidimensional burden of CCA, including psychological distress, symptom profiles, and socioeconomic impacts. It also summarizes evidence from related gastrointestinal cancers to explain the biological mechanisms through which lifestyle modifications may improve prognosis, including immunomodulation, inflammatory cascades, and metabolic reprogramming. Furthermore, we propose an integrated framework for behavioral medicine and hepatobiliary care, emphasizing the pivotal role of behavioral interventions in enhancing CCA prevention and clinical outcomes.

Membrane fusion is central to biological function and bioengineering, yet few design rules exist that enable proteins to be programmed to drive fusion at defined membrane interfaces. Here, we show that cholera toxin B-subunit (CTB), a naturally occurring glycolipid-binding pentamer, can be re-engineered into a programmable membrane fusogen by assembling two CTB units through rationally designed coiled-coil linkers attached to the CTA2 peptide that threads through the CTB pentamer. Using discrete parallel and antiparallel coiled-coil architectures, we generated CTB dimers with defined orientations and examined their ability to drive fusion of giant unilamellar vesicles containing the CTB ligand ganglioside GM1. Fusion efficiency was evaluated using a fluorescence resonance energy transfer (FRET)-based lipid mixing assay, while flow cytometry, confocal microscopy, and quartz crystal microbalance with dissipation monitoring (QCM-D) provided mechanistic insights. Both parallel and antiparallel CTB dimers induced cross-linking and full fusion; strikingly, fusogenic efficiency was governed primarily by the length of the CTA2 linker rather than coiled-coil orientation. These findings establish a generalizable strategy for engineering lectin-based fusogens with tunable activity, defining linker geometry as a key design parameter and advancing the development of programmable membrane fusion platforms for drug delivery and synthetic cell systems.

Achillea arabica Kotschy, known locally as "Thafera'a" in Saudi Arabia, has been widely used in traditional medicine for treating various human ailments, including diabetes and skin inflammation. In the current investigation, we sought to unravel the phytochemical profile, antioxidant, antidiabetic, and anti-inflammatory activities of A. arabica ethanolic extract (AAEE) using in vitro and in silico approaches. The extract contained substantial total phenolic and flavonoid content (TPC = 87.15 ± 1.15 mg GAE/g DE and TFC = 26.2 ± 0.15 mg QE/g DE). Furthermore, UHPLC-QTOF-MS2 analysis exhibited a broad spectrum of metabolites, chiefly phenolic acids and flavonoids. Key compounds included chlorogenic acid, isorhamnetin, kaempferide, Kaempferol-3-O-glucoside, cyanidin-3-O-glucoside, delphinidin-3-O-β-glucopyranoside, naringenin and apigenin. This rich phytochemical profile underpinned the extract's potent bioactivities, as demonstrated by its ability to scavenge DPPH• radicals (IC50 = 135.99 ± 0.87 µg/mL) and ABTS+• radicals (IC50 = 422.02 ± 11.02 µg/mL), reduce metals (FRAP EC50 = 548.70 ± 0.06 µmol Trolox/g dry extract), inhibit α-amylase enzyme (IC50 = 233.65 ± 5.03 µg/mL), and suppression of protein denaturation (IC50 = 138.33 ± 2.23 µg/mL). Docking analysis showed strong binding of flavonoids to the target proteins with energies of -8.3 to -9.8 kcal/mol, while 200 ns molecular dynamics confirmed stable binding of the 1OSE-cosmosiin complex. ADMET predictions indicated favorable pharmacokinetic and safety profiles for naringenin and apigenin, and DFT calculations supported these findings by revealing suitable electronic properties. These results demonstrate that A. arabica is recognized as a significant source of biologically active metabolites with therapeutic potency, validating its traditional medicinal use and warranting further in vivo and clinical investigations to confirm its effectiveness.

Exposure to environmental stressors such as hypoxia or heat has emerged as an effective strategy to stimulate physiological adaptations, enhance sport/physical performance and promote health. Both environmental stressors activate shared molecular pathways, notably through the stabilisation of hypoxia-inducible factor-1 alpha and the induction of heat shock proteins, which mediate cellular protection and systemic molecular adaptation. However, hypoxia and heat differ in several aspects, including induction modalities, monitoring methods, metrics used to assess physiological strain and substantial intra- and inter-individual variability. Together, these differences challenge direct comparison and/or combination. Therefore, this current opinion highlights research gaps by critically presenting the common and distinct physiological responses and adaptations to hypoxia and heat exposure. It also emphasises the importance of monitoring internal physiological strain rather than external environmental stress to better account for individual variability. Finally, we propose future research perspectives to address current methodological challenges.

Through integrated transcriptomic and metabolomic analyses, we systematically assessed the role of calcium signaling pathways in adaptation of eelgrass to high-salinity environments. Phenylpropanoid biosynthesis is a crucial metabolic pathway through which calcium signaling involves to salt adaptability of eelgrass. There is a close crosstalk between calcium signaling and nitric oxide in eelgrass. Zostera marina L. (eelgrass), a representative marine submerged angiosperm, exhibits unique traits for salt adaptation. In previous studies, we found that the calcium signaling pathway in eelgrass was activated under high salt conditions, but the specific role of it in adaptation of eelgrass to salt environments is still unclear. In this study, we utilized ethylene glycol tetraacetic acid to inhibit calcium signaling, thereby to find differentially expressed genes and differential accumulated metabolites in eelgrass. Through integrated transcriptomic and metabolomic analyses, we systematically assessed the role of calcium signaling pathways in adaptation of eelgrass to high-salinity environments. Specifically, calcium signaling in roots adjusts homeostasis through cell wall regulation and plant hormone signaling pathways, contributing to osmotic regulation and antioxidant defenses; In stems, calcium signaling primarily mediates ion transport and osmotic regulation; In leaves, the antioxidant defense system would be activated as a compensatory mechanism to alleviate salt stress damage after inhibiting calcium signaling. Notably, phenylpropanoid biosynthesis is a crucial metabolic pathway through which calcium signaling is involved in salt adaptability of eelgrass. Additionally, there is a close crosstalk between calcium signaling and nitric oxide in eelgrass: calcium signaling regulates the expression of nitric oxide synthase, while nitric oxide also influences the expression of several calcium sensor proteins during calcium signaling transduction. These studies provide valuable insights into the role of calcium signaling in eelgrass, contributing to the understanding of the evolutionary processes of marine higher plants, and offering a theoretical foundation for the improved cultivation of salt-tolerant terrestrial crops.

The island of Cyprus is located in the Eastern Mediterranean, where leishmaniasis is endemic. Although human visceral and cutaneous leishmaniasis (VL and CL) cases have already been documented on the island, there are limited data on the Leishmania species in northern Cyprus. In this report, we present a CL case diagnosed by both microscopic examination and quantitative real-time PCR (qPCR). The patient, a 79-year-old man residing in northern Cyprus, developed an ulcerative lesion on his left leg. The lesion was surgically excised for histopathological examination, and tissue sections were stained with hematoxylin and eosin (H&E). Microscopic examination of H&E-stained tissue sections revealed Leishmania amastigotes. To confirm the diagnosis and identify Leishmania species at the molecular level, DNA was extracted from the paraffin-embedded tissue sections. Following deparaffinization, qPCR targeting the Leishmania-specific Internal Transcribed Spacer 1 region (located between SSU and 5.8S rRNA genes) was performed. In the qPCR assay, the infecting agent was identified as a member of the L. donovani complex, presumptively L. infantum, based on the melting curve analysis. Our findings provide molecular evidence for the presence of leishmaniasis in northern Cyprus and contribute to addressing the lack of molecular data in the region. Our study also suggests that, due to the zoonotic nature of the identified pathogen, continuous vector and reservoir control programs should be implemented in the region to prevent the spread of the disease.

Biofilms are structured microbial communities that thrive on diverse surfaces in natural, industrial, and host environments. The biofilm lifestyle underpins microbial survival, shapes ecosystem function, and drives persistent infections; yet, for many microbes, the molecular determinants of biofilm development remain poorly defined. Here, we introduce "label-free analysis of biofilms" (LFAB), an imaging method that integrates time-lapse, low-magnification brightfield microscopy with regional optical density measurements to quantify biofilm biomass. Unlike conventional assays, LFAB enables real-time, non-perturbative, and high-throughput monitoring of biofilm dynamics. We validated LFAB across diverse microbial species and observed a strong correlation with traditional biofilm quantification methods. Applying LFAB to Streptococcus pneumoniae, a major human pathogen whose biofilm lifecycle underpins colonization and infection, we uncovered reproducible patterns of microcolony biofilm expansion and growth. LFAB-enabled screening of a transposon mutant library revealed that biofilm formation in S. pneumoniae is shaped by genes spanning carbohydrate metabolism, cell wall synthesis, adhesion, and surface interactions. Further analysis identified choline-binding protein A (CbpA) and its associated two-component regulator, as well as the peptidoglycan hydrolase LytB, as key drivers of microcolony biofilm dynamics. Together, these findings establish LFAB as a broadly applicable platform for dissecting biofilm biology and reveal new regulators of biofilm development in a clinically important pathogen. Biofilms are structured communities of microorganisms that attach to surfaces and persist within a self-produced matrix. The biofilm lifestyle underlies microbial survival in nature, contributes to industrial biofouling, and drives many chronic infections. Despite the importance of biofilms, high-throughput measurements of biofilm growth dynamics are challenging using existing tools, which are often disruptive or are not scalable. To overcome this limitation, we developed "label-free analysis of biofilms" (LFAB), a brightfield-based imaging platform that enables real-time, non-perturbative, and scalable quantification of biofilm biomass. LFAB is broadly applicable across species and correlates strongly with traditional assays. Applying LFAB to Streptococcus pneumoniae, a major human pathogen, we performed a mutagenesis screen, uncovering new genetic regulators of biofilm formation in this organism. These findings advance understanding of S. pneumoniae pathogenesis and establish LFAB as a powerful approach for dissecting the molecular basis of microbial community growth.

Fluorescent sensors have emerged as indispensable tools for detecting various fields, including visualising biological processes in molecular biology, clinical diagnostics, biotechnology, and environmental monitoring, due to their high sensitivity, selectivity, excellent biocompatibility, ease of use, and low cost. However, conventional fluorophores, which exhibit diminished fluorescence upon aggregation/detection, that is, in the aggregated state, predominantly suffer from aggregation-caused quenching (ACQ); thus, limit their potential in sensing technologies. To overcome, the new aggregation-induced emission (AIE)-luminogens have been extensively applied in biomedical imaging, optoelectronics, stimuli-responsive systems, drug delivery, and chemical sensing, as AIEgens display greater fluorescence upon aggregation, offering a powerful solution. This review provides a comprehensive, systematic overview of the latest advancements in organic AIE-based fluorescent luminogens. It begins with the fundamentals of AIE and their use in ion sensing, followed by a discussion of the detection of explosives and bioactive molecules. We then summarized by highlighting the diverse range of approaches to establishing an association between the structures of AIEgens and their sensing performance, and finally discussed the current challenges and future opportunities in this rapidly growing research area. We hope this review will spark new ideas and inspire new endeavors in this emerging research area, thereby further promoting state-of-the-art progress in sensing.

The spontaneous transition from α-helix to β-sheet in proteins is a transformative structural event essential for diverse biological functions, yet its dysregulation is a hallmark of protein misfolding diseases. Controlling this transition with molecular precision remains a significant challenge in chemical biology. Here, we report the development of lysine-targeted small hydrophobic chemical constructs (HCCs) designed to bypass native folding pathways and induce α-to-β structural remodeling across a spectrum of model proteins. Through a screening of four HCCs, we identified an activated N,N-dimethyl leucine derivative as a potent, dose-dependent inducer of this conformational switch. Using ion mobility-mass spectrometry and Fourier transform infrared spectroscopy, we demonstrate that these chemical modifications effectively recapitulate the transition from helical architectures to β-sheet-rich assemblies. Beyond structural remodeling, we show that this chemically induced transition drives significant functional shifts, including the precise modulation of cytochrome c catalytic activity and the regulation of amyloidogenic aggregation in lysozyme. Our findings establish HCCs as a versatile platform for interrogating protein conformational landscapes and provide a synthetic strategy to manipulate protein topology. This approach opens new avenues for protein engineering and offers deep insights into the fundamental principles governing protein homeostasis and the molecular basis of proteotoxicity.

Type 2 diabetes mellitus (T2DM) is a chronic metabolic disorder characterized by insulin resistance and hyperglycaemia, which are associated with a high risk of developing several complications, including cognitive dysfunction. Cognitive decline is particularly prevalent in T2DM, encompassing deficits in memory, executive function and information processing speed, which ultimately impact quality of life. The molecular mechanistic and cellular pathways linking T2DM and cognitive dysfunction are complex and multifactorial, involving hyperglycaemia-induced oxidative distress, chronic inflammation, vascular dysfunction and impaired insulin signalling in the brain. In this review, we examine how these interconnected pathways compromise key neuronal and vascular processes essential to maintaining brain proper functioning. Moreover, we explore how emerging evidence suggests that dietary nitrate, found abundantly in vegetables like beetroot and spinach, may offer innovative therapeutic benefits for individuals with T2DM. Finally, we discuss pre-clinical and clinical evidence, addressing challenges specific to T2DM populations that may influence the outcomes of nitrate interventions and highlighting future perspectives for leveraging dietary nitrate as a therapeutic innovative strategy to improve cognitive health in T2DM. Emerging evidence suggests that once ingested, dietary nitrate may act as a bioprecursor of nitric oxide (˙NO), playing a pivotal role in promoting glucose homeostasis, mitigating oxidative distress and inflammation and improving vascular function, mechanisms that collectively counteract the drivers of cognitive decline in T2DM. Dietary nitrate represents a promising nutritional strategy to target mechanisms underlying T2DM-associated cognitive dysfunction. Nevertheless, further studies are required to clarify its therapeutic efficacy, optimal intervention protocols and long-term impact on cognitive health in T2DM.

Behavioral flexibility, the ability to adjust behavioral strategies in response to changing environmental contingencies and internal demands, is fundamental to cognitive functions. Despite a large body of pharmacology and lesion studies, the precise neurophysiological mechanisms that underlie behavioral flexibility are still under active investigations. This work is aimed to determine the role of a brainstem-to-prefrontal cortex circuit in flexible rule switching. We trained mice to perform a set-shifting task in which they learned to switch attention to distinguish complex sensory cues. Using chemogenetic inhibition, we selectively targeted genetically defined locus coeruleus (LC) neurons or their input to the medial prefrontal cortex (mPFC). We revealed that suppressing either the LC or its mPFC projections severely impaired switching behavior, establishing the critical role of the LC-mPFC circuit in supporting attentional switching. To uncover the neurophysiological substrates of the behavioral deficits, we paired endoscopic calcium imaging of the mPFC with chemogenetic inhibition of the LC in task-performing mice. We found that mPFC prominently responded to attentional switching and that LC inhibition not only enhanced the engagement of mPFC neurons but also broadened single-neuron tuning in the task. At the population level, LC inhibition disrupted mPFC dynamic changes and impaired the encoding capacity for switching. Our results highlight the profound impact of the ascending LC input on modulating prefrontal dynamics and provide new insights into the cellular and circuit-level mechanisms that support behavioral flexibility.

Prolyl hydroxylase domain enzyme 1 (PHD1) is a key regulator of hypoxic adaptation and metabolic homeostasis, playing an important role in tissue damage and repair. To enable precise pharmacological interrogation of PHD1 function, we developed the first PHD1 degrader using proteolysis-targeting chimera (PROTAC) technology. Our lead compound, SH-26, a cereblon (CRBN)-recruiting PROTAC, induced PHD1 degradation in a concentration-, time-, and ubiquitin-proteasome system (UPS)-dependent manner across multiple cell lines. In an acetaminophen (APAP)-induced acute liver injury (ALI) model, SH-26 demonstrated protective effects, attenuating hepatic inflammation and necrosis without detectable cytotoxicity. Mechanistically, SH-26-mediated PHD1 degradation attenuated APAP-triggered reactive oxygen species (ROS) accumulation, mitochondrial dysfunction, and NLRP3 inflammasome activation, leading to robust in vivo protection against ALI. Collectively, our work identifies SH-26 as the first effective PHD1 degrader and demonstrates its utility as a chemical tool to dissect the pathological role of PHD1 in ALI.

Central carbon metabolism (CCM) is the primary metabolic hub of the cell, governing energy production and providing precursors essential for a myriad of biosynthetic pathways. Developing analytical tools that can identify and quantify intermediates of these metabolic reactions is crucial for studying cell metabolism in biomedical and biotechnological applications. This study proposes a liquid chromatography (LC)-high-resolution (HR) mass spectrometry (MS) method, covering the CCM of mammalian cell systems. Cells were extracted using a one-step liquid extraction, recovering the hydrophilic metabolites. A stable isotope dilution approach was employed, utilizing a U-13C-yeast internal standard (IS). A LC-HRMS metabolomics method using hydrophilic interaction liquid chromatography (HILIC) coupled to a Zeno-time-of-flight (ZenoTOF) MS was implemented for metabolite semi-quantification. A total of 82 CCM metabolites is reported, of which 77 were confirmed with authentic standards, and for 63 , linearity ranges were obtained. IS normalization enhanced overall robustness, from sample preparation to metabolite semi-quantification. To study the effects on CCM by 5 chemical inhibitors (2-deoxy-D-glucose, etomoxir, UK-5099, rotenone, and 3-nitropropionic acid), our HILIC-HR-TOF-MS method was used. The approach proved efficient in capturing altered metabolite concentrations, within implicated metabolic reactions, as a consequence of inhibitor exposure. Our HILIC-HR-TOF-MS metabolome method is efficient in mapping changes in metabolic intermediates of the CCM in mammalian cells. This approach holds potential for analysing a variety of biological samples across a range of applications, from drug development to biomedicine.

Hemolysins are a varied group of bacterial toxins that play a significant role in making microbes more harmful by disrupting host cell membranes and affecting host-pathogen interactions. Both Gram-positive and Gram-negative bacteria produce hemolysins, which help them acquire nutrients, avoid the immune system, damage tissue, and spread. This review offers a detailed look at bacterial hemolysins, emphasizing their classification, structural differences, and how they work at a molecular level. It discusses the role of hemolysins in how microbes' function and cause disease, as well as their interactions with host cell responses. The review summarizes current methods for detecting and characterizing hemolysins, highlighting progress in both analytical and molecular techniques. Furthermore, it examines recent advancements in targeting hemolysins through strategies that reduce their harmful effects and therapeutic inhibition. By connecting mechanisms with new intervention strategies, this review stresses the significance of hemolysins in microbial biology and their potential as targets for new antimicrobial methods.

Sugar crops, including but not limited to sugarcane, sugar beet, sweet sorghum and stevia, are major sources of sugar production in the world. However, conventional breeding approaches, limited by long breeding cycles, low efficiency and restricted capacity to improve complex traits in sugar crops, are increasingly insufficient to address the challenges posed by climate change and the demands of sustainable agriculture. This review systematically summarizes recent advances in biotechnology and molecular breeding that have transformed sugar crop improvement. Recently, high-throughput sequencing technologies have generated extensive multi-omics resources. Concurrently, numerous functional genes and genetic elements with substantial breeding potential have been identified and cloned, offering precise targets for the key agronomic traits in sugar crops. Marker-assisted selection has been successfully implemented to enhance disease resistance, while genomic selection has demonstrated well for the evaluation and selection of complex quantitative traits. Importantly, genetic transformation systems have enabled precise manipulation of target genes and facilitated the creation of novel germplasm. In the future, the integration of multi-omics data, artificial intelligence, high-throughput phenotyping and precision genome editing into an intelligent breeding framework will be essential for achieving breeding by design and developing climate-adaptive and smart cultivars. Ultimately, these technological innovations will expand the role of sugar crops beyond traditional sugar production, positioning them as a central platform for sustainable biomanufacturing and providing critical support for global sugar security, energy transition and the development of the bioeconomy.

Arbuscular mycorrhizal (AM) fungi change phosphorus (P) uptake and plant growth. The degree of change is defined as mycorrhizal dependency. Mycorrhizal dependency differs among cultivars and among different levels of soil P availability. The purpose of this study was to study the effect of AM fungus colonization on Allium fistulosum (A. fistulosum) with different mycorrhizal dependency grown at different levels of soil P availability. Twenty cultivars of A. fistulosum were grown with or without (control) AM fungus Rhizophagus spp. strain R-10 for 82 days. Three cultivars of A. fistulosum with different mycorrhizal dependency were inoculated and grown in soils fertilized at the rate of 0.43, 0.87, 2.18, and 4.36 g P kg-1 soil (P1, P2, P3, and P4, respectively) with or without (control) the AM fungus for 82 days. AM colonization, root length, shoot dry weight, and P concentration were determined. AM colonization increased shoot P content and shoot dry weight. Mycorrhizal dependencies were different among 20 cultivars and Mogamigawa, Shonan, and Kannonhosonegi were used as high, middle and low mycorrhizal dependency cultivars, respectively. Shoot P content of Mogamigawa and Shonan cultivars was higher in the inoculated plants than that in the uninoculated plants at P1, P2 and P3 soil fertilization rates. Shoot P content of the Kannonhoso was higher in the inoculated plants than that in the uninoculated plants at P1. Shoot dry weight of the Mogamigawa and Shonan was higher in the inoculated plant than that in the uninoculated plant at P1. These results suggest that selection of an appropriate cultivar and soil P availability are important factors in determining possible mutualistic, commensalistic, and parasitic relationships between the AM fungus and the host plant.

Triple-negative breast cancer (TNBC) lacks targeted therapies and is driven by dysregulated signaling networks that promote migration, invasion, and survival. Connexin43 (Cx43), a gap junction protein essential for maintaining normal mammary epithelial homeostasis, becomes aberrantly phosphorylated and mislocalized in breast cancer, contributing to disease progression. Because the tyrosine kinases Pyk2 and Src regulate Cx43 and multiple pro-tumorigenic pathways, we investigated whether their combined inhibition could suppress malignant behaviors in TNBC. In MDA-MB-231 cells, the Pyk2 inhibitor PF4618433 and Src inhibitor Saracatinib modestly reduced metabolic activity at high concentrations; however dual treatment produced a dose-dependent and synergistic reduction in viability. In migration and invasion assays, each inhibitor reduced motility, however dual inhibition produced the strongest suppression. Cx43 knockdown impaired baseline migration and invasion and altered the response to Pyk2/Src inhibition, indicating that Cx43 modulates sensitivity to these agents. PF4618433 increased Cx43 plaque formation without changing total protein levels. Mechanistically, Pyk2 inhibition reduced phosphorylation of Cx43 at Y265 and decreased levels of TAZ, p-Erk1/2, p130Cas, and Notch1, whereas Src inhibition only reduced p-Erk1/2. Dual treatment did not further decrease these signaling nodes but nonetheless produced stronger functional inhibition of viability and motility, and the shared regulation of p-Erk1/2 by Pyk2 and Src may help explain how compensatory Pyk2 activation limits the effectiveness of Src-targeted therapies. Together, these findings show that coordinated Pyk2 and Src inhibition restores Cx43 organization and disrupts multiple malignant traits in TNBC cells, supporting this combination as a promising therapeutic strategy.