Pancreatic cancer (PC) remains one of the most lethal malignancies across the world, which is due to delayed diagnosis and resistance to current therapies. The interactions between pancreatic tumor cells and their tumor microenvironment (TME) allow cancer cells to escape from anti-cancer therapies, leading to difficulties in treating PC. With endocrine function and lipid storage capacity, adipose tissue can maintain energy homeostasis. Direct or indirect interaction between adipocytes and PC cells leads to adipocyte dysfunction characterized by morphological change, fat loss, abnormal adipokine secretion, and fibroblast-like transformation. Various adipokines released from dysfunctional adipocytes have been reported to promote proliferation, invasion, metastasis, stemness, and chemoresistance of PC cells via different mechanisms. Additional lipid outflow from adipocytes can be taken into the TME and thus alter the metabolism in PC cells and surrounding stromal cells. Besides, the trans-differentiation potential enables adipocytes to turn into various cell types, which may give rise to an inflammatory response as well as extracellular matrix reorganization to modulate tumor burden. Understanding the molecular basis behind the protumor functions of adipocytes in PC may offer new therapeutic targets. Pancreatic cancer (PC) is one of the most lethal malignancies in the world Kras-driven pancreatic tumorigenesis Adipose tissue regulates energy homeostasis through its lipid storage and endocrine functions Adipose tissue is a specialized connective tissue containing two major fractions, lipid-rich adipocytes, and the stromal vascular fraction. Adipocytes store body fat in the form of triglycerides within lipid droplets in their cytoplasm. The stromal vascular fraction consists of various cell types, including endothelial cells, preadipocytes, adipose-derived mesenchymal stem cells (ADSCs), and immune cells. The remarkable heterogeneity of adipose tissue contributes to its multifunctionality and cooperative role in modulating energy balance. Both fractions of adipose tissue are surrounded by the ECM, which serves as a scaffold supporting multiple biological processes, such as cell adhesion, signal transduction, and the formation of new blood vessels Adipose tissue modulates energy homeostasis by lipid reservation and endocrine secretion de novo lipogenesis. These newly synthesized fats are then delivered and incorporated into WAT for storage by lipoproteins. To enhance energy storage capacity, adipocytes change through two mechanisms. Firstly, they can incorporate additional lipids content into their lipid droplets leading to an enlargement of their volume, a process known as adipocyte hypertrophy. Secondly, adipocytes can also increase in number by recruiting precursor cells and promoting their proliferation, referred to as hyperplasia. These two processes collectively contribute to the overall growth of adipose tissue mass. In contrast, systemic energy demands, energy deficiency, or catabolic stimulation serve as external stimuli that initiate the process of lipolysis, whereby stored lipid droplets within adipocytes undergo breakdown . Perilipin plays as a gatekeeper in the regulation of lipolysis by forming a complex with CGI-58 on the surface of lipid droplets. When lipolysis is activated, Perilipin undergoes phosphorylation, leading to the dissociation of CGI-58 from the complex. This action facilitates the subsequent binding of CGI-58 to adipose tissue triglyceride lipase (ATGL), enabling the breakdown of triglycerides (TGs) into diglycerides (DGs) and free fatty acids (FFAs). Phosphorylated Perilipin also recruits hormone-sensitive lipase (HSL) to the surface of lipid droplets, working together to break down DGs into FFAs and monoglycerides (MGs). Monoglyceride lipase (MGL) manages the final step in the lipolysis process that breaks down MGs into glycerol and FFAs WAT governs metabolic actions in distant organs by releasing numerous adipokines into the circulation Unlike WAT, BAT is characterized by the presence of multiple small lipid droplets and high levels of mitochondria. BAT has a substantial contribution to thermoregulation, allowing the body to adjust and maintain its temperature in response to various physical conditions. The high expression of mitochondria uncoupling protein 1 (UCP1) enables BAT to specialize in heat generation. UCP1 acts as a proton pump that balances the ion gradient across the mitochondrial inner membrane during oxidative phosphorylation. Instead of ATP synthesis, this process converts transmembrane potential into heat production Myf5). Brown adipocytes are derived from the +Myf5 lineage, sharing the same transcriptional profiles as skeletal muscle cells. In contrast, beige and white adipocytes are developed from -Myf5 progenitor cells. A transcriptional complex composed of BMP-7 signals, PR domain containing 16 as well as CCAAT/enhancer-binding protein (C/EBP) beta drives Myf5+ precursors into a thermogenic program with co-activation of peroxisome proliferator-activated receptor gamma (PPAR-γ) and PPAR-γ co-activator 1 alpha (PGC-1α), facilitating the formation of brown adipocytes. Partial -Myf5 progenitors also undergo a thermogenic process, but they transform into beige-adipocytes with specific genes expression like CD137, transmembrane protein 26, and T-box 1 -Myf5 population turns into white pre-adipocytes through BMP2 and BMP4 signaling e novo differentiation, beige adipocytes can be also transdifferentiated from white adipocytes under certain conditions like cold exposure, β3-adrenergic signals triggered by the sympathetic neuron system, and chronic inflammation in various pathological processes Multipotent MSCs have been suggested as a major source of adipocyte generation, in which numerous critical pathways are involved, such as the TGF-β/bone morphogenic protein (BMP) pathway, Wingless-related integration site (Wnt) signaling, fibroblast growth factors (FGFs) signaling, Hedgehogs signaling, and Notch signaling Figure 1). Inflamed adipose tissue may also undergo morphological transition to other cell types which results in the loss of their original function. In this section, we sum up the findings of how PC induces functional defects in adipose tissue, and how the latter conforms to promote PC growth.Exogenous signals and stimuli can greatly affect adipocyte functions and features. During cancer progression, uncontrolled cancer cell proliferation brings high nutrient demands that break normal energy balance de novo lipogenesis; instead, they take up exogenous FFAs to maintain lipid homeostasis under hypoxic stress Figure 1) Abnormal lipid metabolism in adipocytes has been found in the development of various cancers, including PC Kras mutation In addition to upregulated lipolysis, WAT may undergo beige adipocyte-transition during chronic inflammation, leading to inefficient energy use through UCP1 expression. IL-6 and parathyroid-hormone-related protein, a hormone frequently upregulated in PC with CreERT; KrasG12D/+fElas) transgenic mice, a high-fat diet increased the activity of Kras, a substantial oncogenic factor in PC development, while the numbers of both PanIN and PC tumors were increased during the intervention G12D; Trp53R172H/+Pdx1-Cre; LSL-Kras) transgenic mice in vitro. The above studies support the idea that lipid resources derived from adipose tissue promote PC progression. Lipids not only serve as substrates for ATP production, but also play central roles in cell membrane construction, signaling transduction, and posttranslational protein modification, all of which are necessary for tumor expansion. PC patients with obesity suffer increased tumor burden and faster disease progression compared with those without obesity Besides tumor cells, noncancerous cells in the TME are also affected by abnormal lipid metabolism during cancer progression. PC has a highly immunosuppressive TME where immune cells fail to destroy tumor cells or even transit into a pro-tumor phenotype, exemplified by tumor-associated macrophages (TAMs). Under long-term stimulation with cytokines such as IL-4, IL-10, and IL-13 in the TME, cytotoxic M1 macrophages are polarized into the M2 phenotype with anti-inflammation, pro-angiogenic, and tissue remodeling abilities to promote tumor growth + T lymphocytes recognize specific antigenic peptides presented on the tumor cells by human leukocyte antigen class I and are activated to kill malignant cells through secreting cytotoxic granules or cytokines like interferon and TNF + T cells + T cells in a lipid-rich environment + T cells CD8Adipocytes can also secrete glutamine to preserve PC cell proliferation in nutrient-poor conditions Adipose tissue produces a variety of hormones called adipokines to regulate the metabolic processes throughout the body. However, adipocyte functions can be impaired under pathological conditions. In obesity, adipocytes undergo hypertrophy and hyperplasia, leading to excessive secretion of cytokines and the recruitment of immune cells, which results in low-grade inflammation, and further compromising adipocyte function. This is accompanied by elevated serum levels of leptin, resistin, hepatocyte growth factor (HGF), and TNF-α, along with the decreased release of adiponectin, insulin-like growth factors (IGF), and type two cytokines, which hinder systemic catabolic pathways, insulin sensitivity, and immune regulation Figure 2).During PC tumorigenesis, the high levels of pro-inflammatory cytokines in the TME bring adipose tissue to an inflammatory state, causing tissue fibrosis and even adipocyte death Leptin is a key hormone that regulates appetite and food intake, with additional functions involved in immune response and angiogenesis in vitro study has demonstrated that short-term leptin treatment increases IFN-γ secretion and cytotoxicity of NK cells. However, prolonged leptin stimulation can lead to decreased proliferation and immune function of NK cells + T cells represent the main force against tumor cells. Leptin can also influence the frequency of mitochondrial respiration in CD8+ T cells derived from peripheral lymph nodes near the tumor site, thus enhancing CD8+ T cells' tumor-killing function in a melanoma model Although the effect of leptin on noncancerous cells within the pancreatic TME is poorly understood, previous research has revealed the roles of leptin in regulating both innate immunity and adaptive immunity. An Adiponectin is the most abundant adipokine secreted by mature adipocytes and possesses insulin-sensitizing and anti-inflammatory abilities Previous clinical studies have also found that patients with PC have lower plasma adiponectin levels Resistin is originally secreted from adipose tissue and takes part in obesity, inflammation, and insulin resistance. A clinical study indicated that patients with resistin-positive pancreatic tumors displayed shorter relapse-free survival times G12D/+Kras-driven pancreatic intraepithelial neoplasia development in vivoHGF has been well known to promote epithelial cell proliferation and cell motility IGFs have two isoforms, IGF1 and IGF2, and are produced by the liver and adipose tissue. IGFs participate in adipocyte differentiation and blood glucose regulation, and they have been reported as cancer risk factors in various tumor types Normally, resident macrophages in adipose tissue produce anti-inflammatory factors to maintain an appropriate environment for regular adipose tissue function. However, during cancer progression, systemic inflammation activates macrophages in adipose tissue, leading to the secretion of inflammatory cytokines from adipocytes, which in turn results in immune cell infiltration, intensifying the immune response in adipose tissue Figure 3).Numerous signal pathways that sustain the normal function of adipocytes are interfered with by stimulation of cytokines derived from the pancreatic TME or adipose tissue itself, leading to altered expression of adipogenic and metabolic genes. Over time, adipocytes gradually lose their specific characteristics acquired during their development and undergo phenotypic changes, transitioning into progenitor-like cell types or directly trans-differentiating into pro-tumor fibroblasts. In this section, we will discuss the phenotypic changes observed in adipocytes following their interaction with PC cells and elucidate how these dedifferentiated adipocytes contribute to the progression of PC , which contribute to fibrosis and tissue remodeling within the TME and further intensify the chemoresistance and metastatic potential of PC.Obesity and other metabolic syndromes have been linked to the progression of PC. In addition to the influence of remote subcutaneous fat, fat tissue adjacent to the tumor also plays a vital role in cancer development. In the KPC transgenic mouse model, a high-fat diet significantly increases the percentages of intra-tumoral adipocytes and the incidence of distant metastasis compared with a standard diet ANGPTL4 and SPP1, encoding angiopoietin-like 4 and osteopontin respectively, were found to be upregulated in CM-treated PC cells. Both are ECM-associated glycoproteins involved in stroma structure remodeling for tumor metastasis Conditioned medium (CM) collected from omental fat promotes the proliferation, invasion, migration, and gemcitabine resistance of PC cells Besides the uni-directional signal from adipocytes, the crosstalk between adipocytes and PC cells also brings benefits for tumor growth. Previous studies have shown that adipocytes increase serum amyloid A1 (SAA1) expression in PC, which mediates PC cell migration by upregulating metastasis-related proteins N-cadherin, Snail, and Slug The interaction between adipocytes and PC cells also enhances the expression of MMP-11 in PC cells, which has been reported to inhibit apoptosis and is capable of degrading ECM components for tumor invasion or migration Adipose tissue-derived stem cells (ADSCs) are mesenchymal stem cells that stay in the stromal vascular fraction of adipose tissue and are capable of self-renewal as well as multipotent differentiation. ADSCs near the tumor site can easily interact with surrounding cancer cells. ADSCs excrete stromal cell-derived factor-1 (SDF-1) into the interstitial space and turn on CXC chemokine receptor 4 (CXCR4) signaling in PC cells, consequently triggering the proliferation, invasion, and migration of PC IL6, CXCL1, and LIF genes are characterized by a secretory phenotype and are able to stimulate STAT3 activation in PC cells. myCAFs with high ACTA2 (α-SMA) and TPM1 expression are usually adjacent to cancer cells and contribute to ECM production In addition to the impact of secreted factors from ADSCs on PC growth, the inherent pluripotency of ADSCs also enables them to undergo phenotypic changes that promote PC development. When exposed to external stimuli, ADSCs can differentiate into various cell types. Specifically, TGF-β released by pancreatic tumors activates ADSCs, leading to the downregulation of S100A4 expression and their subsequent transformation into CAF-like cells. These transformed cells acquire an enhanced ability to produce ECM components that support PC cell migration APOD is the representative marker of the ADSCs population, together with other genes like CFD, CXCL12, MGP, and PTGDS expressed predominantly. During transformation, SFRP4, a Wnt pathway regulator, and RARRES1, which is known for regulating the proliferation and differentiation of ADSCs were both highly expressed. At the same time, the levels of PLA2G2A proteins were elevated during this transition process. PLA2G2A participates in the hydrolysis of phospholipids, leading to the production of FFAs that provide additional energy resources for cancer metastasis. At the end of the transition, ADSCs lose the expression of SFRP4 and RARRES1 and turn into COL11A1-expressing CAFs Given the high potential of ADSCs transiting into pro-tumor CAFs, targeting the factors and mechanisms involved in this process can potentially suppress the development and progression of PC. A single-cell analysis from patients' samples reveals the gene expression pattern during ADSCs-CAFs conversion. Table 1). By understanding the role of adipose tissue in PC progression, novel therapeutic approaches can be explored to effectively counteract this disease.Emerging evidence underscores the vital role of adipose tissue in the development of PC. There is increasing interest in exploring innovative approaches to target dysfunctional adipose tissue, intending to reduce the aggressiveness of tumors and overcome treatment resistance in PC. Strategies such as blocking catabolic pathways, regulating the production of specific molecules known as adipokines and their downstream effects, or preventing the transformation of adipocytes show great promise in this regard. In this section, we provide a summary of the current applications of anti-pancreatic tumor reagents that target different functions of adipose tissue and their impact on the development of PC  treatment has been demonstrated to maintain immune system functions, prevent gemcitabine resistance, and inhibit PC metastasis Overactivated lipolysis induced by proinflammatory cytokines during PC progression leads to the release of FFAs into the circulation or surrounding tumor area. These FFAs can be taken up and accumulated in the pancreatic TME, which not only serves as a source of nutrients for tumor growth but also induces CD8In addition to blocking lipid influx, disturbing the metabolism of PC also has a significant impact on tumor growth. Pancreatic tumors with high ATGL levels show increased desmoplasia On the other hand, targeting the upstream regulator of lipolysis is an effective way to prevent fat wasting during PC progression. Administration of anit-IL-6 receptor antibodies or genetic knockout of IL-6 prohibits lipid storage loss from adipose tissue Mature adipocytes can transit into CAF-like cells under chronic cytokine stimulation to exacerbate desmoplasia, metastasis, and chemo-resistance of PC. It is known that adipocyte dedifferentiation initiates activation of Notch signaling to cut down fatty acid synthesis and PPAR-γ activity that sustains adipogenesis. Wnt5a signaling induced by TGF-β and IL-6 promotes MMPs production and the expression of fibrotic genes in the adipocytes. Treatment with rosiglitazone, a synthetic ligand for PPAR-γ, has successfully reversed the dedifferentiation of adipocytes in vivo studies have demonstrated significant advancements in anti-pancreatic tumor progression and improving survival by employing this approach. Combining targeted anti-adipokine reagents or neutralizing antibodies with chemotherapy drugs has proven successful in overcoming chemoresistance mediated by dysfunctional adipocytes. These findings provide valuable insights and a hopeful outlook for overcoming cancer drug resistance in clinical treatment. They offer valuable guidance for future therapeutic strategies, paving the way for innovative directions in the field of cancer treatment. However, there remains a need to better understand the impact of adipose tissue on pancreatic tumor expansion and the mechanisms underlying the transition of adipocytes.As the significance of lipid metabolism in tumor progression becomes more prominent, an increasing number of studies are now focusing on the role of adipose tissue in the development of PC, especially in obese subjects. Growing evidence suggests a strong connection between adipose tissue and aggressive PC, characterized by enhanced metastasis and chemoresistance. Long-term exposure to proinflammatory cytokines secreted from the pancreatic TME triggers the release of excessive lipid resources and abnormal adipokines from dysfunctional adipose tissue, providing a supportive environment for cancer cell proliferation and the establishment of a tumor-promoting TME. The aberrant transition of mature adipocytes into a cell type exhibiting myCAF characteristics gives rise to severe desmoplasia in the pancreatic TME. Cytokines and growth factors produced by these cells further stimulate the proliferation, invasion, migration, and chemoprotection of PC. Thus, disrupting the communication between adipose tissue and PC cells holds great promise for improving the efficacy of antineoplastic treatments. Indeed, Most studies investigating the involvement of adipose tissue in PC progression have utilized obesity as their disease model. However, emerging evidence suggests that adipose tissue adjacent to the pancreas can promote locoregional recurrence and result in unfavorable clinical outcomes, even in the absence of metabolic disorders. In animal models of cachexia, the loss and remodeling of adipose tissue have been observed during PC development, showing that while obesity can be a contributing factor, it is not a necessary condition for disease progression. Chronic inflammation induced by adipose tissue itself during obesity creates a metabolic imbalance and unhealthy immune response that may not accurately reflect the conditions in patients without metabolic diseases. Therefore, further investigation is needed to understand the complicated communication between adipose tissue and PC. Additionally, different adipose tissue depots exhibit heterogeneous responses to physiological stimulation. For instance, epididymal and mesenteric adipose tissue display a higher inflammation status compared to subcutaneous depots during tumor progression. This indicates that the distance between adipose tissue and the tumor may result in varying degrees of impact. While most studies have focused on peritoneal fat in PC analyses, it is important to consider that proinflammatory cytokines released from TME may influence all fat depots through the systemic circulation. Exploring whether other adipose tissue depots exhibit similar pro-tumor effects as peritoneal fat is an interesting area for future investigations. Moreover, limited attention has been given to the direct effects of adipokines on immune cells rather than cancer cells in PC models. The immune escape continues to pose a significant challenge in the treatment of PC, and exploring the potential role of adipokines in the development of treatment resistance in PC cells could yield valuable insights for overcoming this difficulty. Future research focusing on the direct impact of adipokines on immune cells may uncover novel strategies to enhance immune responses and improve treatment outcomes in PC.in vitro. The lack of in vivo evaluation hinders better assessment of their efficacy and safety, which is essential before clinical translation. Although the journey towards the clinical use and approval of these therapeutics may be long, acquiring a more comprehensive understanding of the molecular mechanisms through which adipose tissue promotes pancreatic tumors and associated wasting symptoms will undoubtedly contribute to the development of more effective anti-cancer strategies.Nowadays, increasing evidence reveal the connection between adipose tissue and the clinical outcomes of PC, highlighting adipose tissue as a promising target for therapeutic interventions. However, it is important to note that most intervention methods targeting adipose-tumor interactions have primarily been tested


A recent study from Yan et al.  providesHTT gene located in the 4th chromosome of the human genome  is a devastating monogenetic neurodegenerative disorder that is caused by an expanded CAG repeat in the exon 1 of the n genome . The pren genome . Thus, HHTT) or its transcript has been considered as promising strategies for HD treatment. For example, anti-sense oligonucleotides (ASOs) that suppress the HTT mRNA have been investigated in the HD mouse models and then entered the clinical trials  to navigate a nuclease (Cas9) to the target genome site and cleave it, triggering genome editing by generating double-stranded breaks (DSBs) and the subsequent DNA repair. CRISPR has been applied in both plants and animals, including rodents and non-human primates (NHP)  . Importaes (NHP) . Recentles (NHP) .HTT correction. They applied the AAVs to the striatum of HD KI pigs by stereotaxic injection, and the injected AAVs successfully “hunt out” the CAG repeat expansion with remarkably high efficiency and corrected it with a normal 20×CAG by homology directed repair (HDR) following CRISPR-mediated DNA cleavage. This genetic correction led to unprecedented phenotypic rescue that has never been observed in HD animal models previously. At the molecular level, the authors detected a correction of the transcriptome towards the wild-type (WT) after AAV injection, and the expression of mutant HTT protein (mHTT) was also drastically decreased. At the cellular level, the in vivo neurodegeneration and glia activation were significantly ameliorated. At the whole animal level, the injected pigs exhibited alleviated HD-relevant symptoms such as gait abnormalities and shortened lifespan. Overall, the study demonstrates that CRIPSR-mediated gene correction is an extremely promising new approach to fundamentally treat HD.In the Yan et al. study, the authors used their previous established HD KI pig model harboring 150×CAG repeats to test the potential therapeutic effects of genome editing by the CRISPR-mediated knock-in approach. They used the adeno-associated virus (AAV) to package the CRISPR elements, including Cas9, gRNAs, and the homologous recombination template for mMore specifically, there are several highlights of this study: (i) The gene therapy of HD in large animal models. Almost all the HD gene therapy studies were performed in mouse model, which typically lacked obvious neurodegeneration to mimic human HD. The pig models exhibit HD-like phenotypes with higher fidelity and are much closer to the patient’s symptoms, providing an ideal tool for HD therapeutic studies; (ii) Remarkable therapeutic effects after the gene editing by a single intracranial or intravenous injection of AAVs. Unlike the ASO-based gene therapy approaches that require multiple intrathecal injections per year, this study showed that a single peripheral injection could potentially treat HD, providing a much more desirable scenario for potential clinical application; (iii) Potential safety issues were systematically evaluated, including deep-sequencing and whole-genome sequencing that reveal minimal off-target effects and strong animal safety data that encourages further clinical trials.p53 activation (Scientists have been seeking for a fundamental HD therapy for decades without a final success. The Yan et al. study provides a great new hope for the HD patients by leading a new orientation of HD treatment approach. This promising study also inspires many future studies. The CAG repeat replacement is very promising but its efficiency remains to be improved. Besides potential clinical trials utilizing the reported approach, other genome-editing technologies could be considered to tackle HD or further improve the current approach. In light of the recent study showing that CRISPR may lead to endogenous DNA damage response arising from aberrant tivation , inactivtivation  are also


The landscape of financial technology is undergoing a continuous evolution, driven by the relentless advancement of information technology. In this transformative milieu, digital finance has emerged as a novel financial paradigm, offering a blueprint for fostering inclusive finance. With a particular emphasis on its implications for Small and Medium-sized Enterprises (SMEs), this article harnesses a comprehensive dataset spanning the years 2011 to 2021, encompassing digital inclusive finance and SMEs listed on the SME board. Employing fixed effects models, this study performs a regression analysis to verify the driving effect of digital inclusive finance on SMEs’ innovation activities. The findings unequivocally affirm the potency of inclusive finance in ameliorating the longstanding financing constraints that have historically constrained the growth trajectory of SMEs. Furthermore, the study elucidated the nuanced nature of the promotional impact of digital inclusive finance on SMEs, contingent upon their distinct property rights and technological attributes. Specifically, the empirical findings unveil a discernible pattern wherein digital inclusive finance exerts a conspicuously stronger promotional effect on non-state-owned enterprises and high-tech SMEs’ endeavors in technological innovation. The conclusions derived from this research furnish a salient point of reference for governmental authorities engaged in the formulation and advancement of digital inclusive finance strategies, thereby imparting strategic guidance for the cultivation of innovation and holistic development within the SME sector. In the contemporary epoch characterized by the global new generation science and technology revolution, China finds itself immersed in a milieu of intense international competition within the realms of science and technology. This panorama unfolds against the backdrop of a pivotal juncture in its economic evolution . ConsequBeyond the aforementioned challenges, the process of innovation transformation necessitates a substantial infusion of investment. This intricate journey entails substantial monetary outlays and a continuous inflow of resources, encompassing requisites such as equipment, human capital, and distribution networks , 8. AddiSMEs, positioned as the long-tail constituents within the intricate fabric of the market economy system, find themselves particularly susceptible when embarking upon innovative financing undertakings. Their vulnerability stems from encountering formidable financing constraints, a consequence of their marginalization within the traditional bastions of financial institutions. Moreover, their forays into technological innovation often encounter limited efficacy within the conventional financial markets, further exacerbating their funding challenges . In a trConsidering this overarching backdrop, this article conducts an empirical expedition, forging a tangible link between the realms of digital inclusive finance, SME innovation endeavors, and the intricate fabric of financing constraints. The study’s primary thrust revolves around unraveling the intricate web of incentives nurtured by digital inclusive finance, particularly its role in catalyzing SMEs’ investments in technological innovation. A principal focus rests upon unveiling the operative mechanisms through which the burgeoning landscape of digital inclusive finance orchestrates a twofold transformation: the alleviation of the pervasive quandaries associated with financing constraints and the concomitant cultivation of a conducive milieu for augmenting SMEs’ investments in innovation, meticulously scrutinized at the microcosmic level of enterprises.Given the intrinsic heterogeneity prevalent within the landscape of SMEs, which invariably begets a spectrum of financing constraints, this study undertakes a discerning exploration into the manifold ramifications of the operational mechanism. Delving into the intricacies of innovation across diverse SME typologies, this study adroitly probes this mechanism’s influence, casting a penetrating gaze through the prisms of property rights and technological attributes. The fundamental goal inherent in this investigation is to distill a reservoir of perspicacious insights and cogent decision-making benchmarks, thereby orchestrating a symphony of endeavors aimed at propelling the growth trajectory of digital inclusive finance. In tandem, this paper endeavors to fortify the bedrock of SMEs’ innovation capabilities, engendering a symbiotic partnership between digital inclusivity and enhanced innovative prowess.The foundation of competence generation lies in the allocation and utilization of resources, as expounded by the resource-based theory , 16. TheIn an era marked by the seamless integration of digital technology and financial services, barriers that have historically impeded the progress of financial services are gradually yielding ground . DispersThe first impact mechanism of digital inclusive finance on SME innovation resides in its capability to diminish the barriers to entry for financial services, diversify avenues for financing, and augment reservoirs of funds for innovation investment , 24–26. The second conduit by which digital inclusive finance propels SME innovation rests in its capacity to mitigate the quandary of information asymmetry between financial institutions and SMEs, thereby engendering a decline in the outlay associated with innovation financing . With thThe third instrumental mechanism facilitated by advanced digital technology involves financial institutions harnessing expansive datasets from diverse platforms. This data-driven approach encompasses the gathering, organization, and analysis of behavioral data spanning various industries and enterprises. This methodology efficaciously mitigates the predicaments of inadequate credit and financial information, along with the elevated credit default risk that SMEs encounter when soliciting funds from banks , 28. LevBuilding upon the manifold benefits of digital inclusive finance, which encompass its extensive coverage, reduced transaction costs, and heightened capital efficiency, the debilitating financing constraints that have hampered SMEs find effective mitigation . With itHypothesis 1: Digital inclusive finance exerts a positive driving effect in fostering technological innovation activities among SMEs.Hypothesis 2: By alleviating the financing constraints faced by SMEs, digital inclusive finance propels and sustains their technological innovation endeavors.Within the research sample comprising SMEs, individual firms exhibit inherent characteristics that set them apart, and diverse categories of SMEs may encounter varying institutional influences in their financing endeavors , 32. ConExamining the property rights of SMEs unveils significant distinctions in the financing landscape between non-state-owned and state-owned entities . The ownAgainst the backdrop of advancing digital inclusive finance, financial institutions harness emergent technologies such as cloud computing and big data analytics to undertake comprehensive assessments of enterprises’ credit standing, financial health, and operational status. This endeavor, in turn, mitigates information asymmetry and ameliorates financing limitations experienced by non-state-owned SMEs . MoreoveAdditionally, discernible disparities in strategic orientation and organizational management models between non-state-owned and state-owned enterprises come to the fore , 32. ThiMoreover, scholarship implies that state-owned enterprises exhibit relative inflexibility in their operational structure and manifest a lack of adaptability , potentiHypothesis 3: The impact of digital inclusive finance on innovation investment varies across SMEs with distinct ownership structures, with its influence more pronounced in fostering innovation investment among non-state-owned SMEs.Delving into technical attributes, a conspicuous dichotomy emerges in the R&D intensity and funding constraints observed between high-tech and non-high-tech SMEs . High-teHypothesis 4: Digital inclusive finance engenders disparate impetuses on the innovation investment tendencies of SMEs hinging on their technological attributes, wielding a more conspicuous sway in augmenting innovation investment among high-tech SMEs.This study centers on the examination of China’s listed SMEs, with a temporal scope encompassing the years spanning from 2011 to 202l. The dataset employed to gauge digital inclusive finance derives from the CSMAR database, wherein meticulous data refinement procedures were instituted. This entailed the culling of delisted entities, companies evincing anomalous or incomplete data, and financial enterprises. To enhance data fidelity, a 2.5% truncation process was employed, targeting continuous variables within the sample dataset to attenuate the influence of outliers. Consequent to these measures, an unbalanced panel data set of 6543 SMEs emanating from 862 companies materialized, serving as the bedrock for the ensuing investigation.In gauging enterprises’ innovation capability and prowess, extant scholarly literature outlines three principal methodological avenues. The first entails the selection of an innovation input index as a gauge of enterprises’ innovation capacity. This index comprehensively mirrors SMEs’ subjective inclination toward participating in innovative undertakings and serves as a proxy indicator for the technological innovation extent of these entities , 44. PerThe second method revolves around employing innovation output indicators to quantify firms’ innovation capacity. Each such output indicator, to a certain degree, symbolizes a firm’s technological innovation competence . Notableinnovation input is employed to gauge the latter [the total R&D expenditure of enterprises serves as a proxy variable to encapsulate firms’ innovation input levels. To ensure scale uniformity, the R&D expenditure indicator is dimensionless and presented as a ratio of total R&D expenditure to total operating revenue for the given year [Moreover, the multifaceted factors impacting firms’ innovation levels necessitate consideration . These fe latter . Specifiven year .To investigate the influence of digital inclusive finance on SMEs’ innovation capacity, we incorporate the Digital Inclusive Finance Index (DIFI), established by the Digital Finance Research Center of Peking University, as the principal explanatory variable . This inDue to the intrinsic attributes of high investment and elevated risk associated with innovation, conventional financial frameworks encounter challenges in adequately catering to the capital requisites of small and medium-sized enterprises (SMEs) in pursuit of innovative endeavors. The pronounced constraints on financing exert a profound inhibitory influence on the capacity of SMEs to engender technological innovation. In light of contemplating the potential of digital inclusive finance to ameliorate the quandary of "financing difficulty" confronted by SMEs, the present study opts to position financing constraints as an intermediary construct. This strategic choice propels a deeper exploration into the potential of digital inclusive finance to facilitate technological innovation within SMEs, mediated through the mitigation of financing constraints.Within the purview of assessing financing constraints, diverse indices are extant, among which the KZ Index , WW IndeAccordingly, the present research underscores the salience of the SA Index as a surrogate metric for gauging the magnitude of financing constraints. Aligned with the methodological praxis advocated by Hadlock & Pierce (2009), the SA Index is computed as SA = -0.737 × Size + 0.043 × Size^2–0.04 × Age. In this formulation, ’Size’ denotes the organizational scale, while ’Age’ encapsulates the temporal trajectory of the enterprise , while the independent variables consist of the digital inclusive finance index (DIFI) and its constituent sub-indices . Furthermore, the model includes several control variables, encompassing enterprise size (SIZE), return on assets (ROA), enterprise growth (GROW), fixed asset share (FAS), enterprise age (Age), independent director ratio (Ind), two-job combination , and asset-liability ratio (Lev). The model is structured as follows:i and t correspond to the enterprise and year, respectively. The constant term is denoted as α, while ε represents the error term. The coefficients are represented by β1,β2,……β7. The dependent variable, INNOV, signifies the level of innovation investment undertaken by the firm. The explanatory variables consist of the total digital finance index (DIFI) along with its sub-indices . Moreover, CV represents the set of control variables mentioned earlier. To mitigate the impact of the industry in which the enterprise is located on innovation, this study uses a fixed effects model, controlling for time effects (Year) and industry effects (Industry).In the provided equations, the variables In order to gain a deeper understanding of the mechanism through which digital inclusive finance influences innovation in small and medium-sized enterprises (SMEs), the present study introduces financing constraints (denoted as SA) as an intermediary variable. The absolute value of SA is employed as a proxy indicator for financing constraints. This approach is undertaken to empirically investigate whether digital inclusive finance facilitates the promotion of technological innovation activities within SMEs by alleviating their financing constraints. The research employs a stepwise approach to examine and analyze the influencing mechanism of how digital inclusive finance fosters innovation within SMEs. The regression model is specified as follows:α0,β0,γ0" represents the intercept term, "ε" denotes the error term, and "α1,β1,γ1,γ2" signifies the regression coefficients. The variables and indicators are defined as described in the preceding text. According to the principles of the mediation effects model, the total effect “α1”, direct effect “γ1”, and indirect effect “β1γ2” should satisfy the following equation: α1 = γ1+βγ2. In consideration of the theoretical analysis and research assumptions outlined earlier, if the coefficient of "α1" is positive, it signifies that the development of digital inclusive finance, on the whole, fosters the advancement of technological innovation in SMEs. Concurrently, if the coefficient of "β1" is negative, it indicates that the development of digital finance mitigates the financing constraints faced by SMEs. Furthermore, if the coefficient of "γ2" is negative, it implies that the reduction of financing constraints can enhance the level of technological innovation among SMEs [In Eqs , 3), an, an3), aong SMEs .This section presents descriptive statistics for the variables under investigation, with summarized findings presented in the total digital inclusive finance index (DIFI), the coefficient of coverage breadth index (DUD), the usage depth index (DSS), and the digital service support index (DCB) all exhibit positive coefficients. Furthermore, the associated p-values for these variables are less than 0.01 or 0.05, underscoring their statistically significant and positive effects. These empirical results, detailed in This study employs regression analysis to explore the relationship between the development of digital inclusive finance and the innovation level of SMEs. The explanatory variables consist of the total digital inclusive finance index and individual dimension indices. The regression outcomes, as presented in Conversely, the coefficient of return on assets (ROA) displays a significant negative relationship. This implies that enterprises with more robust profitability might display reduced inclination and incentive for innovation. Additionally, these enterprises could be constrained by external financing limitations, prompting them to adopt a more cautious approach toward innovation investment. Instead, they might prioritize retaining profits for internal use rather than engaging in innovative endeavors. Furthermore, the coefficient linked to leverage (Lev) showcases a significant negative correlation. Enterprises carrying higher leverage encounter difficulties in meeting repayment obligations, which subsequently curtails their access to innovative financing opportunities. Consequently, these enterprises are prone to exhibit diminished levels of investment in innovation. Cumulatively, the regression analysis pertaining to the control variables contributes valuable insights into the determinants influencing the innovation capabilities of SMEs.In accordance with the testing procedure of the intermediary effect, the initial step involves scrutinizing the aggregate impact of inclusive digital finance on innovation within the realm of SMEs. The outcomes of this analysis, delineated in the first column of The discernible reduction in the coefficient associated with the nexus between digital inclusive finance and innovation, upon the inclusion of considerations pertaining to financing constraints, can be ascribed to the prevailing reliance of a majority of SMEs upon constricted internal financial resources. Consequently, these entities encounter they encounter impediments in bridging the financing chasm requisite for the sustenance of their R&D activities . In contIn order to scrutinize the divergent effects of digital inclusive finance on investments in innovation across diverse enterprise types, this study employs a classification framework rooted in property rights and technological attributes for the purpose of categorizing SMEs. Subsequent to this classification, a collective regression analysis is undertaken, with the ensuing outcomes being meticulously delineated in The research findings, outlined in Columns (3) and (4) within In order to fortify the integrity of the findings, this study employed two rigorous tests for robustness. Firstly, the dependent variable underwent substitution with the count of patent applications submitted by SMEs, encompassing various patent types such as inventions, designs, and utility models. This test aimed to scrutinize the impact of digital inclusive finance on the innovative endeavors of SMEs through an alternative metric. Secondly, in acknowledgment of the sway exerted by the COVID-19 pandemic, the scope of the dependent variable was confined to data until 2019, facilitating robustness testing within this constrained timeframe. The outcomes of these examinations are expounded in To address potential concerns related to endogeneity and enhance the resilience of the findings, this study employs a set of strategies designed to mitigate bias stemming from endogeneity. Two principal sources of endogeneity are meticulously examined within the analysis. Firstly, the issue of reverse causality is addressed, wherein the technological innovation within SMEs might conceivably influence the progression of digital inclusive finance . To counWithin this study, a fixed-effects model has been adeptly employed, with data spanning from 2011 to 2021, amalgamating the digital inclusive finance index with panel data encompassing SMEs listed on stock exchanges for empirical analysis. The resultant findings robustly substantiate the pivotal role played by digital inclusive finance in propelling SME innovation. Noteworthy facets such as coverage, depth of utilization, and the extent of digital service provision within the purview of digital inclusive finance all demonstrate a positive contribution to the advancement of innovation. This contribution is notably manifested through the alleviation of financing constraints encountered by SMEs, consequently fostering heightened levels of innovation activity. Heterogeneity analysis underscores that digital inclusive finance distinctly affects non-state-owned SMEs and high-tech SMEs with respect to their innovation endeavors. The preeminence of digital inclusive finance in elevating the innovation capacities of these specific categories of enterprises is a testament to its potential to redress conventional financial resource disparities. These segments, facing more acute financing constraints in comparison to others, observe a more substantial impetus towards innovation driven by digital inclusive finance. In light of these research outcomes, the following policy recommendations are posited:Firstly, cultivating healthy development and easing financing constraints: At the government level, an active impetus should be lent to the cultivation of digital inclusive finance. This could entail encouraging the digitization and modernization of traditional financial services, accompanied by the provision of a diverse array of financial products and services meticulously tailored to the technological innovation and growth of SMEs. Financial institutions should embrace digital transformation, intensifying their support for digital technology to facilitate the expansion of inclusive finance. Convenient and reasonably priced financial services ought to be extended to SMEs, efficaciously mitigating their financing restrictions. Regulatory frameworks for digital inclusive finance should be refined, with a comprehensive comprehension of financial technology, collaborative regulatory mechanisms established, and prudent expansion of regulated digital inclusive finance ensured. This approach safeguards its status as a valuable instrument catering to the innovative financing aspirations of SMEs.Secondly, tailored policies for heterogeneous SME innovation: Discerning that different enterprises grapple with varying financing challenges and that the influence of digital inclusive finance on innovation is divergent, targeted policies are recommended. A pronounced elevation in innovation levels is observed for non-state-owned and high-tech SMEs due to the pivotal role of digital inclusive finance in reconciling traditional financial resource discrepancies. Recognizing this, digital inclusive finance institutions should intimately acquaint themselves with the developmental idiosyncrasies of these enterprises and proffer bespoke financial products that ameliorate financing expenses, bolster innovation financing efficiency, and expedite innovation undertakings. For instance, harnessing digital technology for precise big data models, assessing the value of innovation projects, and enhancing SMEs’ access to financing can mitigate industry-based biases, thereby engendering a just financing milieu that stimulates innovation ardor.Thirdly, augmenting SME self-improvement and prudent resource allocation: In an epoch characterized by rapid digital advancements, SMEs should enhance their adeptness in interfacing with digital inclusive finance to fuel their own progress. Augmenting enterprise operation and financial management systems, including comprehensive financial research and development project information disclosure, can alleviate financing impediments and streamline innovation financing efficiency. concurrently, SMEs should bolster their proficiency in leveraging digital inclusive finance. This necessitates bolstering financial literacy, making informed decisions about the allocation of digital inclusive finance resources, and cherry-picking fitting financial products and services in alignment with their distinct attributes and financing requisites.


JCI, Sun and colleagues detail a mechanism by which local activation of the nuclear receptor liver receptor homolog-1  directly induces de novo asparagine synthesis and expression of asparagine synthetase (ASNS) in response to injury and show that this response restrains hepatic damage. This work opens up several avenues for inquiry, including the potential for asparagine supplementation to ameliorate acute hepatic injury.Acute hepatic injury is observed in response to various stressors, including trauma, ingestion of hepatic toxins, and hepatitis. Investigations to date have focused on extrinsic and intrinsic signals required for hepatocytes to proliferate and regenerate the liver in response to injury, though there is a more limited understanding of induced stress responses promoting hepatocyte survival upon acute injury. In this issue of the  JCI, Sun, et al. advance our understanding of such prosurvival signals in response to hepatocyte injury, delineating a mechanism by which hepatocyte activation of the nuclear receptor liver receptor homolog-1 (LRH-1) rewires metabolic networks to drive asparagine synthesis from glutamine, a process that limits the extent of hepatocellular death and liver injury without affecting proliferation  also expressed asparagine synthetase (ASNS), but lacked glutaminase, glutamine’s primary catabolic enzyme in the cell. Sun et al. demonstrated that ASNS, the enzyme required to synthesize asparagine from glutamine and aspartate, was maximally induced upon sublethal dosing of carbon tetrachloride (CCl4) or acetaminophen 24 hours after administration. The observation that genetic ablation of hepatic ASNS enhanced liver damage in both models confirmed that this response was adaptive. Intriguingly, ASNS was not induced through classical cellular stress response pathways  but rather through the activity of LRH-1. Hepatic LRH-1–knockout mice exhibited low ASNS expression and increased damage in response to chemically induced hepatic injury. Conversely, two mouse models of LRH-1 gain-of-function — the activating LRH-1 K289R point mutation and knockout of hepatic small heterodimer protein (SHP), a negative regulator of LRH-1 — exhibited upregulated ASNS expression and showed substantial protection from hepatic injury. ChIP and cotransfection assays confirmed that LRH-1 bound directly to the ASNS promoter and increased transcriptional activity. Since genetic ablation of hepatic ASNS alone could not indicate the relative impact of either depletion of substrate or generation of product, Sun et al. confirmed, via amino acid profiling and evaluating the consequences of i.v. injection, that the ASNS product asparagine, but not glutamate or valine, was indeed the protective metabolite  and the constitutive androstane receptor (CAR) expression have been shown to affect liver regeneration following injury, linking both metabolic signals — i.e., bile acids — and xenobiotic metabolism to injury and repair , 14. PPAUnlike ASNS’s product asparagine, its substrate glutamine has been intensively studied in both cellular and clinical contexts. Interest in glutamine stems from early observations by Bergstrom and colleagues  that freCollectively, the results by Sun and colleagues identify an endogenous hepatoprotective program relying on de novo asparagine synthesis induced by LRH-1 in the hepatocyte to promote cell survival in response to injury . These r


The whole-body iodine-131 scintigraphy is an imaging technique in monitoring patients with a history of thyroid cancer. Although the rate of false positives is negligible, it is not nonexistent. We report the case of an intervened and treated patient for thyroid cancer with good clinical and biochemical response. Scintigraphic findings were consistent with unsuspected bone metastasis. Fused SPECT/CT data allowed accurate diagnosis of giant diaphragmatic hernia associated with intrathoracic stomach, a very rare pathology that can lead to false positive results. During regular follow-up, a whole-body 131I scintigraphy was performed 12 months after treatment ends in order to evaluate the persistence of thyroid remainders. Whole-body 131I scan  was performed due to discordance between biochemical findings (low hormone levels) and the clinical picture of intense back pain and functional imaging consistent with spine bone metastasis. A severe thoracolumbar scoliosis that determines a giant diaphragmatic hernia was revealed thanks to multimodality scan . TherebThe giant diaphragmatic hernia associated with intrathoracic stomach is a very rare entity  that se131I scintigraphy has proven to be a minimally invasive and safe technique that allows the diagnosis metastasis and recurrence after thyroidectomy when performed at 6-12 months of thyroid remainders ablation with radioiodine, as well as in monitoring during the long-term follow-up. However, the ATA and ETA guidelines state that low-risk individuals who have had a first post-radioactive iodine remnant ablation whole-body scan with an undetectable Tg level and a negative anti-Tg antibody level as well as a negative neck ultrasound do not require routine follow-up whole-body scan. Even if false positives in 131I body scans are extremely rare, they have been reported due to contamination by body fluids, ectopic thyroid tissue, infectious and inflammatory processes, benign and malignant tumors, serous cysts and even pulmonary bronchiolitis  of 131I  demonst131I SPECT/CT  was performed in order to clarifying our clinical and radiological inconsistency. Fused images, SPECT alone and CT alone  revealed severe kyphoscoliosis associated with giant diaphragmatic hernia and intrathoracic stomach. In this way, hypermetabolic foci corresponded to physiological radiotracer uptake of the gastric mucosa.A It is known that SPECT/CT was highly accurate in patients who underwent a single challenge of radioiodine therapy . In our


Traditional cell biological techniques are not readily suitablefor studying lipid signaling events because genetic perturbationsare much slower than the interconversion of lipids in complex metabolicnetworks. For this reason, novel chemical biological approaches havebeen developed. One approach is to chemically modify a lipid witha so-called “caging group” that renders it inactive,but this cage can be removed photochemically inside cells to releasethe bioactive molecule. These caged compounds offer unique advantagesfor studying the kinetics of cellular biochemistry and have been extensivelyused in the past. However, a limitation of conventional caged compoundsis their ability to diffuse freely inside the cell, which does notpermit localized activation below optical precision. This poses achallenge for studying lipid signaling as lipid function inside cellsis tightly linked to their parent membrane. An ideal lipid probe should,therefore, be restricted to a single organelle membrane or preferentiallyto a single leaflet. We first demonstrated the plasma-membrane-specificphotorelease of fatty acids by employing sulfonated caging groups.Using these caged fatty acid probes we demonstrated that lipid localizationdetermines signaling outcome. Generalizing this approach, we designeda so-called “click cage” that can be coupled to lipidsand offers the possibility to attach organelle targeting groups viaclick chemistry. Using this strategy, we have synthesized plasma membrane,lysosomal, mitochondria, and endoplasmic-reticulum-targeted lipidsthat can be used to dissect organelle-specific signaling events. Toreduce the synthetic effort associated with generating caged compounds,we designed a coumarin triflate reagent that allows the direct functionalizationof phosphate- or carboxylate-containing compounds. With this novelreagent, we synthesized a small library of photocaged G-protein-coupledreceptor (GPCR) ligands to study the underlying lipid signaling dynamics.Most recently, we have focused on quantifying the kinetics of lipidsignaling for different diacylglycerol (DAG) species using plasma-membrane-targetedcaged DAGs. Using this approach, we quantitatively measured lipid–proteinaffinities and lipid transbilayer dynamics in living cells. Afteranalyzing DAGs with different acyl chain length and saturation degree,we discovered that affinities can vary by up to an order of magnitude.This finding clearly shows that cells are able to distinguish betweenindividual DAG species, thereby demonstrating that lipid diversitymatters in cellular signal processing. Although the recent advanceshave yielded valuable tools to study lipid signaling, challenges remainon specifically targeting the different leaflets of organelle membranes.Furthermore, it is necessary to simplify the experimental approachesrequired for parametrizing and corroborating quantitative kineticmodels of lipid signaling. In the future, we envision that the applicationof leaflet-specific caged lipids to model membrane systems will beof crucial importance for understanding lipid asymmetry. NadlerA.; YushchenkoD. A.; MüllerR.; SteinF.; FengS.; MulleC.; CartaM.; SchultzC.Exclusive Photoreleaseof Signaling Lipids at the Plasma Membrane. Nat. Commun.2015, 6 ( (1), ), 1005626686736.1 Development of plasma membrane specific caging groups for fattyacids. Shows that fatty acid localization and function are linked.Release of arachidonic acid in the plasma membrane triggers Ca2+ oscillations in MIN6 cells, release in internal membranesresults in downregulation.WagnerN.; StephanM.; HöglingerD.; NadlerA.AClick Cage: Organelle-Specific Uncagingof Lipid Messengers. Angew. Chem. Int. Ed.2018, 57 ( (40), ), 13339–13343.2 Streamlined the synthesis of organelle-specific caginggroups by introducing a butynyl unit in place of an ethyl group atthe tertiary aromatic amine of the diethylaminocoumarin caging group.WagnerN.; SchuhmacherM.; LohmannA.; NadlerA.A Coumarin Triflate Reagent Enables One-Step Synthesisof Photo-Caged Lipid Metabolites for Studying Cell Signaling. Chem. Eur. J.2019, 25 ( (68), ), 15483–1548731461184.3 Show the in situ synthesisof diethylaminocoumarin-triflate to directly functionalize nativelipids with a photocaging group at the phosphate or carboxylate moieties.SchuhmacherM.; GrasskampA. T.; BarahtjanP.; WagnerN.; LombardotB.; SchuhmacherJ. S.; SalaP.; LohmannA.; HenryI.; ShevchenkoA.; CoskunÜ.; WalterA. M.; NadlerA.Live-Cell Lipid Biochemistry Reveals a Role of DiacylglycerolSide-ChainComposition for Cellular Lipid Dynamics and Protein Affinities. Proc. Natl. Acad. Sci.2020, 117 ( (14), ), 7729–773832213584.4 By making useof different caged diacylglycerol species (DAGs), the authors showthat the structural differences in DAGs are relevant for PKC signalspecification. Importantly, the authors calculated that protein-affinitiesfor two native DAGs differ by an order of magnitude.5 To perform these differentfunctions, numerous kinds of lipids are synthesized by the cell. Theycan be subdivided in categories, including fatty acids, glycerolipids,phosphoglycerolipids, sphingolipids, and sterols.6 Each lipid category is composed of several lipid classesthat differ in their specific hydrophilic headgroup. Individual lipidclasses in turn feature numerous lipid species that differ in acylchain length and saturation degree. Taken together, this providesmammalian cells with a chemical space in which several thousands ofdifferent lipid species can be realized.8 Mass spectrometricanalysis has confirmed that mammalian cells do, in fact, produce manyhundreds, if not thousands, of the theoretically possible lipid species.9 The realization of the chemical complexity ofthe cellular lipidome has given rise to the term lipid diversity andin turn triggered questions about the biological roles of these molecules.Lipids perform three main functions incells. They constitute limitingmembranes of individual organelles and the entire cell, they are theprimary cellular means for long-term energy storage, and they arekey components in signaling pathways as receptor ligands and secondmessengers.11 For example, phosphatidylcholines are the main membrane-forminglipids,12 and cardiolipins ensure the functioningof the unique inner mitochondrial membrane.13 Furthermore, we roughly know how lipid classes are distributed betweenorganelles5 and even individual membraneleaflets.14 However, lipidomic analysesof diseased tissue,15 plasma samples,17 and also genetically modified cells18 often reveal significant changes on the level of individual lipidspecies rather than entire lipid classes. Such findings strongly suggestthe existence of cellular regulatory mechanisms that control the levelof individual, molecularly distinct lipid species. In turn, biologicalfunction would be associated with lipid species, rather than withentire lipid classes. However, mechanistic analysis of the role ofindividual lipid species in cell biology is still severely constrainedby the available methodological repertoire that can be brought tobear on the problem. Unlike proteins, lipids cannot be faithfullyvisualized by attaching fluorescent tags—the chemical changesintroduced by such modifications far outweigh the chemical differencesbetween individual lipid species from the same class. Furthermore,genetic perturbations are a very indirect way to modulate lipid levels,as the consequences of removing components of the lipid handling machineryare difficult to predict.The cell biology of lipids is best understood at the level of lipidclasses. Various phosphatidylinositides contribute to defining organelleidentities and play a role in signaling cascades.21 photocaged22 and photoswitchable lipids28 for modulating lipid levels, and a number of applications of bioorthogonalchemistry for the visualization of lipid metabolism and localizationand the study of the lipid–protein interactome.34 Within this broader push by the chemical biology community towardthe development of more accurate methods for studying lipids, ourlaboratory has focused on developing photocaged lipid probes thatallow one to perturb the levels of individual lipids in an organellein a specific and quantitative manner. The present Account detailsour efforts over the last years in this space.Because traditional cell biologicaltechniques are less suitedto study the functions of lipids, numerous chemical biology approacheshave been developed in order to expand the methodological repertoire.These tools include chemical dimerizer approaches,38 They are applied to biological samples in their inactive form andare activated by photorelease of the bioactive molecule through photolysis.Photolysis is initiated during the course of an experiment, e.g.,a fluorescence microscopy time lapse or an electrophysiological recording.Because the photolysis reaction is typically orders of magnitude fasterthan the investigated biological process, and because the cellularresponse is triggered only by the unmodified bioactive compound afterphotolysis, caged compounds offer unique advantages for studying thekinetics of cellular biochemistry and have been extensively used inthat role for decades.Cagedcompounds are bioactive molecules that are rendered inactiveby attaching a photoremovable protecting group that inhibits theirbiological activity.42 They are either applied extracellularly or converted into bioactivatableprodrugs through, e.g., functionalization with acetoxymethyl (AM)groups to convert them to AM esters that are cell permeable and allowdelivery into the cytoplasm prior to photoactivation. In both cases,they are able to rapidly diffuse through space (as their parent molecules),and the question of localized activation on submicron scales is largelyirrelevant since rapid, largely unhindered diffusion in three dimensionsis an inherent element of their mode of action. Thus, the precisionthat can be achieved by optical means is typically sufficient for initiating local perturbations in cellbiological experiments , a lipid that acts as a second messenger during cell signalingat the plasma membrane.44 DAG can also serveas the key intermediate for glycerophospholipid biosynthesis in themembrane of the endoplasmic reticulum45 and is a catabolic intermediate during the mobilization of energystored in the form of triacylglycerides in lipid droplets.46This is not strictly true for the application of caged lipids.Lipids act mostly in cellular membranes, and the investigation oftheir biological functions necessitates taking the cellular architectureinto account. The membranes of cellular organelles feature distinctlipid compositions that furthermore differ in the degree of asymmetriclipid distribution between the membrane leaflets.To avoid affecting multiple unrelated processes in the sameexperiment,lipid photoactivation should preferentially be restricted to a singleorganelle membrane, ideally to a single leaflet. Distinct cellularmembranes come as close as 15–20 nm in membrane contact sites,whereas individual membrane leaflets have an average width of 2.5nm. These dimensions suggest that photoactivation in specific cellularmembranes is not possible using current optical technology if thecaged lipid is distributed through all cellular membranes. The problemof localized lipid uncaging thus necessitates a chemical solution—cagedlipids need to be prelocalized in the membrane/compartment of interestprior to photoactivation. Since, in this case, photoactivation canonly take place where the caged compound resides, off-target lightwill not have any effects if careful controls for phototoxicity arein place.1 and has been driven forward byour laboratory and others  since then. Here, we focusmainly on our contributions to the field, retrace the developmentand applications of prelocalized caged lipids during the last years,and offer a perspective for future directions.The development of organelle-specific caged lipidswas initiatedby one of us (A.N.) together with colleagues in the Schultz laboratoryin 201553 and the realization that simple fatty acids,such as arachidonic acid, fulfill highly specialized functions insmall signaling compartments.54The decisionto develop organelle-specific approaches for modulatingthe levels of molecularly distinct lipid species with light was motivatedby our finding that individual diacylglycerol species have differentabilities to trigger localized signaling responses in uncaging experimentsKa value of the sulfonated compound—asmall but sufficient portion of the carboxylated caging group is protonatedat any given time and can, thus, passively cross the plasma membranewith a nonzero rate, which is not the case for the sulfonated probe.Wefirst set out to develop strategies that would enable us todeliver caged lipid probes specifically to the plasma membrane. Wefocused on two fatty acids, arachidonic acid and oleic acid, as firstproof-of-principle examples to demonstrate the feasibility of thisapproach. Both molecules serve as first and second messengers in cellularsignaling besides their role as crucial intermediates in lipid biogenesisand energy metabolism. Since any chemical probe has to move acrossthe plasma membrane to reach the cytoplasm, we reasoned that modificationsof the caging group could block transbilayer movement and would allowus to generate probes that exclusively localize to the outer leafletof the plasma membrane prior to uncaging–provided that endocytosisis sufficiently slow. Specifically, the introduction of negative chargeson the utilized caging group should prevent caged lipid probes fromcrossing the plasma membrane, as the hydrophobic side chains wouldincorporate into the outer leaflet, but flipping would be thermodynamicallyhighly unfavorable 2a. To acUsing these probes, we found that plasma membrane localizationwas largely maintained in Hela and MIN6 cells for 30–60 minafter the initial loading procedure, after which significant probeinternalization by endocytosis became observable 2b. The mPlasma-membrane-specific caged lipids can be used in combinationwith their nonspecific counterparts to investigate whether the intracellularlocalization of the lipid messenger photorelease is relevant for thesignaling outcome. We chose to address this question in two biologicalsystems: calcium signaling in insulin-secreting beta cells (usingthe MIN6 model cell line) and the modulation of synaptic transmissionin mossy fiber to CA3 synapses, which we analyzed in mouse brain slices.Beta cells exhibit spontaneous calcium transients if glucose is presentin the medium, which often display oscillatory patterns. Upon uncagingof either arachidonic acid or oleic acid on the plasma membrane, calciumtransients were both more pronounced and more frequent 2e,f. In In a complementaryset of experiments, we used arachidonic aciduncaging at the plasma membrane and internal membranes in mossy fiberto CA3 synapses and performed whole-cell voltage clamp recordingsof excitatory postsynaptic currents. We found that photorelease ofarachidonic acid at the plasma membrane resulted in sustained potentiationof synaptic transmission, whereas photorelease on internal membraneshad a significantly smaller effect. This finding is in line with thedirect action of arachidonic acid on presynaptic Kv channels.Taken together, this first study on organelle-specific lipid uncagingdemonstrated that plasma-membrane-targeted probes can be obtainedby decorating the caging group with sulfonates, which prevents lipidflipping. We showed that these probes can be used to monitor uncagingphotoreactions using fluorescence microscopy because they remain inthe exoplasmic leaflet of all cellular membranes. These first biologicalapplications clearly demonstrated that the site of lipid photoreleaseis of great importance for ultimate signaling outcome. We furtherdemonstrated that the photorelease of arachidonic acid can be eitherstimulatory (on the plasma membrane) or inhibitory for MIN6 calcium oscillations. Together, these findings highlightedthe need for developing finely tailored lipid probes designed to perturband study individual cellular compartments.48 However, both probe types and other theoreticallyaccessible probes require dedicated synthetic routes, thereby limitingtheir application potential. This speaks to a general problem in chemicalbiology. As probes become ever more sophisticated to account for therequirements of complex cell biological experiments, the syntheticeffort to generate probes increases. This leads to a point where probesyntheses are only carried out for a designated project and not repeatedfor follow-up studies. Thus, streamlined strategies for generatinghighly specialized probes are warranted.Our next aim wasto increase the flexibility of organelle-targetedlipids. Besides our work on plasma-membrane targeted lipids, the Riezmannlaboratory reported probes that could be directed to the mitochondria.2 On the basis of the DEAC scaffold, the “clickcage” is equipped with a butynyl unit in the place of an ethylgroup at the tertiary aromatic amine  ligands, including lysophosphatidicacid, platelet activating factor, and prostaglandin E2. After synthesizingan entire set of photocaged GPCR ligands  are generated and signal at theplasma membrane after cell surface receptor activation. They recruiteffector proteins to the plasma membrane, mostly through DAG-C1 domaininteractions. In turn, these effector proteins subsequently propagatethe cellular signal to nodes further downstream in the signaling network.In a secondary mode of action, DAGs can also function as an agonistfor plasma-membrane-localized calcium channels, including the transientreceptor potential (TRP) channels TRPC3 and TRPC6.4 including the arachidonate-containing variantcaged 1-stearoyl-2-arachidonoyl-sn-glycerol (cgSAG),which is considered to be the archetypical signaling DAG; the oleicacid containing caged species 1-stearoyl-2-oleoyl-sn-glycerol (cgSOG), which corresponds to one of the most prevalentDAG species in mammalian cells; and the short chain, soluble cagedDAG analogue 1,2-dioctanoyl-sn-glycerol (cgDOG),which does not correspond to a native DAG species but has neverthelessbeen heavily used in cell biological experiments , which faithfully replicates thephotoreaction but does not induce signaling responses at the plasmamembrane  and novel  protein kinase C isoforms, important signaling effector proteinsthat all contain DAG-binding C1 domains , a fusion protein composedof a C1 domain, EGFP, and a nuclear exclusion signal, which was includedto prevent plasma membrane recruitment kinetics from being obscuredby the slow release of a nuclear protein pool 4f. In orWe next conductedDAG uncaging experiments in a light dose-dependentmanner and acquired protein recruitment traces for a wide range ofinitial levels of photoreleased DAGs to, in effect, establish a quantitative,kinetic dose–response curve for DAG-driven protein recruitmentto the plasma membrane. We used these data to conduct a parameteroptimization for a minimal, kinetic model that describes DAG transbilayermovement, DAG–protein affinities, and DAG turnover. Despitethe fact that the model contained only four free parameters, we foundthat it could explain the obtained data very well, and we were ableto identify values for rate constants and affinities for all analyzedDAGs 4g. Wefo52 Leaflet-specific tools for theother organelles would, thus, be of great interest. Furthermore, whilephotoreactions can be quantified in principle in living cells, itrequires significant experimental effort. Ideally, methods shouldbe developed to infer the initial photoreaction yield from fittingkinetic models preferentially on a single cell level. Finally, weand all other laboratories in the field have had a strong focus onsignaling lipids. While this reflects the fact that small absoluteconcentration changes will lead to strong cellular responses and minimizethe probability of adverse effects of the induced photoreactions,it also constitutes a systematic bias with regard to the investigatedpart of the cellular lipidome. Thus, tools for studying the role ofperceived “structural” lipid species should constitutea primary focus of future probe development efforts. The applicationof such probes should not be limited to cells but expanded to modelmembrane systems to complement applications of photoswitchable lipids.Leaflet-specific caged lipid probes, in particular, could prove invaluablefor generating realistic model membrane systems for the study andultimately understanding of lipid asymmetry.In summary, our work over the last years has led to the generationof caged lipid probes that now can be used to modulate the levelsof native lipid species in an organelle and, in some cases, in a leaflet-specificmanner in living cells. By developing modular synthetic routes andnew synthetic methodologies, we have made these tools readily accessibleto explore the chemical space of the diverse cellular lipidome. Incombination with quantitative microscopy approaches, the organelle-specificuncaging of lipids can be employed to perform quantitative lipid biochemistryin living cells. Such a capability is essential for studying the biologicalfunction of lipids on the species level, as shown in the example ofsignaling DAGs. Despite these advances, major hurdles remain. Fornow, only plasma membrane lipids can be modulated in a leaflet-specificmanner, as demonstrated by us and the Frank laboratory for the outerleaflet.