Subcellular Distribution Research Articles

Characterization and phylogenetic analysis of vomeronasal receptors in the female muskrat (Ondatra zibethicus)

Vomeronasal receptors (VRs) play a crucial role in recognizing pheromones, which are essential for social chemical communication. The male muskrat (Ondatra zibethicus) secretes musk, which contains pheromones as a reproductive signal, and the female can recognize it through the VNO to mediate social communication behavior. This study aimed to identify the genomic information of VRs (OzVRs) in the female muskrat and elucidate their physicochemical properties and evolutionary relationship. Six predominantly expressed OzVR genes were identified using the RACE technique, and a comprehensive analysis was conducted on their gene structure, subcellular distribution, functional predictions, and mRNA levels, revealed that all OzVRs were transmembrane proteins. Phylogenetic analysis clustered OzVR genes into two clades (V1Rs: OzV1R21, OzV1R81, OzV1R105; V2Rs: OzV2R33, OzV2R44, OzV2R60). Physiochemically, OzV1Rs were basic proteins, while OzV2Rs exhibited weakly acidic character. Among them, OzV1R81 and OzV2R44 were identified as hydrophobicitystable proteins, with the remainder categorized as hydrophobicity-unstable proteins. Promoters analysis revealed the involvement of transcription factors and complexes, including Ahr::Arnt, Runx1, Arnt, and TFAP2A, in regulating the expression of the OzVR genes. Conserved domain and motif analyses demonstrated a high conservation of the VRs superfamily in rodents, with many conserved domains linked to pheromone binding. Functional predictions confirmed that OzVRs were associated with pheromones detection. Finally, the expression patterns of OzVR genes in different tissues and seasons indicated that OzVRs have the highest level of expression in the vomeronasal organ, and OzV1Rs notably higher in the breeding season than that in the non-breeding season, however the expression levels of OzV2Rs were higher in the non-breeding season. This study provided insights into the phylogenetic relationships, gene structure, physicochemical properties, promoter binding sites, functions and gene expression patterns of OzVRs, offering a theoretical reference for further examination of VR gene functions and a foundation for understanding chemical signaling mechanisms in the muskrat.

5143 Disulfide Bonds of Thyroid Peroxidase Are Critical Elements for Subcellular Localization, Proteasome-Dependent Degradation, and Enzyme Activity

Abstract Disclosure: H. Iwasaki: None. H. Suwanai: None. K. Kanekura: None. N. Satoshi: None. F. Yakou: None. H. Sakai: None. K. Ishii: None. N. hara: None. R. Suzuki: None. Background: Congenital hypothyroidism (CH) is caused by mutations in cysteine residues including Cys655 and Cys825 that form disulfide bonds in thyroid peroxidase (TPO). It is highly likely that disulfide bonds play an important role in TPO activity. However, no study has comprehensively analyzed cysteine mutations that form disulfide bonds in TPO. In this study, we induced mutations in cysteine residues involved in the formation of disulfide bonds and analyzed their effect on subcellular localization, degradation, and enzyme activities to evaluate the importance of disulfide bonds in TPO. Methods: Vector plasmid TPO mutants C655F and C825R in CH patients were transfected into HEK293 cells. TPO activity and protein expression levels were measured by Amplex red assay and western blotting. The same procedure was performed in the presence of proteasome inhibitor MG132. Subcellular localization was determined by immunocytochemistry and flow cytometry. The location of all disulfide bonds within TPO was predicted by in silico analysis. All TPO mutations associated with disulfide bonds were induced. TPO activity and protein expression levels were also measured in all TPO mutants associated with disulfide bonds using the Amplex red assay and western blotting. Results: C655F and C825R showed significantly decreased activity and protein expression compared to the wild-type (P < 0.05). In the presence of a proteasome inhibitor MG132, the protein expression level of TPO was increased to the level comparable with that of the wild-type but its activity did not. The degree of subcellular distribution of TPO to the cell surface in the mutant was lower than that in the wild-type TPO. Twenty-four cysteine residues were involved in the formation of 12 disulfide bonds in TPO, and all TPO mutants harboring an amino acid substitution in each cysteine showed significantly reduced TPO activity and protein expression levels. Furthermore, the differences in TPO activity depended on the position of the disulfide bond. Conclusions: All 12 disulfide bonds play an important role in the activity of TPO. Furthermore, the mutations lead to misfolding, degradation and membrane insertion. Presentation: 6/2/2024

Differential effect of plakoglobin in restoring the tumor suppressor activities of p53-R273H vs. p53-R175H mutants.

The six most common missense mutations in the DNA binding domain of p53 are known as "hot spots" and include two of the most frequently occurring p53 mutations (p53-R175H and p53-R273H). p53 stability and function are regulated by various post-translational modifications such as phosphorylation, acetylation, sumoylation, methylation, and interactions with other proteins including plakoglobin. Previously, using various carcinoma cell lines we showed that plakoglobin interacted with wild-type and several endogenous p53 mutants (e.g., R280K, R273H, S241F, S215R, R175H) and restored their tumor suppressor activities in vitro. Since mutant p53 function is both mutant-specific and cell context-dependent, we sought herein, to determine if plakoglobin tumor suppressive effects on exogenously expressed p53-R273H and p53-R175H mutants are similarly maintained under the same genetic background using the p53-null and plakoglobin-deficient H1299 cell line. Functional assays were performed to assess colony formation, migration, and invasion while immunoblotting and qPCR were used to examine the subcellular distribution and expression of specific proteins and genes that are typically regulated by or regulate p53 function and are altered in mutant p53-expressing cell lines and tumors. We show that though, plakoglobin interacted with both p53-R273H and p53-R175H mutants, it had a differential effect on the transcription and subcellular distribution of their gene targets and their overall oncogenic properties in vitro. Notably, we found that plakoglobin's tumor suppressive effects were significantly stronger in p53-R175H expressing cells compared to p53-R273H cells. Together, our results indicate that exploring plakoglobin interactions with p53-R175H may be useful for the development of cancer therapeutics focused on the restoration of p53 function.

The effect of biochar amendment on Cd accumulation in Bidens pilosa L: Changing Cd subcellular distribution, cell wall polysaccharide Cd-binding capacity and composition

Biochar can reduce Cd uptake by plants, thereby reducing its biotoxicity, but the mechanisms involved at the subcellular level have not been thoroughly elucidated. In this work, we explored the effect of maize straw biochar on Cd accumulation by Bidens pilosa L. and its mechanism at subcellular levels. After 90 days of potting experiment, the subcellular fractions were extracted by differential centrifugation, and the polysaccharide fractions of root cell walls were extracted by leaching centrifugation, and then the Cd content of each fraction was determined. Results showed that Cd was preferentially distributed in cell walls of three organs. Additionally, biochar addition resulted in a greater distribution of Cd from cell wall to soluble fractions and organelles in stems. These results suggested that cell wall immobilization and intracellular compartmentalization were critical detoxification mechanisms tackling Cd stress with biochar addition of Bidens pilosa L. Pectin was the main sink where Cd was stored. And galacturonic acid content in pectin occupied the highest ratio among the three polysaccharide fractions. After biochar addition, in hemicellulose the change of Cd content was consistent with the change of galacturonic acid content. These results suggested that the galacturonic acid in hemicellulose played an important role in Cd binding. Biochar addition reduced the bioavailability of soil Cd and improved the growth environment, thus inducing Bidens pilosa L. to change the composition of root cell wall in response to Cd stress.

Stress resistance, antiaging, and neuroprotective activities of baicalein 5,6-dimethyl ether and Alnus rugosa extract in Caenorhabditis elegans model.

The leaf extract of Alnus rugosa (AR) together with the isolated compound baicalein 5,6-dimethyl ether (BME) were investigated for their antioxidant, radical scavenging, antiaging, and neuroprotective properties using the Caenorhabditis elegans model. The stress resistance and antiaging potential of AR and BME were assessed in wild-type N2 and transgenic C. elegans strains CF1553, TJ356, and BA17. Transgenic CL4176 expressing the human amyloid-beta peptide (Aβ) was used as a model for Aβ toxicity, whereas transgenic AM141 expressing polyQ aggregates was employed as a model for Huntington's disease. An in silico molecular docking study using Discovery Studio 4.5 was performed to elucidate the putative binding mode of BME to the active sites of Daf-2 protein, involved in longevity and oxidative stress resistance in C. elegans. BME and AR significantly delayed the appearance of oxidative stress markers in wild-type N2 and transgenic strains TJ356 and CF1553, affecting the DAF-16/FOXO transcription factor subcellular distribution and inducing expression of the sod-3 antioxidative gene. Pretreatment with AR significantly reduced the aging marker lipofuscin accumulation in BA17 worms, its effect was greater than that of epigallocatechin gallate, suggesting a potential antiaging effect. Neuroprotective effects of AR and BME were confirmed in AM141 transgenic worms, inducing a significant reduction in the score of polyQ40::GFP aggregates. Moreover, BME (25 µg/mL) resulted in a significant delay in Aβ-induced paralysis in CL4176 worms. In silico molecular modeling revealed that BME exhibited good fitting scores within the active sites of the Daf-2 protein. AR and BME exert beneficial effects in the modulation of age-related markers and attenuation of neurotoxicity in neurodegenerative disorders. Hence, AR and BME could be recognized as promising antioxidant and neuroprotective natural drug candidates that could be included in neuro-nutraceuticals.

Lipid Droplets Big and Small: Basic Mechanisms That Make Them All.

Lipid droplets (LDs) are dynamic storage organelles with central roles in lipid and energy metabolism. They consist of a core of neutral lipids, such as triacylglycerol, which is surrounded by a monolayer of phospholipids and specialized surface proteins. The surface composition determines many of the LD properties, such as size, subcellular distribution, and interaction with partner organelles. Considering the diverse energetic and metabolic demands of various cell types, it is not surprising that LDs are highly heterogeneous within and between cell types. Despite their diversity, all LDs share a common biogenesis mechanism. However, adipocytes have evolved specific adaptations of these basic mechanisms, enabling the regulation of lipid and energy metabolism at both the cellular and organismal levels. Here, we discuss recent advances in the understanding of both the general mechanisms of LD biogenesis and the adipocyte-specific adaptations controlling these fascinating organelles.

Bioaccumulation and toxicological effects of dietborne arsenic exposure on the apple snail (Pomacea canaliculata)

An eight-compartment physiologically based pharmacokinetic (PBPK) model was used to simulate the bioaccumulation and distribution of arsenic (As) within the apple snail (Pomacea canaliculata) following the ingestion of As-contaminated lettuce. The bioaccumulation results revealed that the shell contained the majority (67.21 %) of the total As content, with the liver and the head-foot containing approximately 11.14 % and 10.45 % of the total As content in the snail, respectively. Modeling quantified the process of intestine-stomach absorption of dietborne As and revealed its crucial role in the subsequent distribution of As within the body. The liver is the primary metabolic site, whereas the shell is the primary storage site. Exposure to dietborne As leads to pronounced physiological and biochemical alterations in apple snails. Total protein levels decreased by 24.06 %, superoxide dismutase (SOD) activity decreased by 24.43 %, malondialdehyde (MDA) content increased by 47.51 %, glutathione (GSH) content decreased by 46.99 %, and glutathione S-transferase (GST) activity decreased by 42.22 %. Furthermore, the subcellular-level results indicated that dietborne As exposure altered subcellular distribution in the liver. Additionally, dietborne As exposure significantly reduced the abundance of gut microbiota in apple snails.

Different stoichiometric ratios of Ca and Cd affect the Cd tolerance of Capsicum annuum L. by regulating the subcellular distribution and chemical forms of Cd

The effect of calcium (Ca)–cadmium (Cd) interactions on the plant Cd bioaccumulation process may be closely related to the ecological Ca/Cd stoichiometry in the substrate. However, owing to the complexity of plant absorption, accumulation mechanisms and influencing factors, the mechanism of Ca-mediated Cd bioaccumulation and Cd tolerance in Capsicum is still unclear. In this study, the bioaccumulation, subcellular distribution and chemical forms of Cd in Capsicum were analysed via pot experiments to reveal the Ca-mediated Cd bioaccumulation process and its detoxification mechanism under different Ca/Cd stoichiometric ratios. The results revealed that an increase in the substrate Ca/Cd ratio promoted the accumulation of Cd in the roots; restricted the transport of Cd to the stems, leaves and peppers; and promoted the accumulation of Cd in the aboveground leaves but decreased its accumulation in edible parts. Cd was enriched mainly in the cell wall and cell-soluble fraction in each tissue and was enriched in only 1 %–13 % of the organelles. The accumulation of Cd in the cell wall and cell-soluble fractions of roots treated with different Ca concentrations increased by 56.57 %–236.98 % and 64.41 %–442.14 %, respectively. The carboxyl, hydroxyl and amino groups on the root cell wall play important roles in binding and fixing Cd2+. Moreover, the increase in the Ca content also increased the proportion of pectin and protein-bound Cd (F-NaCl), insoluble phosphate-bound Cd (F-C) and insoluble oxalate-bound Cd (F-HCl) in the roots, stems and leaves and reduced the proportion of highly active chemical forms such as inorganic acid salt-bound Cd (F-E) and water-soluble phosphate-bound Cd (F-W). Our study revealed that the bioaccumulation of Cd in Capsicum was influenced by the Ca/Cd ratio and that Ca could alleviate Cd stress by regulating the subcellular distribution and chemical form ratio of Cd in different tissues where the cell wall plays an important role in Cd tolerance and detoxification.

G protein-gated inwardly rectifying K+ (GIRK/Kir3) channels: Molecular, cellular, and subcellular diversity.

G protein-gated inwardly rectifying K+ (GIRK/Kir3) channels are mainly expressed in excitable cells such as neurons and atrial myocytes, where they can respond to a wide variety of neurotransmitters. Four GIRK subunits have been found in mammals (GIRK1-4) and act as downstream targets for various Gαi/o-linked G protein-coupled receptors (GPCRs). Activation of GIRK channels produces a postsynaptic efflux of potassium from the cell, responsible for hyperpolarization/inhibition of the neuron. A growing body of evidence suggests that dysregulation of GIRK signalling can lead to excessive or deficient neuronal excitability, which contributes to neurological diseases and disorders. Therefore, GIRK channels are proposed as new pharmacological targets. The function of GIRK channels in neurons is not only determined by their biophysical properties but also by their cellular and subcellular localization patterns and densities on the neuronal surface. GIRK channels can be located within several subcellular compartments, where they have many different functional implications. This subcellular localization changes dynamically along the neuronal surface in response to drug intake. Ongoing research is focusing on determining the proteins that form macromolecular complexes with GIRK channels and are responsible for fast and precise signalling under physiological conditions, and how their alteration is implicated in pathological conditions. In this review, the distinct regional, cellular, and subcellular distribution of GIRK channel subunits in the brain will be discussed in view of their possible functional and pathological implications.

Foliar application of silicon and selenium reduce the toxicity of cadmium to soybeans

Silicon (Si) and selenium (Se), two environmental protection materials, which are beneficial to plant growth and stress resistance, can also alleviate crop stress induced by heavy metals. However, the effects of Si, Se and their interactions in reducing cadmium (Cd) toxicity and the related mechanisms require further elucidation. Hence, this study implemented a foliar application of Si and Se on soybean (Glycine max L.) that subjected to Cd-induced stress with four treatments (sole/combined application of Si, Se, no fertilizer treatment). The results demonstrated that Si and Se showed effective mitigation of Cd toxicity on soybeans mainly by promoting growth, enhancing photosynthesis, maintaining root vigor, improving antioxidant capacity, alleviating oxidative damage, altering the storage form, subcellular distribution of Cd in soybeans, and was more noticeable when combined overall (Si + Se＞Se＞Si). Si + Se increased root activity by 28% and CAT activity in leaves by 130.65%. Overall, the combined application of Si and Se exhibited a pronounced synergistic effect in enhancing the healthy growth of soybean plants under Cd pollution, with a more prominent impact observed following the second fertilization.

Unveiling responses and mechanisms of spice crop chive exposure to three typical pesticides using metabolomics combined with transcriptomics, physiology and biochemistry

Pesticides are frequently used to control target pests in the production of spice crops such as chives (Allium ascalonicum). However, little information is available on the responses and underlying mechanisms of pesticide exposure in this crop. Our findings revealed that the uptake, transportation, and subcellular distribution of three typical pesticides—the fungicide pyraclostrobin (PAL), insecticide acetamiprid (ATP), and herbicide pendimethalin (PND) in chives, as well as their physiological, biochemical, metabolic, and transcriptomic responses—were dependent on pesticide properties, especially hydrophobicity. The distribution of PAL and PND in chives decreased in the order root > stem > leaf, but the distribution order of ATP was the opposite. The proportion of PAL and PND in the solid phase of the root cells gradually increased, but ATP mainly existed in the cell-soluble component, indicating that the latter had an upward translocation ability and thus mainly accumulated in the leaves. Malondialdehyde levels in chive leaves were not significantly affected by exposure to these pesticides; however, the activities of superoxide dismutase (SOD) and catalase (CAT) in chive leaves increased significantly. Moreover, these pesticides exhibited critical differences in chive responses through the interaction of metabolites and regulation of differentially expressed genes. PAL dramatically influenced five carbohydrate metabolic pathways (34.35 %), disturbing the starch-to-sucrose balance. ATP strongly affected five amino acid (AC) metabolic pathways (33.38 %), enhancing four free amino acid levels. PND notably affected eight fatty acid (FA) metabolic pathways (25.38 %), increasing two unsaturated and decreasing one saturated FA. Simultaneously, PND, ATP, and PND accumulated in the chives could be detoxified through metabolic pathways mediated by cytochrome P450 (P450) and glycosyltransferase (GT)/glutathione S-transferase (GST), producing phase I (7, 4, and 5) and II (11, 13, and 10) metabolites, respectively. This study provides important molecular insights into the responses and underlying mechanisms of spice crop exposure to pesticides.

Subcellular Level Spatial Transcriptomics with PHOTON.

The subcellular localization of RNA is closely linked to its function. Many RNA species are partitioned into organelles and other subcellular compartments for storage, processing, translation, or degradation. Thus, capturing the subcellular spatial distribution of RNA would directly contribute to the understanding of RNA functions and regulation. Here, we present PHOTON (Photoselection of Transcriptome over Nanoscale), a method which combines high resolution imaging with high throughput sequencing to achieve spatial transcriptome profiling at subcellular resolution. We demonstrate PHOTON as a versatile tool to accurately capture the transcriptome of target cell types in situ at the tissue level such as granulosa cells in the ovary, as well as RNA content within subcellular compartments such as the nucleolus and the stress granule. Using PHOTON, we also reveal the functional role of m6A modification on mRNA partitioning into stress granules. These results collectively demonstrate that PHOTON is a flexible and generalizable platform for understanding subcellular molecular dynamics through the transcriptomic lens.

Zinc oxide nanoparticles reduce cadmium accumulation in hydroponic lettuce (Lactuca sativa L.) by increasing photosynthetic capacity and regulating phenylpropane metabolism

Due to the continuous production of industrial wastes and the excessive use of chemical fertilizers and pesticides, severe cadmium (Cd) pollution in soil has occurred globally. This study investigated the impacts of incorporating zinc oxide nanoparticles (ZnONPs) into hydroponically grown lettuce (Lactuca sativa) under cadmium stress conditions, to seek effective methods to minimize Cd buildup in green leafy vegetables. The results showed that 1 mg/L of Cd significantly inhibited lettuce growth, decreasing in leaves (29 %) and roots (33 %) biomass. However, when lettuce was exposed to 2.5 mg/L ZnONPs under cadmium stress, the growth, chlorophyll content, net photosynthetic rate (Pn), stomatal conductance (Gs), actual photochemical efficiency of PSII (φPSII), and activity of key enzymes in photosynthesis were all significantly enhanced. Furthermore, ZnONPs significantly decreased the accumulation of Cd in lettuce leaves (36 %) and roots (13 %). They altered the subcellular distribution and chemical morphology of Cd in lettuce by modifying the composition of cell walls (such as pectin content) and the levels of phenolic compounds, resulting in a reduction of 27 % in Cd translocation from roots to leaves. RNA sequencing yielded 45.9 × 107 and 53.4 × 107 clean reads from plant leaves and roots in control (T0), Cd (T1), Cd+ZnONPs (T2), and ZnONPs (T3) treatment groups respectively, and 3614 and 1873 differentially expressed genes (DEGs) were identified. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis identified photosynthesis, carbon fixation, and phenylpropanoid metabolism as the main causes of ZnONPs-mediated alleviation of Cd stress in lettuce. Specifically, the DEGs identified included 12 associated with photosystem I, 13 with photosystem II and 23 DEGs with the carbon fixation pathway of photosynthesis. Additionally, DEGs related to phenylalanine ammonia-lyase, caffeoyl CoA 3-O-methyltransferase, peroxidase, 4-coumarate-CoA ligase, hydroxycinnamoyl transferase, and cytochrome P450 proteins were also identified. Therefore, further research is recommended to elucidate the molecular mechanisms by which ZnONPs reduce Cd absorption in lettuce through phenolic acid components in the phenylpropanoid metabolism pathway. Overall, treatments with ZnONPs are recommended to effectively reduce Cd accumulation in the edible portion of lettuce.

Reduced copper uptake and efflux by the mussel Mytilus coruscus after Cu exposure: Implication for biomonitoring

The hard-shell mussels Mytilus coruscus have been extensively employed in pollution biomonitoring. Earlier studies indicated that metal concentrations in Mytilus coruscus may not accurately reflect the true metal contamination levels in the sampling areas, possibly due to their modified metal uptake and efflux. Given the likelihood of mussels in the field being exposed to intermittent metal contaminants, this study investigated whether different Cu pre-exposures significantly affected its uptake and efflux upon Cu exposure. We found significant reduction in Cu uptake rate constant (ku) and efflux rate constant (ke) in the mussels with varying Cu pre-exposure regimes. Specifically, the ku decreased from 1.55 ± 0.37 L g−1 d−1 in the control group to 0.65 ± 0.19 after 5 days and 0.53 ± 0.28 after 15 days of exposure to 20 μg L−1 Cu, respectively, and then was further reduced to as low as 0.096 ± 0.046 L g−1 d−1 following a 5-day exposure at 50 μg L−1 Cu. Similarly, the ke decreased from 0.18 ± 0.020 to 0.15 ± 0.015 d−1 following 5–15 days of exposure to 20 μg L−1 Cu, and further decreased to 0.081 ± 0.023 d−1 after a 5-day exposure at 50 μg L−1 Cu. Our subcellular distribution analysis underscored the critical role of the metallothionein-like protein (MTLP) fraction in modifying both Cu ku and ke during the rapid-depuration phase (ke1), whereas the metal-rich granule (MRG) fraction influenced the ke during the second depuration phase (ke2). This study demonstrated that environmental assessments utilizing biomonitoring species should consider the exposure of these organisms to ensure accurate interpretations of metal contamination in marine ecosystems and enhance the effectiveness of these species in environmental monitoring. This crucial factor is often overlooked, potentially skewing data and leading to misinterpretations of environmental health and pollution levels.

The subcellular distribution of miRNA isoforms, tRNA-derived fragments, and rRNA-derived fragments depends on nucleotide sequence and cell type

BackgroundMicroRNA isoforms (isomiRs), tRNA-derived fragments (tRFs), and rRNA-derived fragments (rRFs) represent most of the small non-coding RNAs (sncRNAs) found in cells. Members of these three classes modulate messenger RNA (mRNA) and protein abundance and are dysregulated in diseases. Experimental studies to date have assumed that the subcellular distribution of these molecules is well-understood, independent of cell type, and the same for all isoforms of a sncRNA.ResultsWe tested these assumptions by investigating the subcellular distribution of isomiRs, tRFs, and rRFs in biological replicates from three cell lines from the same tissue and same-sex donors that model the same cancer subtype. In each cell line, we profiled the isomiRs, tRFs, and rRFs in the nucleus, cytoplasm, whole mitochondrion (MT), mitoplast (MP), and whole cell. Using a rigorous mathematical model we developed, we accounted for cross-fraction contamination and technical errors and adjusted the measured abundances accordingly. Analyses of the adjusted abundances show that isomiRs, tRFs, and rRFs exhibit complex patterns of subcellular distributions. These patterns depend on each sncRNA’s exact sequence and the cell type. Even in the same cell line, isoforms of the same sncRNA whose sequences differ by a few nucleotides (nts) can have different subcellular distributions.ConclusionsSncRNAs with similar sequences have different subcellular distributions within and across cell lines, suggesting that each isoform could have a different function. Future computational and experimental studies of isomiRs, tRFs, and rRFs will need to distinguish among each molecule’s various isoforms and account for differences in each isoform’s subcellular distribution in the cell line at hand. While the findings add to a growing body of evidence that isomiRs, tRFs, rRFs, tRNAs, and rRNAs follow complex intracellular trafficking rules, further investigation is needed to exclude alternative explanations for the observed subcellular distribution of sncRNAs.

Subcellular distribution of the β-N-methylamino-L-alanine-containing proteins in marine diatoms

Neurotoxin β-N-methylamino-L-alanine (BMAA) has been implicated as a major inducer of human neurodegenerative diseases. In recent years, marine diatoms were verified to produce BMAA-containing proteins. It will be an important cue to elucidate the subcellular distribution of BMAA in marine diatoms for disclosing its biosynthesis pathway. In this study, three species of Thalassiosira (T. andamanica, T. allenii and T. minima) were used to investigate the subcellular distribution of BMAA in organelles. Results showed that the crushing efficiency of diatoms was species-specific and increased with the rise of ultrasonic intensity of 22, 50 and 100 W (pulse = 0.2 s/s, 4 min), of which T. andamanica and T. allenii obtained the lowest and highest crushing efficiency, respectively. Interestingly, although T. allenii and T. minima were more efficiently crushed at 50 W and 100 W power (pulse = 0.2 s/s), their organelles were largely fragmented, which was verified by cytochrome c oxidase (CCO) enzyme analysis and transmission electron microscopy (TEM) observation. Their organelles were not fragmented only at 22 W. However, the crushing efficiency of T. andamanica was more reliable, and its organelles were essentially intact and only damaged at 100 W. Analysis of the BMAA-containing proteins showed that these proteins exclusively distribute in the Golgi and endoplasmic reticulum (ER) organelles. The nearly intact membranes of nucleus, mitochondria, Golgi and ER organelles testified that the absence of BMAA in other organelles was not caused by damage of nucleus or mitochondria. Results demonstrated that the BMAA-containing proteins were produced and accumulated in the ER and Golgi of diatoms.

FACT inhibitor CBL0137, administered in an optimized schedule, potentiates radiation therapy for glioblastoma by suppressing DNA damage repair.

Standard-of-care for glioblastoma remains surgical debulking followed by temozolomide and radiation. However, many tumors become radio-resistant while radiation damages surrounding brain tissue. Novel therapies are needed to increase the effectiveness of radiation and reduce the required radiation dose. Drug candidate CBL0137 is efficacious against glioblastoma by inhibiting histone chaperone FACT, known to be involved in DNA damage repair. We investigated the combination of CBL0137 and radiation on glioblastoma. In vitro, we combined CBL0137 with radiation on U87MG and A1207 glioblastoma cells using the clonogenic assay to evaluate the response to several treatment regimens, and the Fast Halo Assay to examine DNA repair. In vivo, we used the optimum combination treatment regimen to evaluate the response of orthotopic tumors in nude mice. In vitro, the combination of CBL0137 and radiation is superior to either alone and administering CBL0137 two hours prior to radiation, having the drug present during and for a prolonged period post-radiation, is an optimal schedule. CBL0137 inhibits DNA damage repair following radiation and affects the subcellular distribution of histone chaperone ATRX, a molecule involved in DNA repair. In vivo, one dose of CBL0137 is efficacious and the combination of CBL0137 with radiation increases median survival over either monotherapy. CBL0137 is most effective with radiation for glioblastoma when present at the time of radiation, immediately after and for a prolonged period post-radiation, by inhibiting DNA repair caused by radiation. The combination leads to increased survival making it attractive as a dual therapy.

FACT inhibitor CBL0137, administered in an optimized schedule, potentiates radiation therapy for glioblastoma by suppressing DNA damage repair.

Standard-of-care for glioblastoma remains surgical debulking followed by temozolomide and radiation. However, many tumors become radio-resistant while radiation damages surrounding brain tissue. Novel therapies are needed to increase the effectiveness of radiation and reduce the required radiation dose. Drug candidate CBL0137 is efficacious against glioblastoma by inhibiting histone chaperone FACT, known to be involved in DNA damage repair. We investigated the combination of CBL0137 and radiation on glioblastoma. In vitro, we combined CBL0137 with radiation on U87MG and A1207 glioblastoma cells using the clonogenic assay to evaluate the response to several treatment regimens, and the Fast Halo Assay to examine DNA repair. In vivo, we used the optimum combination treatment regimen to evaluate the response of orthotopic tumors in nude mice. In vitro, the combination of CBL0137 and radiation is superior to either alone and administering CBL0137 two hours prior to radiation, having the drug present during and for a prolonged period post-radiation, is an optimal schedule. CBL0137 inhibits DNA damage repair following radiation and affects the subcellular distribution of histone chaperone ATRX, a molecule involved in DNA repair. In vivo, one dose of CBL0137 is efficacious and the combination of CBL0137 with radiation increases median survival over either monotherapy. CBL0137 is most effective with radiation for glioblastoma when present at the time of radiation, immediately after and for a prolonged period post-radiation, by inhibiting DNA repair caused by radiation. The combination leads to increased survival making it attractive as a dual therapy.

The partitioning of fatty acids between membrane and storage lipids controls ER membrane expansion.

The biogenesis of membrane-bound organelles involves the synthesis, remodelling and degradation of their constituent phospholipids. How these pathways regulate organelle size, remains still poorly understood. Here we demonstrate that a lipid degradation pathway inhibits the expansion of the endoplasmic reticulum (ER) membrane. Phospholipid diacylglycerol acyltransferases (PDATs) use endogenous phospholipids as fatty acyl donors to generate triglyceride stored in lipid droplets. The significance of this non-canonical triglyceride biosynthetic pathway has remained elusive. We find that the activity of the yeast PDAT Lro1 is regulated by a membrane- proximal domain facing the luminal side of the ER bilayer. To reveal the biological roles of PDATs, we engineered an Lro1 variant with derepressed activity. We show that active Lro1 mediates the retraction of ER membrane expansion driven by phospholipid synthesis. Furthermore, the subcellular distribution and membrane turnover activity of Lro1 are controlled by diacylglycerol, produced by the activity of Pah1, a conserved member of the lipin family. Collectively, our findings reveal a lipid metabolic network that regulates endoplasmic reticulum biogenesis by converting phospholipids into storage lipids.

Subcellular Drug Distribution: Exploring Organelle-Specific Characteristics for Enhanced Therapeutic Efficacy.

The efficacy and potential toxicity of drug treatments depends on the drug concentration at its site of action, intricately linked to its distribution within diverse organelles of mammalian cells. These organelles, including the nucleus, endosome, lysosome, mitochondria, endoplasmic reticulum, Golgi apparatus, lipid droplets, exosomes, and membrane-less structures, create distinct sub-compartments within the cell, each with unique biological features. Certain structures within these sub-compartments possess the ability to selectively accumulate or exclude drugs based on their physicochemical attributes, directly impacting drug efficacy. Under pathological conditions, such as cancer, many cells undergo dynamic alterations in subcellular organelles, leading to changes in the active concentration of drugs. A mechanistic and quantitative understanding of how organelle characteristics and abundance alter drug partition coefficients is crucial. This review explores biological factors and physicochemical properties influencing subcellular drug distribution, alongside strategies for modulation to enhance efficacy. Additionally, we discuss physiologically based computational models for subcellular drug distribution, providing a quantifiable means to simulate and predict drug distribution at the subcellular level, with the potential to optimize drug development strategies.