An ultra-long-acting L-asparaginase synergizes with an immune checkpoint inhibitor in starvation-immunotherapy of metastatic solid tumors.

Metastasis stands as the primary contributor to mortality associated with tumors. Chemotherapy and immunotherapy are frequently utilized in the management of metastatic solid tumors. Nevertheless, these therapeutic modalities are linked to serious adverse effects and limited effectiveness in preventing metastasis. Here, we report a novel therapeutic strategy named starvation-immunotherapy, wherein an immune checkpoint inhibitor is combined with an ultra-long-acting L-asparaginase that is a fusion protein comprising L-asparaginase (ASNase) and an elastin-like polypeptide (ELP), termed ASNase-ELP. ASNase-ELP's thermosensitivity enables it to generate an in-situ depot following an intratumoral injection, yielding increased dose tolerance, improved pharmacokinetics, sustained release, optimized biodistribution, and augmented tumor retention compared to free ASNase. As a result, in murine models of oral cancer, melanoma, and cervical cancer, the antitumor efficacy of ASNase-ELP by selectively and sustainably depleting L-asparagine essential for tumor cell survival was substantially superior to that of ASNase or Cisplatin, a first-line anti-solid tumor medicine, without any observable adverse effects. Furthermore, the combination of ASNase-ELP and an immune checkpoint inhibitor was more effective than either therapy alone in impeding melanoma metastasis. Overall, the synergistic strategy of starvation-immunotherapy holds excellent promise in reshaping the therapeutic landscape of refractory metastatic tumors and offering a new alternative for next-generation oncology treatments.

Efficiency of acetate-based isopropanol synthesis in Escherichia coli W is controlled by ATP demand.

BACKGROUND: Due to increasing ecological concerns, microbial production of biochemicals from sustainable carbon sources like acetate is rapidly gaining importance. However, to successfully establish large-scale production scenarios, a solid understanding of metabolic driving forces is required to inform bioprocess design. To generate such knowledge, we constructed isopropanol-producing Escherichia coli W strains. RESULTS: Based on strain screening and metabolic considerations, a 2-stage process was designed, incorporating a growth phase followed by a nitrogen-starvation phase. This process design yielded the highest isopropanol titers on acetate to date (13.3 g L-1). Additionally, we performed shotgun and acetylated proteomics, and identified several stress conditions in the bioreactor scenarios, such as acid stress and impaired sulfur uptake. Metabolic modeling allowed for an in-depth characterization of intracellular flux distributions, uncovering cellular demand for ATP and acetyl-CoA as limiting factors for routing carbon toward the isopropanol pathway. Moreover, we asserted the importance of a balance between fluxes of the NADPH-providing isocitrate dehydrogenase (ICDH) and the product pathway. CONCLUSIONS: Using the newly gained system-level understanding for isopropanol production from acetate, we assessed possible engineering approaches and propose process designs to maximize production. Collectively, our work contributes to the establishment and optimization of acetate-based bioproduction systems.

Monitoring Autophagy During Drosophila Oogenesis.

Macroautophagy (autophagy hereafter) is an evolutionarily conserved mechanism that maintains the health of cells by degrading toxic proteins and damaged organelles within the lysosomes. Tissues like ovary are made up of heterogeneous cell types and each cell type has distinct levels of autophagy. Studying autophagy in a cell-type specific manner helps better understand the role of autophagy during oogenesis. Here, we describe assays for monitoring autophagy during oogenesis in Drosophila using the two protein markers, Atg8a and Ref(2)P.

The superiority of innovative spiral-interdigital microelectrode pattern in increasing the sensitivity of tracing synchronization via serum starvation in cellular metabolism.

In order to investigate the changes in the properties of the cell culture solution in the effect of cell synchronization via cell starvation (for 12, 24, and 36 h), a new spiral-interdigital pattern of microelectrode as a biosensor has been proposed. Then, to test its superiority, the results of this spiral-interdigital pattern with the results of the commercial pattern have been compared. The cells were selected from breast cancer standard lines (MDA-MB-231). Changes in CV peaks of the secretions were recorded by the spiral-interdigital pattern, in which increasing the interactive surface with homogenous electric paths had been considered by simulation before fabrication. The results of the simulation and experimental procedures showed a meaningful correlation. The occurrence of CV oxidative peaks at about 0.1-0.4 V and reductive peaks at approximately 0 V in the spiral-interdigital biosensor in the starved MDA-MB-231 cell line has been observed. The starvation situation resembles one that does not cause meaningful cell apoptosis or necrosis, and this method is only used to make the cells synchronized. Also, no peak is observed in normal cell growth conditions. In addition, by using the commercial design of the electrodes, no peak is observed in any of the conditions of normal and synchronized growth of the cells. Therefore, it seems that the observed peaks are caused by the agents that are secreted in the cell culture solution in a synchronized situation. Moreover, the design of the new spiral-interdigital electrode can significantly increase the sensitivity of the sensor to receive these peaks due to more space and a uniform electric field.

FOXO3 Activates MFN2 Expression to Maintain the Autophagy Response in Cancer Cells Under Amino Acid Deprivation.

The lack of amino acids triggers the autophagic response. Some studies have shown such starvation conditions also induce mitochondrial fusion, revealing a close correlation between the two processes. Although Mitofusin-2 (MFN2) has been demonstrated to play a role in fusion regulation, its role in the autophagic response and the variables that activate MFN2 under stress remain unknown. In this investigation, we screened and confirmed that forkhead box protein O3 (FOXO3) participates in MFN2's expression during short periods of starvation. Luciferase reporter test proved that FOXO3 facilitates MFN2's transcription by binding to its promoter region, and FOXO3 downregulation directly depresses MFN2's expression. Consequently, inhibiting the FOXO3-MFN2 axis results in the loss of mitochondrial fusion, disrupting the normal morphology of mitochondria, impairing the degradation of substrates, and reducing autophagosome accumulation, ultimately leading to the blockage of the autophagy. In conclusion, our work demonstrates that the FOXO3-MFN2 pathway is essential for adaptive changes in mitochondrial morphology and cellular autophagy response under nutritional constraints.

MANF facilitates breast cancer cell survival under glucose-starvation conditions via prkn-mediated mitophagy regulation.

During tumor expansion, breast cancer (BC) cells often experience reactive oxygen species accumulation and mitochondrial damage because of glucose shortage. However, the mechanism by which BC cells deal with the glucose-shortage-induced oxidative stress remains unclear. Here, we showed that MANF (mesencephalic astrocyte derived neurotrophic factor)-mediated mitophagy facilitates BC cell survival under glucose-starvation conditions. MANF-mediated mitophagy also promotes fatty acid oxidation in glucose-starved BC cells. Moreover, during glucose starvation, SENP1-mediated de-SUMOylation of MANF increases cytoplasmic MANF expression through the inhibition of MANF's nuclear translocation and hence renders mitochondrial distribution of MANF. MANF mediates mitophagy by binding to PRKN (parkin RBR E3 ubiquitin protein ligase), a key mitophagy regulator, in the mitochondria. Under conditions of glucose starvation, protein oxidation inhibits PRKN activity; nevertheless, the CXXC motif of MANF alleviates protein oxidation in RING II-domain of PRKN and restores its E3 ligase activity. Furthermore, MANF-PRKN interactions are essential for BC tumor growth and metastasis. High MANF expression predicts poor outcomes in patients with BC. Our results highlight the prosurvival role of MANF-mediated mitophagy in BC cells during glucose starvation, suggesting MANF as a potential therapeutic target.

Starvation Ketoacidosis in Pregnancy: An Unusual Presentation and Brief Literature Review.

Starvation ketoacidosis in pregnant patients is a rare but life-threatening condition that requires prompt diagnosis and timely treatment. A 35-year-old pregnant woman at 33 weeks' gestation was admitted for abdominal pain with poor oral intake. She was diagnosed with perforated appendicitis and underwent emergent laparotomy. During the procedure and afterwards, she was found to have an anion gap metabolic acidosis. She was treated with a dextrose infusion with a fixed-rate insulin with correction of metabolic parameters. Women in late pregnancy are at increased risk for ketosis from increased relative insulin resistance and enhanced lipolysis. There is no consensus on optimal management of starvation ketoacidosis of pregnancy; however, carbohydrate administration is a cornerstone of treatment. We chose simultaneous administration of carbohydrates with insulin to overcome any inherent insulin resistance and to suppress lipolysis with rapid resolution of the patient's metabolic derangements.

Nitrogen limitation-induced adaptive response and lipogenesis in the Antarctic yeast Rhodotorula mucilaginosa M94C9.

The extremotolerant red yeast Rhodotorula mucilaginosa displays resilience to diverse environmental stressors, including cold, osmolarity, salinity, and oligotrophic conditions. Particularly, this yeast exhibits a remarkable ability to accumulate lipids and carotenoids in response to stress conditions. However, research into lipid biosynthesis has been hampered by limited genetic tools and a scarcity of studies on adaptive responses to nutrient stressors stimulating lipogenesis. This study investigated the impact of nitrogen stress on the adaptive response in Antarctic yeast R. mucilaginosa M94C9. Varied nitrogen availability reveals a nitrogen-dependent modulation of biomass and lipid droplet production, accompanied by significant ultrastructural changes to withstand nitrogen starvation. In silico analysis identifies open reading frames of genes encoding key lipogenesis enzymes, including acetyl-CoA carboxylase (Acc1), fatty acid synthases 1 and 2 (Fas1/Fas2), and acyl-CoA diacylglycerol O-acyltransferase 1 (Dga1). Further investigation into the expression profiles of RmACC1, RmFAS1, RmFAS2, and RmDGA1 genes under nitrogen stress revealed that the prolonged up-regulation of the RmDGA1 gene is a molecular indicator of lipogenesis. Subsequent fatty acid profiling unveiled an accumulation of oleic and palmitic acids under nitrogen limitation during the stationary phase. This investigation enhances our understanding of nitrogen stress adaptation and lipid biosynthesis, offering valuable insights into R. mucilaginosa M94C9 for potential industrial applications in the future.

Growth Phase Contribution in Dictating Drug Transport and Subcellular Accumulation inside Escherichia coli.

Depending upon nutrient availability, bacteria transit to multiple growth phases. The transition from the active to nongrowing phase results in reduced drug efficacy and, in some cases, even multidrug resistance. However, due to multiple alterations in the cell envelope, probing the drug permeation kinetics during growth phases becomes perplexing, especially across the Gram-negative bacteria's complex dual membrane envelope. To advance the understanding of drug permeation during the life cycle of Gram-negative bacteria, we sought to address two underlying objectives: (a) how changes are occurring inside the bacterial envelope during growth and (b) how the drug permeation and accumulation vary across both the membranes and in subcellular compartments during growth. Both objectives are met with the help of nonlinear optical technique second-harmonic generation spectroscopy (SHG). Specifically, using SHG, we probed the transport kinetics and accumulation of a quaternary ammonium compound (QAC), malachite green, inside Escherichia coli in various growth phases. Further insight about another QAC molecule, propidium iodide, is accomplished using fluorescence microscopy. Results indicate that actively growing cells have faster drug transport and higher cytoplasmic accumulation than slow- or nongrowing cells. In this regard, the rpoS gene plays a crucial role in limiting drug transport across the saturation phase cultures. Moreover, within a particular growth phase, membrane permeability undergoes gradual changes much before the subsequent growth phase commences. These outcomes signify the importance of reporting the growth phase and rate in drug efficacy studies.

DoSPX1 and DoMYB37 regulate the expression of DoCSLA6 in Dendrobium officinale during phosphorus starvation.

BACKGROUND: Dendrobium officinale Kimura et Migo (D. officinale) is parasitic on rocks or plants with very few mineral elements that can be absorbed directly, so its growth and development are affected by nutritional deficiencies. Previous studies found that phosphorus deficiency promotes polysaccharides accumulation in D. officinale, the expression of DoCSLA6 (glucomannan synthase gene) was positively correlated with polysaccharide synthesis. However, the molecular mechanism by which the low phosphorus environment affects polysaccharide accumulation remains unclear. RESULTS: We found that DoSPX1 can reduce phosphate accumulation in plants and promote the expression of PSIs genes, thereby enhancing plant tolerance to low phosphorus environments.Y1H and EMSA experimental show that DoMYB37 can bind the promoter of DoCSLA6. DoSPX1 interact with DoMYB37 transiently overexpressed DoSPX1 and DoMYB37 in D. officinale protocorm-like bodies, decreased the Pi content, while increased the expression of DoCSLA6. CONCLUSIONS: The signaling pathway of DoSPX1-DoMYB37-DoCSLA6 was revealed. This provides a theoretical basis for the accumulation of polysaccharide content in D. officinale under phosphorus starvation.

Effect of Dietary Restriction on Gut Microbiota and Brain-Gut Short Neuropeptide F in Mud Crab, Scylla paramamosain.

Aquatic animals frequently undergo feed deprivation and starvation stress. It is well-known that the gut microbiota and the gut-brain short neuropeptide F (sNPF) play essential roles in diet restriction. Therefore, investigating the responses of the gut microbiota and sNPF can enhance our understanding of physiological adaptations to feed deprivation and starvation stress. In this study, we examined the alterations in the gut microbiota of juvenile mud crabs under feed deprivation and starvation conditions. The results reveal differences in the richness and diversity of gut microbiota among the satisfied, half food, and starvation groups. Moreover, the microbial composition was affected by starvation stress, and more than 30 bacterial taxa exhibited significantly different abundances among the three feeding conditions. These results indicate that the diversity and composition of the gut microbiota are influenced by diet restriction, potentially involving interactions with the gut-brain sNPF. Subsequently, we detected the location of sNPF in the brains and guts of mud crabs through immunofluorescence and investigated the expression profile of sNPF under different feeding conditions. The results suggest that sNPF is located in both the brains and guts of mud crabs and shows increased expression levels among different degrees of diet restriction during a 96 h period. This study suggested a potential role for sNPF in regulating digestive activities and immunity through interactions with the gut microbiota. In conclusion, these findings significantly contribute to our understanding of the dynamic changes in gut microbiota and sNPF, highlighting their interplay in response to diet restriction.

Regulation of bacterial stringent response by an evolutionarily conserved ribosomal protein L11 methylation.

Lysine and arginine methylation is an important regulator of enzyme activity and transcription in eukaryotes. However, little is known about this covalent modification in bacteria. In this work, we investigated the role of methylation in bacteria. By reanalyzing a large phyloproteomics data set from 48 bacterial strains representing six phyla, we found that almost a quarter of the bacterial proteome is methylated. Many of these methylated proteins are conserved across diverse bacterial lineages, including those involved in central carbon metabolism and translation. Among the proteins with the most conserved methylation sites is ribosomal protein L11 (bL11). bL11 methylation has been a mystery for five decades, as the deletion of its methyltransferase PrmA causes no cell growth defects. Comparative proteomics analysis combined with inorganic polyphosphate and guanosine tetra/pentaphosphate assays of the ΔprmA mutant in Escherichia coli revealed that bL11 methylation is important for stringent response signaling. In the stationary phase, we found that the ΔprmA mutant has impaired guanosine tetra/pentaphosphate production. This leads to a reduction in inorganic polyphosphate levels, accumulation of RNA and ribosomal proteins, and an abnormal polysome profile. Overall, our investigation demonstrates that the evolutionarily conserved bL11 methylation is important for stringent response signaling and ribosomal activity regulation and turnover. IMPORTANCE: Protein methylation in bacteria was first identified over 60 years ago. Since then, its functional role has been identified for only a few proteins. To better understand the functional role of methylation in bacteria, we analyzed a large phyloproteomics data set encompassing 48 diverse bacteria. Our analysis revealed that ribosomal proteins are often methylated at conserved residues, suggesting that methylation of these sites may have a functional role in translation. Further analysis revealed that methylation of ribosomal protein L11 is important for stringent response signaling and ribosomal homeostasis.

Early fasting does not impact gonadal size nor vasa gene expression in the European seabass Dicentrarchus labrax.

Primordial germ cells (PGCs) play a crucial role in sexual development in fish, with recent studies revealing their influence on sexual fate. Notably, PGC number at specific developmental stages can determine whether an individual develops as male or female. Temperature was shown to impact PGC proliferation and the subsequent phenotypic sex in some fish species. Here, we aimed at testing the role of food deprivation on gonad development in the European seabass Dicentrarchus labrax, a species displaying a polygenic sex determination system with an environmental influence. We subjected larvae to two periods of starvation to investigate whether restricting growth affects both gonadal size and vasa gene expression. We first confirmed by immunohistochemistry that Vasa was indeed a marker of PGCs in the European seabass, as in other fish species. We also showed that vasa correlated positively with fish size, confirming that it could be used as a marker of feminization. However, starvation did not show any significant effects on vasa expression nor on gonadal size. It is hypothesized that evolutionary mechanisms likely safeguard PGCs against environmental stressors to ensure reproductive success. Further research is needed to elucidate the intricate interplay between environmental cues, PGC biology, and sexual differentiation in fish.

Role of cultivation parameters in carbohydrate accretion for production of bioethanol and C-phycocyanin from a marine cyanobacterium Leptolyngbya valderiana BDU 41001: A sustainable approach.

The investigation aimed to augment carbohydrate accumulation in the marine cyanobacterium Leptolyngbya valderiana BDU 41001 to facilitate bioethanol production. Under the standardised physiochemical condition (SPC), i.e. 90 µmol photon m-2 s-1 light intensity, initial culture pH 8.5, 35 °C temperature and mixing at 150 rpm increased the carbohydrate productivity ∼70 % than the control, while a 47 % rise in content was obtained under the nitrate (N)-starved condition. Therefore, a two-stage cultivation strategy was implemented, combining SPC at the 1st stage and N starvation at the 2nd stage, resulting in 80 % augmentation of carbohydrate yield, which enhanced the bioethanol yield by ∼86 % as compared to the control employing immobilised yeast fermentation. Moreover, biomass utilisation was maximised by extracting C-phycocyanin, where a ∼77 % rise in productivity was recorded under the SPC. This study highlights the potential of L. valderiana for pilot-scale biorefinery applications, advancing the understanding of sustainable biofuel production.

Polyphosphate-kinase-1 dependent polyphosphate hyperaccumulation for acclimation to nutrient loss in the cyanobacterium, Synechocystis sp. PCC 6803.

Polyphosphate is prevalent in living organisms. To obtain insights into polyphosphate synthesis and its physiological significance in cyanobacteria, we characterize sll0290, a homolog of the polyphosphate-kinase-1 gene, in the freshwater cyanobacterium Synechocystis sp. PCC 6803. The Sll0290 protein structure reveals characteristics of Ppk1. A Synechocystis sll0290 disruptant and sll0290-overexpressing Escherichia coli transformant demonstrated loss and gain of polyphosphate synthesis ability, respectively. Accordingly, sll0290 is identified as ppk1. The disruptant (Δppk1) grows normally with aeration of ordinary air (0.04% CO2), consistent with its photosynthesis comparable to the wild type level, which contrasts with a previously reported high-CO2 (5%) requirement for Δppk1 in an alkaline hot spring cyanobacterium, Synechococcus OS-B'. Synechocystis Δppk1 is defective in polyphosphate hyperaccumulation and survival competence at the stationary phase, and also under sulfur-starvation conditions, implying that sulfur limitation is one of the triggers to induce polyphosphate hyperaccumulation in stationary cells. Furthermore, Δppk1 is defective in the enhancement of total phosphorus contents under sulfur-starvation conditions, a phenomenon that is only partially explained by polyphosphate hyperaccumulation. This study therefore demonstrates that in Synechocystis, ppk1 is not essential for low-CO2 acclimation but plays a crucial role in dynamic P-metabolic regulation, including polyP hyperaccumulation, to maintain physiological fitness under sulfur-starvation conditions.

Improvement of starvation resistance via periodic fasting is genetically variable in Drosophila melanogaster.

Organisms subjected to periodic nutrient limitation early in life exhibit improvements in aspects of survival, including resistance to some environmental stressors. Recent findings indicate that forms of periodic fasting such as intermittent fasting and time restricted feeding can improve starvation resistance. However, it remains unclear to what extent this survival improvement persists across different genetic backgrounds. In this study, we examine fasting-induced starvation resistance across a broad survey of wild-derived lineages and document genetic variation within this trait. We adopt a standard dietary intervention and show improvement to starvation resistance within a common laboratory lineage, replicating previous results. Next, we examine fasting-induced starvation resistance across isofemale lines collected across latitudes and in different seasons, and among inbred lines derived from flies collected on different continents. We discover genetic variation of fasting-induced starvation resistance, and show that fasting improved starvation resistance as often as it worsened starvation resistance. Fasted flies generally showed reduced fat concentration, and their starvation survival varied with sex, season of collection, and geographic origin. While specific lineages common to the laboratory can show a specific fasting-induced phenotype, we show that this result is not consistent across genetic backgrounds, reinforcing the idea that phenotypes observed in historic laboratory strains may not be conserved across a species.

An acridone based fluorescent dye for lipid droplet tracking and cancer diagnosis.

Lipid droplets (LDs) are spherical organelles that localize in the cytosol of eukaryotic cells. Different proteins are embedded on the surface of LDs, so LDs play a vital role in the physiological activities of cells. The dysregulation of LDs is associated with various human diseases, such as diabetes and obesity. Therefore, it is essential to develop a fluorescent dye that labels LDs to detect and monitor illnesses. In this study, we developed the compound BDAA12C for staining LDs in cells. BDAA12C exhibits excellent LD specificity and low toxicity, enabling us to successfully stain and observe the fusion of LDs in A549 cancer cells. Furthermore, we also successfully distinguished A549 cancer cells and MRC-5 normal cells in a co-culture experiment and in normal and tumour tissues. Interestingly, we found different localizations of BDAA12C in well-fed and starved A549 cancer cells and consequently illustrated the transfer of fatty acids (FAs) from LDs to mitochondria to supply energy for ß-oxidation upon starvation. Therefore, BDAA12C is a promising LD-targeted probe for cancer diagnosis and tracking lipid trafficking within cells.

Opportunity for genome engineering to enhance phosphate homeostasis in crops.

Plants maintain cellular homeostasis of phosphate (Pi) through an integrated response pathway regulated by different families of transcription factors including MYB, WRKY, BHLH, and ZFP. The systemic response to Pi limitation showed the critical role played by inositol pyrophosphate (PP-InsPs) as signaling molecule and SPX (SYG1/PHO81/XPR1) domain proteins as sensor of cellular Pi status. Binding of SPX to PP-InsPs regulates the transcriptional activity of the MYB-CC proteins, phosphate starvation response factors (PHR/PHL) as the central regulator of Pi-deficiency response in plants. Vacuolar phosphate transporter, VPT may sense the cellular Pi status by its SPX domain, and vacuolar sequestration is activated under Pi replete condition and the stored Pi is an important resource to be mobilized under Pi deficiency. Proteomic approaches led to new discoveries of proteins associated with Pi-deficient response pathways and post-translational events that may influence plants in achieving Pi homeostasis. This review provides current understanding on the molecular mechanisms at the transcriptional and translational levels for achieving Pi homeostasis in plants. The potential strategies for employing the CRISPR technology to modify the gene sequences of key regulatory and response proteins for attaining plant Pi homeostasis are discussed.

Ca2+ as an essential signaling molecule controlling Snf1-mediated Atg1 activation.

Macroautophagy/autophagy is essential for maintaining glucose homeostasis, but the mechanisms by which cells sense glucose starvation and initiate autophagy are not yet fully understood. Recently, we reported that the assembly of a Ca2+-triggered Snf1-Bmh1/Bmh2-Atg11 complex initiates autophagy in response to glucose starvation. Our research reveals that during glucose starvation, the efflux of vacuolar Ca2+ increases cytoplasmic Ca2+ levels, which activates the protein kinase Rck2. Rck2-mediated phosphorylation of Atg11 enhances its interaction with Bmh1 and Bmh2. This interaction recruits the Snf1-Sip1-Snf4 complex, which is located on the vacuolar membrane, to the phagophore assembly site (PAS), leading to the activation of Atg1 and the initiation of autophagy. In summary, we have identified a previously unrecognized signaling pathway involved in glucose starvation-induced autophagy, where Ca2+ acts as a fundamental signaling molecule that links energy stress to the formation of the autophagy initiation complex.Abbreviation: AMPK: AMP-activated protein kinase; ATG: autophagy related; co-IP: co-immunoprecipitation; MAPK: mitogen-activated protein kinase; PAS: phagophore assembly site; ULK1: unc-51 like autophagy activating kinase 1.

Search for Expression Marker Genes That Reflect the Physiological Conditions of Blossom End Enlargement Occurrence in Cucumber.

Blossom end enlargement (BEE) is a postharvest deformation that may be related to the influx of photosynthetic assimilates before harvest. To elucidate the mechanism by which BEE occurs, expression marker genes that indicate the physiological condition of BEE-symptomatic fruit are necessary. First, we discovered that preharvest treatment with a synthetic cytokinin, N-(2-Chloro-4-pyridyl)-N'-phenylurea (CPPU), promoted fruit growth and suppressed BEE occurrence. This suggests that excessive assimilate influx is not a main cause of BEE occurrence. Subsequently, the expression levels of seven sugar-starvation marker genes, CsSEF1, AS, CsFDI1, CsPID, CsFUL1, CsETR1, and CsERF1B, were compared among symptomatic and asymptomatic fruits, combined with and without CPPU treatment. Only CsSEF1 showed a higher expression level in asymptomatic fruits than in symptomatic fruits, regardless of CPPU treatment. This was then tested using fruits stored via the modified-atmosphere packaging technique, which resulted in a lower occurrence of BEE, and the asymptomatic fruits showed a higher CsSEF1 expression level than symptomatic fruits, regardless of the packaging method. CsSEF1 codes a CCCH-type zinc finger protein, and an increase in the expression of CsSEF1 was correlated with a decrease in the fruit respiration rate. Thus, CsSEF1 may be usable as a BEE expression marker gene.