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RESEARCH HIGHLIGHTSAvian leukosis virus subgroup J (ALV-J) is an alpharetrovirus that infects chickens, causing immunosuppression and a decrease in production performance, leading to substantial economic losses in the poultry industry. ALV-J is also well-known for its oncogenic properties, inducing tumours such as myelomas and haemangiomas in infected chickens. TRIM45 has been identified as a potential tumour suppressor; however, the relationship between TRIM45 expression and ALV-J infection remains to be elucidated. This study aimed to dissect the molecular characteristics of the chicken TRIM45 gene and its modulation during ALV-J infection, as well as its influence on viral replication. We found that the chicken TRIM45 RING domain is significantly different from that of humans and other mammals. TRIM45 is expressed in all chicken tissues, with the highest levels in the heart. Subcellular localization studies indicated a cytoplasmic distribution of TRIM45, forming aggregates within cells. Our findings demonstrate that ALV-J infection significantly upregulates TRIM45 expression in DF-1 cells. To assess the functional role of TRIM45 in ALV-J replication, we employed both gene silencing and overexpression strategies. Strikingly, the overexpression of TRIM45, including a mutant lacking the RING domain, was found to markedly suppress ALV-J replication. In contrast, TRIM45 knockdown via siRNA resulted in an enhanced viral replication, highlighting the importance of TRIM45 limiting ALV-J replication. Mechanistically, overexpression of TRIM45 induces apoptosis in infected cells, independent of its RING domain function. In conclusion, our study demonstrates that chicken TRIM45 acts as a negative regulator of ALV-J replication in vitro by promoting apoptosis in infected cells.Chicken TRIM45 RING domain and protein localization significantly differ from humans.TRIM45 negatively regulates ALV-J replication in vitro.TRIM45 inhibits ALV-J replication by inducing apoptosis in infected cells.Chicken TRIM45 RING domain and protein localization significantly differ from humans.TRIM45 negatively regulates ALV-J replication in vitro.TRIM45 inhibits ALV-J replication by inducing apoptosis in infected cells. [ABSTRACT FROM AUTHOR]

As cell signaling research has advanced, it has become clearer that signal transduction has complex spatiotemporal regulation that goes beyond foundational linear transduction models. Several technologies have enabled these discoveries, including fluorescent biosensors designed to report live biochemical signaling events. As genetically encoded and live-cell compatible tools, fluorescent biosensors are well suited to address diverse cell signaling questions across different spatial scales of regulation. In this review, methods of examining spatial signaling regulation and the design of fluorescent biosensors are introduced. Then, recent biosensor developments that illuminate the importance of spatial regulation in cell signaling are highlighted at several scales, including membranes and organelles, molecular assemblies, and cell/tissue heterogeneity. In closing, perspectives on how fluorescent biosensors will continue enhancing cell signaling research are discussed.

ACE2, part of the angiotensin-converting enzyme family and the renin–angiotensin–aldosterone system (RAAS), plays vital roles in cardiovascular and renal functions. It is also the primary receptor for SARS-CoV-2, enabling its entry into cells. This project aimed to study ACE2's cellular trafficking and maturation to the cell surface and assess the impact of various drugs and compounds on these processes. We used cellular and biochemical analyses to evaluate these compounds as potential leads for COVID-19 therapeutics. Our screening assay focused on ACE2 maturation levels and subcellular localization with and without drug treatments. Results showed that ACE2 maturation is generally fast and robust, with certain drugs having a mild impact. Out of twenty-three tested compounds, eight significantly reduced ACE2 maturation levels, and three caused approximately 20% decreases. Screening trafficking inhibitors revealed significant effects from most molecular modulators of protein trafficking, mild effects from most proposed COVID-19 drugs, and no effects from statins. This study noted that manipulating ACE2 levels could be beneficial or harmful, depending on the context. Thus, using this approach to uncover leads for COVID-19 therapeutics requires a thorough understanding ACE2's biogenesis and biology. [ABSTRACT FROM AUTHOR]

Ischemic stroke associated with atherosclerosis is globally named atherothrombotic stroke. Presently, the underlying pathogenic genes promoting carotid atherosclerotic plaques transfer from a stable to unstable state remains elusive. This study aims to find the hub genes disturbing the stability of plaques and explore the primary cells affected by these hub genes.

Background: Interferon regulatory factors (IRF-1 and IRF-2) are transcription factors widely implicated in various cellular processes, including regulation of inflammatory responses to pathogens, cell proliferation, oncogenesis, differentiation, autophagy, and apoptosis. Methods: We have studied the expression of IRF-1, IRF-2 mRNAs by RT-PCR, cellular localization of the proteins by immunofluorescence, and expression of mRNAs of genes regulated by IRF-1, IRF-2 by RT-PCR in mouse bone marrow cells (BMCs) and mesenchymal stem cells (MSCs). Results: Higher level of IRF-1 mRNA was observed in BMCs and MSCs compared to that of IRF-2. Similarly, differential expression of IRF-1 and IRF-2 proteins was observed in BMCs and MSCs. IRF-1 was predominantly localized in the cytoplasm, whereas IRF-2 was localized in the nuclei of BMCs. MSCs showed nucleo-cytoplasmic distribution of IRF-1 and nuclear localization of IRF-2. Constitutive expression of IRF-1 and IRF-2 target genes: monocyte chemoattractant protein-1 (MCP-1), vascular cell adhesion molecule-1 (VCAM-1), cyclooxygenase-2 (COX-2), matrix metalloproteinase-9 (MMP-9), and caspase-1 was observed in both BMCs and MSCs. MSCs showed constitutive expression of the pluripotency-associated factors, Oct3/4 and Sox-2. Lipopolysaccharide (LPS)-treatment of MSCs induced prominent cellular localization of IRF-1 and IRF-2. Conclusions: Our results suggest that IRF-1 and IRF-2 exhibit differential expression of their mRNAs and subcellular localization of the proteins in BMCs and MSCs. These cells also show differential levels of constitutive expression of IRF-1 and IRF-2 target genes. This may regulate immune-responsive properties of BMCs and MSCs through IRF-1, IRF-2-dependent gene expression and protein–protein interaction. Regulating IRF-1 and IRF-2 may be helpful for immunomodulatory functions of MSCs for cell therapy and regenerative medicine. [ABSTRACT FROM AUTHOR]

Cytoplasmic Dynein is a multiple-subunit macromolecular motor protein involved in the transport process of cells. The Dynein intermediate chain (DIC) is one of the subunits of Dynein-1. In our previous studies, we showed that Pt-DIC may play an important role in the nuclear deformation of spermiogenesis in Portunus trituberculatus. Lamin B is essential for maintaining nuclear structure and functions. Surprisingly, Pt-Lamin B was expressed not only in the perinucleus but also in the pro-acrosome during spermiogenesis in P. trituberculatus. Studies have also shown that Dynein-1 can mediate the transport of Lamin B in mammals. Thus, to study the relationship of Pt-DIC and Pt-Lamin B in the spermatogenesis of P. trituberculatus, we knocked down the Pt-DIC gene in P. trituberculatus by RNAi. The results showed that the distribution of Pt-DIC and Pt-Lamin B in spermiogenesis was abnormal, and the colocalization was weakened. Moreover, we verified the interaction of Pt-DIC and Pt-Lamin B via coimmunoprecipitation. Therefore, our results suggested that both Pt-DIC and Pt-Lamin B were involved in the spermatogenesis of P. trituberculatus, and one of the functions of Dynein-1 is to mediate the transport of Lamin B in the spermiogenesis of P. trituberculatus. [ABSTRACT FROM AUTHOR]

The SORL1 gene encodes LR11/SorLA, a protein that binds β-amyloid precursor protein (APP) and drives its intracellular trafficking. SORL1 mutations, occurring frequently in a subset of familial cases of Alzheimer's disease (AD), have been documented, but their pathogenic potential is not yet clear and questions remain concerning their putative influence on the physiopathological processing of APP. We have assessed the influence of two SORL1 mutations that were described as likely disease-causing and that were associated with either benign (SorLA924) or severe (SorLA511) AD phenotypes. We examined the influence of wild-type and mutants SorLA in transiently transfected HEK293 cells expressing either wild-type or Swedish mutated APP on APP expression, secreted Aβ and sAPPα levels, intracellular Aβ 40 and Aβ42 peptides, APP-CTFs (C99 and C83) expressions, α-, β- and γ-secretases expressions and activities as well as Aβ and CTFs-degrading enzymes. These paradigms were studied in control conditions or after pharmacological proteasomal modulation. We also established stably transfected CHO cells expressing wild-type SorLA and established the colocalization of APP and either wild-type or mutant SorLA. SorLA mutations partially disrupt co-localization of wild-type sorLA with APP. Overall, although we mostly confirmed previous data concerning the influence of wild-type SorLA on APP processing, we were unable to evidence significant alterations triggered by our set of SorLA mutants, whatever the cells or pharmacological conditions examined. Our study , however, does not rule out the possibility that other AD-linked SORL1 mutations could indeed affect APP processing, and that pathogenic mutations examined in the present study could interfere with other cellular pathways/triggers in AD. [ABSTRACT FROM AUTHOR]

ACE2, part of the angiotensin-converting enzyme family and the renin–angiotensin–aldosterone system (RAAS), plays vital roles in cardiovascular and renal functions. It is also the primary receptor for SARS-CoV-2, enabling its entry into cells. This project aimed to study ACE2’s cellular trafficking and maturation to the cell surface and assess the impact of various drugs and compounds on these processes. We used cellular and biochemical analyses to evaluate these compounds as potential leads for COVID-19 therapeutics. Our screening assay focused on ACE2 maturation levels and subcellular localization with and without drug treatments. Results showed that ACE2 maturation is generally fast and robust, with certain drugs having a mild impact. Out of twenty-three tested compounds, eight significantly reduced ACE2 maturation levels, and three caused approximately 20% decreases. Screening trafficking inhibitors revealed significant effects from most molecular modulators of protein trafficking, mild effects from most proposed COVID-19 drugs, and no effects from statins. This study noted that manipulating ACE2 levels could be beneficial or harmful, depending on the context. Thus, using this approach to uncover leads for COVID-19 therapeutics requires a thorough understanding ACE2’s biogenesis and biology.

Although Brassica juncea has demonstrated potential as a hyperaccumulator crop, it was not entirely clear how cadmium (Cd) accumulates in plants. Here, we found that BjNRAMP1 (Natural Resistance-Associated Macrophage Protein 1) plays a crucial role in the accumulation of Cd and manganese (Mn) through its expression in yeast and Arabidopsis thaliana. The high concentration of Cd exposure could induce the expression of BjNRAMP1. The ectopic expression of BjNRAMP1 in yeast led to higher accumulation of Cd and Mn compared to the vector control. BjNARAMP1 was localized to the plasma membrane and expressed in the vascular system of roots, leaves, and flowers. The overexpression of BjNRAMP1 in A. thaliana resulted in an increased accumulation of Cd in both roots and shoots, which inhibited the normal growth of transgenic lines. Moreover, Mn uptake in roots was activated by the increase in Cd stress. Together, our results indicated that BjNRAMP1 significantly contributes to the uptake of Mn and Cd in B. juncea. [ABSTRACT FROM AUTHOR]

Classical major histocompatibility complex (MHC) class II molecules play an essential role in immune system. In this study, MHC IIα (Pf‐MHC IIα) and MHC IIβ (Pf‐MHC IIβ) homology genes from pufferfish (Takifugu obscurus) were cloned and their functional characterization in response to bacterial challenge was identified. The nucleotide sequences of the open reading frames (ORFs) of pufferfish Pf‐MHC IIα and Pf‐MHC IIβ were 708 bp and 750 bp, encoding 235 aa and 249 aa, respectively. The structure of Pf‐MHC IIα or Pf‐MHC IIβ contained a signal peptide, an α1/β1 domain, an α2/β2 domain, a transmembrane region and a cytoplasmic region. Multiple sequence alignment and phylogenetic analysis showed that Pf‐MHC IIα and Pf‐MHC IIβ molecules had the highest similarity with Fugu rubripes (Takifugu rubripes). Cellular localization analysis indicated that the distribution of Pf‐MHC IIα and Pf‐MHC IIβ was in the lymphocyte membrane and cytoplasm. qRT‐PCR results showed that Pf‐MHC IIα and Pf‐MHC IIβ expressed relatively high in skin, gills and gut. In addition, after stimulation challenge in vitro (lipopolysaccharide, or polyinosinic: polycytidylic acid) and in vivo (A. hydrophila), the mRNA expressions of Pf‐MHC IIα and Pf‐MHC IIβ were significantly up‐regulated in lymphocytes and in tissues of skin, gills, gut and head kidney. Moreover, Pf‐MHC IIα or Pf‐MHC IIβ neutralization reduced the ability of A. hydrophila to induce the expressions of lymphocyte cytokines (TNF‐α, IL‐1β and IL‐10). Overall, it is speculated that Pf‐MHC IIα and Pf‐MHC IIβ may play an important role in the host response against A. hydrophila in pufferfish. [ABSTRACT FROM AUTHOR]

ABSTRACT For many bacterial proteins, specific localizations within the cell have been demonstrated, but enzymes involved in central metabolism are usually considered to be homogenously distributed within the cytoplasm. Here, we provide an example for a spatially defined localization of a unique enzyme complex found in actinobacteria, the hybrid pyruvate/2-oxoglutarate dehydrogenase complex (PDH-ODH). In non-actinobacterial cells, PDH and ODH form separate multienzyme complexes of megadalton size composed of three different subunits, E1, E2, and E3. The actinobacterial PDH-ODH complex is composed of four subunits, AceE (E1p), AceF (E2p), Lpd (E3), and OdhA (E1oE2o). Using fluorescence microscopy, we observed that in Corynebacterium glutamicum, all four subunits are co-localized in distinct spots at the cell poles, and in larger cells, additional spots are present at mid-cell. These results further confirm the existence of the hybrid complex. The unphosporylated OdhI protein, which binds to OdhA and inhibits ODH activity, was co-localized with OdhA at the poles, whereas phosphorylated OdhI, which does not bind OdhA, was distributed in the entire cytoplasm. Isocitrate dehydrogenase and glutamate dehydrogenase, both metabolically linked to ODH, were evenly distributed in the cytoplasm. Based on the available structural data for individual PDH-ODH subunits, a novel supramolecular architecture of the hybrid complex differing from classical PDH and ODH complexes has to be postulated. Our results suggest that localization at the poles or at mid-cell is most likely caused by nucleoid exclusion and results in a spatially organized metabolism in actinobacteria, with consequences yet to be studied. IMPORTANCE Enzymes involved in the central metabolism of bacteria are usually considered to be distributed within the entire cytoplasm. Here, we provide an example for a spatially defined localization of a unique enzyme complex of actinobacteria, the hybrid pyruvate dehydrogenase/2-oxoglutarate dehydrogenase (PDH-ODH) complex composed of four different subunits. Using fusions with mVenus or mCherry and fluorescence microscopy, we show that all four subunits are co-localized in distinct spots at the cell poles, and in larger cells, additional spots were observed at mid-cell. These results clearly support the presence of the hybrid PDH-ODH complex and suggest a similar localization in other actinobacteria. The observation of a defined spatial localization of an enzyme complex catalyzing two key reactions of central metabolism poses questions regarding possible consequences for the availability of substrates and products within the cell and other bacterial enzyme complexes showing similar behavior.

Wilson's disease (WD, MIM#277900) is an autosomal recessive disorder resulting in copper excess caused by biallelic variants in the ATP7B gene (MIM#606882) encoding a copper transporting P‐type ATPase. ATP7B variants of unknown significance (VUS) are detected frequently, sometimes impeding a clear diagnosis. Functional analyses can help to classify these variants as benign or pathogenic. Additionally, variants already classified as (likely) pathogenic benefit from functional analyses to understand their pathomechanism, thus contribute to the development of personalized treatment approaches in the future. We described clinical features of six WD patients and functionally characterized five ATP7B missense variants (two VUS, three yet uncharacterized likely pathogenic variants), detected in these patients. We determined the protein level, copper export capacity, and cellular localization in an in vitro model and potential structural consequences using an ATP7B protein model based on AlphaFold. Our analyses give insight into the pathomechanism and allowed reclassification for the two VUS to likely pathogenic and for two of the three likely pathogenic variants to pathogenic. [ABSTRACT FROM AUTHOR]

Abstract Amorphadiene is the precursor to synthesize the antimalarial drug artemisinin. The production of amorphadiene and artemisinin from metabolically engineered microbes may provide an alternate to plant secondary metabolite extraction. Microbial consortia can offer division of labor, and microbial co-culture system can be leveraged to achieve cost-efficient production of natural products. Using a co-culture system of Y. lipolytica Po1f and Po1g strains, subcellular localization of ADS gene (encoding amorphadiene synthase) into the endoplasmic reticulum, co-utilization of mixed carbon source, and enlargement of the endoplasmic reticulum (ER) surface area, we were able to significantly improve amorphadiene production in this work. Using Po1g/PPtM and Po1f/AaADSERx3/iGFMPDU strains and co-utilization of 5 µM sodium acetate with 20 g/L glucose in YPD media, amorphadiene titer were increased to 65.094 mg/L. The enlargement of the ER surface area caused by the deletion of the PAH1 gene provided more subcellular ER space for the action of the ADS-tagged gene. It further increased the amorphadiene production to 71.74 mg/L. The results demonstrated that the importance of the spatial localization of critical enzymes, and manipulating metabolic flux in the co-culture of Y. lipolytica can be efficient over a single culture for the bioproduction of isoprenoid-related secondary metabolites in a modular manner. Graphical Abstract

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Standard therapies for colorectal cancer cannot eliminate or sufficiently reduce the metastasis process. Photodynamic therapy (PDT) may be an alternative to minimizing this problem. Here, we examined the cellular localization of selected porphyrins and determined whether free-base and manganese (III) metallated porphyrins may limit colon cancer cells' (HT29) or normal colon epithelial cells' (CCD 841 CoTr) motility in vitro. White light irradiation was used to initiate the photodynamic effect. Porphyrin uptake by the cells was determined by porphyrin fluorescence measurements through the use of confocal microscopy. Free-base porphyrin was found in cells, where it initially localized at the edge of the cytoplasm and later in the perinuclear area. The concentrations of porphyrins had no effect on cancer cell migration but had a significant effect on normal cell motility. Due to the low concentrations of porphyrins used, no changes in F-actin filaments of the cellular cytoskeleton were detected. Signal transmission via connexons between neighbouring cells was limited to a maximum of 40 µm for HT29 and 30 µm for CCD 841 CoTr cells. The tested porphyrins differed in their activity against the tumor and normal cells' migration capacity. Depending on the porphyrin used and the type of cells, their migration changed in relation to the control sample. The use of white light may change the activity of the porphyrins relative to the migratory capacity of the cells. The aim of the present study was to analyse the intracellular localization of tested porphyrins and their influence on the mobility of cells after irradiation with harmless white light. [ABSTRACT FROM AUTHOR]

Amorphadiene is the precursor to synthesize the antimalarial drug artemisinin. The production of amorphadiene and artemisinin from metabolically engineered microbes may provide an alternate to plant secondary metabolite extraction. Microbial consortia can offer division of labor, and microbial co-culture system can be leveraged to achieve cost-efficient production of natural products. Using a co-culture system of Y. lipolytica Po1f and Po1g strains, subcellular localization of ADS gene (encoding amorphadiene synthase) into the endoplasmic reticulum, co-utilization of mixed carbon source, and enlargement of the endoplasmic reticulum (ER) surface area, we were able to significantly improve amorphadiene production in this work. Using Po1g/PPtM and Po1f/AaADSERx3/iGFMPDU strains and co-utilization of 5 µM sodium acetate with 20 g/L glucose in YPD media, amorphadiene titer were increased to 65.094 mg/L. The enlargement of the ER surface area caused by the deletion of the PAH1 gene provided more subcellular ER space for the action of the ADS-tagged gene. It further increased the amorphadiene production to 71.74 mg/L. The results demonstrated that the importance of the spatial localization of critical enzymes, and manipulating metabolic flux in the co-culture of Y. lipolytica can be efficient over a single culture for the bioproduction of isoprenoid-related secondary metabolites in a modular manner. [ABSTRACT FROM AUTHOR]

Subtilisin proteases, found in all organisms, are enzymes important in the post-translational steps of protein processing. In Leishmania major and L. donovani, this enzyme has been described as essential to their survival; however, few compounds that target subtilisin have been investigated for their potential as an antileishmanial drug. In this study, we first show, by electron microscopy and flow cytometry, that subtilisin has broad localization throughout the cytoplasm and membrane of the parasite in the promastigote form with foci in the flagellar pocket. Through in silico analysis, the similarity between subtilisin of different Leishmania species and that of humans were determined, and based on molecular docking, we evaluated the interaction capacity of a serine protease inhibitor against both life cycle forms of Leishmania. The selected inhibitor, known as PF-429242, has already been used against the dengue virus, arenaviruses, and the hepatitis C virus. Moreover, it proved to have antilipogenic activity in a mouse model and caused hypolipidemia in human cells in vitro. Here, PF-429242 significantly inhibited the growth of L. amazonensis promastigotes of four different strains (IC50 values = 3.07 ± 0.20; 0.83 ± 0.12; 2.02 ± 0.27 and 5.83 ± 1.2 µM against LTB0016, PH8, Josefa and LV78 strains) whilst having low toxicity in the host macrophages (CC50 = 170.30 µM). We detected by flow cytometry that there is a greater expression of subtilisin in the amastigote form; however, PF-429242 had a low effect against this intracellular form with an IC50 of >100 µM for intracellular amastigotes, as well as against axenic amastigotes (94.12 ± 2.8 µM for the LV78 strain). In conclusion, even though PF-429242 does not affect the intracellular forms, this drug will serve as a tool to explore pharmacological and potentially leishmanicidal targets.

Mortalin (GRP75, HSPA9A), a heat shock protein (HSP), regulates a wide range of cellular processes, including cell survival, growth, and metabolism. The regulatory functions of mortalin are mediated through a diverse set of protein partners associated with different cellular compartments, which allows mortalin to perform critical functions under physiological conditions, including mitochondrial protein quality control. However, alteration of mortalin’s activities, its abnormal subcellular compartmentalization, and its protein partners turn mortalin into a disease-driving protein in different pathological conditions, including cancers. Here, mortalin’s contributions to tumorigenic pathways are explained. Pathology information based on mortalin’s RNA expression extracted from The Cancer Genome Atlas (TCGA) transcriptomic database indicates that mortalin has an independent prognostic value in common tumors, including lung, breast, and colorectal cancer (CRC). Subsequently, the binding partners of mortalin reported in different cellular models, from yeast to mammalian cells, and its regulation by post-translational modifications are discussed. Finally, we focus on colorectal cancer and discuss how mortalin and its tumorigenic downstream protein targets are regulated by a ubiquitin-like protein through the 26S proteasomal degradation machinery. A broader understanding of the function of mortalin and its positive and negative regulation in the formation and progression of human diseases, particularly cancer, is essential for developing new strategies to treat a diverse set of human diseases critically associated with dysregulated mortalin.

A rhodamine B (RhB)-based initiator for atom transfer radical polymerization (ATRP) was synthesized and applied for preparation of poly(2-trimethylammoniumethyl methacrylate) (PChMA), poly(2-hydroxyethyl methacrylate) (PHEMA) and poly(2-trimethylsilyloxyethyl methacrylate) (PHEMATMS). Polymer fluorescence was confirmed by determination of quantum yield by comparative method with piroxicam as the standard exhibiting dependency of emission intensity on the polymer chain hydrophilicity and the kind of solvent. The RhB functionalized polymers were used for biological tests in plant materials except for RhB-PHEMATMS because of weak fluorescence. These two polymers slightly differed in cellular localization. RhB-PChMA was mostly observed in cell walls of root tissues and cotyledon epidermis. It was also observed in cytoplasm and cell organelles of root cap cells and rhizodermis, in contrast with cytoplasm of cotyledon epidermis. RhB-PHEMA was also present in apoplast. A strong signal in protoxylem cell walls and a weak signal in cell walls of rhizodermis and cortex were visible. Moreover, it was also present in cell walls of cotyledon epidermis. However, RhB-PHEMA was mostly observed in cytoplasm and cell organelles of all root tissues and epidermis of cotyledons. Both RhB-polymers did not cause cell death which means that they can be used in living plant material. [ABSTRACT FROM AUTHOR]

Quantum dots play an important role in the in vitro, ex vivo, and in vivo optical imaging. Significant advancements have been made in the areas of targeted specificity, biocompatibility, and surface modification, all of which have a big impact on the in vivo uses of quantum dots. These special qualities have drawn a lot of interest in the biomedical community recently because they make it possible for real-time tissue imaging (bioimaging), diagnostics, single molecule probes, and medication administration, among many other applications. Due to their high brightness, photobleach resistance, multiplexing ability, and high surface-to-volume ratio, quantum dots are excellent candidates for intracellular tracking, diagnostics, in vivo imaging, and therapeutic delivery. The optical properties of quantum dots can be tuned by size and composition. Due to its quick response and high sensitivity, fluorescence imaging has gained popularity recently. It has been difficult to create probes for this modality. By carefully regulating the synthetic processes, it is possible to create QDs with excellent imaging capabilities that emit in the first (650–950 nm) and second (1000–1400 nm) near infrared (NIR) windows. There are still several obstacles to be addressed before any NIR QDs may be used in clinical applications, despite their remarkable optical characteristics and biocompatibility. In this study, we go through the most recent developments in the use of QDs in various forms of biomedical imaging. [ABSTRACT FROM AUTHOR]

Cell signaling fidelity requires specificity in protein-protein interactions and precise subcellular localization of signaling molecules. In the case of protein phosphorylation, many kinases and phosphatases exhibit promiscuous substrate pairing and therefore require targeting interactions to modify the appropriate substrates and avoid cross-talk among different pathways. In the past 10 years, researchers have discovered and investigated how loss of specific interactions and subcellular targeting for the protein kinase A catalytic subunit (PKAc) lead to cortisol-producing adenomas and the debilitating stress disorder adrenal Cushing's syndrome. This article reviews classical studies regarding PKA localization in glucocorticoid-producing adrenal cells and synthesizes recent evidence of disrupted PKA localization and selective regulatory interactions in adrenal pathology., (© 2024 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Chlamydomonas incerta , a genetically close relative of the model green alga Chlamydomonas reinhardtii , shows significant potential as a host for recombinant protein expression. Because of the close genetic relationship between C. incerta and C. reinhardtii , this species offers an additional reference point for advancing our understanding of photosynthetic organisms, and also provides a potential new candidate for biotechnological applications. This study investigates C. incerta's capacity to express three recombinant proteins: the fluorescent protein mCherry, the hemicellulose-degrading enzyme xylanase, and the plastic-degrading enzyme PHL7. We have also examined the capacity to target protein expression to various cellular compartments in this alga, including the cytosol, secretory pathway, cytoplasmic membrane, and cell wall. When compared directly with C. reinhardtii , C. incerta exhibited a distinct but notable capacity for recombinant protein production. Cellular transformation with a vector encoding mCherry revealed that C. incerta produced approximately 3.5 times higher fluorescence levels and a 3.7-fold increase in immunoblot intensity compared to C. reinhardtii . For xylanase expression and secretion, both C. incerta and C. reinhardtii showed similar secretion capacities and enzymatic activities, with comparable xylan degradation rates, highlighting the industrial applicability of xylanase expression in microalgae. Finally, C. incerta showed comparable PHL7 activity levels to C. reinhardtii , as demonstrated by the in vitro degradation of a polyester polyurethane suspension, Impranil ® DLN. Finally, we also explored the potential of cellular fusion for the generation of genetic hybrids between C. incerta and C. reinhardtii as a means to enhance phenotypic diversity and augment genetic variation. We were able to generate genetic fusion that could exchange both the recombinant protein genes, as well as associated selectable marker genes into recombinant offspring. These findings emphasize C. incerta 's potential as a robust platform for recombinant protein production, and as a powerful tool for gaining a better understanding of microalgal biology., Competing Interests: CONFLICT OF INTEREST SM was a founding of and holds an equity stake in Algenesis Inc, a company that could potentially benefit from this work. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Background & Aims: The association between Wilson disease and various ATP7B mutations is well-established; however, the molecular mechanism underlying the functional consequence of these mutations, particularly the splicing mutations, remains unclear. This study focused on the ATP7B c.1543+1G>C variant, to reveal a universal pathogenic mechanism of the ATP7B mutants with altered N-terminus., Methods: The splicing assay and RNA pull-down were performed to explore the mechanism of the aberrant splicing. The ATP7B knockout HuH-7 cell line and Atp7b -/- mice were created, and the functional consequence of the mutant ATP7B were evaluated in vitro and in vivo., Results: The c.1543+1G>C mutation resulted in the skipping of ATP7B exon 3, and the mutant ATP7B showed a loss of trans-Golgi network localization and was degraded via the ubiquitin-proteasome pathway, facilitated by enhanced interactions with COMMD1. Elevated intercellular copper concentration and reduced survival rate were observed in HuH-7 cells expressing mutant ATP7B. Restoration of wild-type ATP7B in Atp7b -/- mice resulted in a substantial improvement in phenotype, whereas mice treated with mutant ATP7B did not demonstrate equivalent benefits., Conclusions: Our research investigated the pathogenicity and mechanism of ATP7B c.1543+1G>C variant, with particular focus on its enhanced interaction with COMMD1 as a potential universal mechanism contributing to the dysfunction of various ATP7B variants. These findings provide a foundation for the development of innovative therapeutic strategies that target abnormal splicing events in a range of hereditary diseases, including Wilson disease., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Recent advances in single cell RNA sequencing (scRNA‐seq) empower insights into cell–cell crosstalk within specific tissues. However, customizable data analysis tools that decipher intercellular communication from gene expression in association with biological functions are lacking. The authors have developed InterCellDB, a platform that allows a user‐defined analysis of intercellular communication using scRNA‐seq datasets in combination with protein annotation information, including cellular localization and functional classification, and protein interaction properties. The application of InterCellDB in tumor microenvironment research is exemplified using two independent scRNA‐seq datasets from human and mouse and it is demonstrated that InterCellDB‐inferred cell–cell interactions and ligand–receptor pairs are experimentally valid. [ABSTRACT FROM AUTHOR]

Preeclampsia, a disorder with a heterogeneous physiopathology, can be attributed to maternal, fetal, and/or placental factors. Long non-coding RNAs (lncRNAs) refer to a class of non-coding RNAs, the essential regulators of biological processes; their differential expression has been associated with the pathogenesis of multiple diseases. The study aimed to identify lncRNAs, expressed in the placentas and plasma of patients who presented with preeclampsia, as potential putative biomarkers of the disease. In silico analysis was performed to determine lncRNAs differentially expressed in the placentas of patients with preeclampsia, using a previously published RNA-Seq dataset. Seven placentas and maternal plasma samples collected at delivery from preterm preeclamptic patients (≤37 gestational weeks of gestation), and controls were used to validate the expression of lncRNAs by qRT-PCR. Six lncRNAs were validated and differentially expressed (p < 0.05) in the preeclampsia and control placentas: UCA1 and HCG4 were found upregulated, and LOC101927355, LINC00551, PART1, and NRAD1 downregulated. Two of these lncRNAs, HCG4 and LOC101927355, were also detected in maternal plasma, the latter showing a significant decrease (p = 0.03) in preeclamptic patients compared to the control group. In silico analyses showed the cytoplasmic location of LOC101927355, which suggests a role in post-transcriptional gene regulation. The detection of LOC101927355 in the placenta and plasma opens new possibilities for understanding the pathogenesis of preeclampsia and for its potential use as a biomarker. [ABSTRACT FROM AUTHOR]

Standard therapies for colorectal cancer cannot eliminate or sufficiently reduce the metastasis process. Photodynamic therapy (PDT) may be an alternative to minimizing this problem. Here, we examined the cellular localization of selected porphyrins and determined whether free-base and manganese (III) metallated porphyrins may limit colon cancer cells’ (HT29) or normal colon epithelial cells’ (CCD 841 CoTr) motility in vitro. White light irradiation was used to initiate the photodynamic effect. Porphyrin uptake by the cells was determined by porphyrin fluorescence measurements through the use of confocal microscopy. Free-base porphyrin was found in cells, where it initially localized at the edge of the cytoplasm and later in the perinuclear area. The concentrations of porphyrins had no effect on cancer cell migration but had a significant effect on normal cell motility. Due to the low concentrations of porphyrins used, no changes in F-actin filaments of the cellular cytoskeleton were detected. Signal transmission via connexons between neighbouring cells was limited to a maximum of 40 µm for HT29 and 30 µm for CCD 841 CoTr cells. The tested porphyrins differed in their activity against the tumor and normal cells’ migration capacity. Depending on the porphyrin used and the type of cells, their migration changed in relation to the control sample. The use of white light may change the activity of the porphyrins relative to the migratory capacity of the cells. The aim of the present study was to analyse the intracellular localization of tested porphyrins and their influence on the mobility of cells after irradiation with harmless white light.

Both paralogs of the calcium-dependent activator protein for secretion (CAPS) are required for exocytosis of synaptic vesicles (SVs) and large dense core vesicles (LDCVs). Despite approximately 80% sequence identity, CAPS1 and CAPS2 have distinct functions in promoting exocytosis of SVs and LDCVs in dorsal root ganglion (DRG) neurons. However, the molecular mechanisms underlying these differences remain enigmatic. In this study, we applied high- and super-resolution imaging techniques to systematically assess the subcellular localization of CAPS paralogs in DRG neurons deficient in both CAPS1 and CAPS2. CAPS1 was found to be more enriched at the synapses. Using – in-depth sequence analysis, we identified a unique CAPS1 N-terminal sequence, which we introduced into CAPS2. This CAPS1/2 chimera reproduced the pre-synaptic localization of CAPS1 and partially rescued synaptic transmission in neurons devoid of CAPS1 and CAPS2. Using immunoprecipitation combined with mass spectrometry, we identified CAPS1-specific interaction partners that could be responsible for its pre-synaptic enrichment. Taken together, these data suggest an important role of the CAPS1-N terminus in the localization of the protein at pre-synapses. [ABSTRACT FROM AUTHOR]

Despite extensive research, there is still no treatment that would lead to remission in all patients with rheumatoid arthritis as our understanding of the affected site, the synovium, is still incomplete. Recently, single-cell technologies helped to decipher the cellular heterogeneity of the synovium; however, certain synovial cell populations, such as endothelial cells or peripheral neurons, remain to be profiled on a single-cell level. Furthermore, associations between certain cellular states and inflammation were found; whether these cells cause the inflammation remains to be answered. Similarly, cellular zonation and interactions between individual effectors in the synovium are yet to be fully determined. A deeper understanding of cell signalling and interactions in the synovium is crucial for a better design of therapeutics with the goal of complete remission in all patients. [ABSTRACT FROM AUTHOR]

Wenlong Sheng,1 Shijun Weng,2 Fei Li,1 Yun Zhang,1 Qiuxia He,1 Wenxiang Sheng,1 Ying Fu,3 Haiyue Yan,4 Kechun Liu1 1Biology Institute, Qilu University of Technology (Shandong Academy of Sciences), Jinan, People’s Republic of China; 2State Key Laboratory of Medical Neurobiology, Institutes of Brain Science, Department of Ophthalmology, Zhongshan Hospital, Fudan University, Shanghai, People’s Republic of China; 3Shandong Science and Technology Exchange Center, Jinan, People’s Republic of China; 4Shandong Institute of Scientific and Technical Information, Jinan, People’s Republic of ChinaCorrespondence: Wenlong Sheng; Kechun Liu Email shengwenlong1121@163.com; liukechun2000@163.comBackground: Melatonin (N-acetyl-5-methoxytryptamine), a significant indoleamine neuromodulator implicated in circadian rhythms and sleep patterns, regulates diverse rhythmic functions via activating its high-affinity G-protein-coupled receptors. However, the detailed cellular expression of the Mel1a receptor in the retina is still a research gap.Methods: The expression of the Mel1a receptor in pigeon retina was assessed using Western blot analysis and immunofluorescent staining. The cellular localization of the Mel1a receptor was studied using double immunofluorescent staining and laser-scanning confocal microscopy.Results: Our data suggested that the Mel1a receptor was extensively expressed in the outer segment of Rho4D2-labeled rod and L/M-opsin-labeled red/green cone and in the somata of the CB-labeled horizontal cell, TH-labeled dopaminergic amacrine cell, ChAT-labeled cholinergic amacrine cell, PV-labeled AII amacrine cell, Brn3a-labeled conventional ganglion cell, melanopsin-containing ganglion cell and CRALBP-labeled Müller glial cell. In addition, the Mel1a receptor was diffusely distributed throughout the full thickness of the inner plexiform layer. However, the outer segment of S-opsin-labeled blue cone, the somata of ChX-10-labeled bipolar cell and outer plexiform layer seemed to lack immunoreactivity of the Mel1a receptor.Conclusion: The finding that multiple types of retinal cells express the Mel1a receptor provides a new neurobiological basis for the participation of melatonin in the regulation of retinal functions through activating the Mel1a receptor.Keywords: circadian rhythm, retinal Mel1a receptor, cellular localization, diurnal animal

Both paralogs of the calcium-dependent activator protein for secretion (CAPS) are required for exocytosis of synaptic vesicles (SVs) and large dense core vesicles (LDCVs). Despite approximately 80% sequence identity, CAPS1 and CAPS2 have distinct functions in promoting exocytosis of SVs and LDCVs in dorsal root ganglion (DRG) neurons. However, the molecular mechanisms underlying these differences remain enigmatic. In this study, we applied high- and super-resolution imaging techniques to systematically assess the subcellular localization of CAPS paralogs in DRG neurons deficient in both CAPS1 and CAPS2. CAPS1 was found to be more enriched at the synapses. Using – in-depth sequence analysis, we identified a unique CAPS1 N-terminal sequence, which we introduced into CAPS2. This CAPS1/2 chimera reproduced the pre-synaptic localization of CAPS1 and partially rescued synaptic transmission in neurons devoid of CAPS1 and CAPS2. Using immunoprecipitation combined with mass spectrometry, we identified CAPS1-specific interaction partners that could be responsible for its pre-synaptic enrichment. Taken together, these data suggest an important role of the CAPS1-N terminus in the localization of the protein at pre-synapses.

With the development of gold nanorods (AuNRs) for a number of biomedical applications, understanding their various biological effects has become important. Surface functionalization of AuNRs is attracting increasing attention with regard to toxicity, cellular uptake, localization, therapeutic potential, and biodistribution. The aim of the study is to synthesis of stable AuNRs functionalized with polyethylenimine (PEI) and polyethylene glycol (PEG), multiparametric investigation of their cellular effects on the dorsal root ganglion (DRG) primary sensory neurones, evaluation of the in vitro/in vivo biodistribution, and toxicity. PEI and PEG surface coatings increased both biocompatibility and biodistribution of the AuNRs. With the near‐infrared laser of the two‐photon microscope, very strong radiations are taken from the nucleolus parts of the neurones particularly and these localizations of the AuNRs‐PEI are confirmed by the transmission electron microscope images. Inductively coupled plasma mass spectrometry analysis shows the presence of AuNRs in liver, spleen, kidney, heart, blood, and brain within a 30 day period. It is observed that the surface coatings of the AuNRs significantly increase the biodistribution and biocompatibility. The surface functionalization, stability, and biocompatibility of the AuNRs are very important parameters for the potential nanotheranostic applications of AuNRs in the next studies. [ABSTRACT FROM AUTHOR]

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Ribonucleases (RNases) are essential for almost every aspect of RNA metabolism. However, despite their important metabolic roles, RNases can also be destructive enzymes. As a consequence, cells must carefully regulate the amount, the activity, and the localization of RNases to avoid the inappropriate degradation of essential RNA molecules. In addition, bacterial cells often must adjust RNase levels as environmental situations demand, also requiring careful regulation of these critical enzymes. As the need for strict control of RNases has become more evident, multiple mechanisms for this regulation have been identified and studied, and these are described in this review. The major conclusion that emerges is that no common regulatory mechanism applies to all RNases, or even to a family of RNases; rather, a wide variety of processes have evolved that act on these enzymes, and in some cases, multiple regulatory mechanisms can even act on a single RNase. [ABSTRACT FROM AUTHOR]

Ascorbate-glutathione (AsA-GSH) cycle plays an important role in tuning beneficial ROS accumulation for intracellular signals and imparts plant tolerance to oxidative stress by detoxifying excess of ROS. Here, we present genome-wide identification of AsA-GSH cycle genes (APX , MDHAR , DHAR , and GR) in several leguminous species and expression analyses in G. max during stress, germination and tissue development. Our data revealed 24 genes in Glycine genus against the maximum of 15 in other leguminous species, which was due to 9 pars of duplicated genes mostly originated from sub/neofunctionalization. Cytosolic APX and MDHAR genes were highly expressed in different tissues and physiological conditions. Germination induced genes encoding AsA-GSH proteins from different cell compartments, whereas vegetative phase (leaves) stimulated predominantly genes related to chloroplast/mitochondria proteins. Moreover, cytosolic APX-1 , 2 , MDHAR-1a , 1b and GR genes were the primary genes linked to senescence and biotic stresses, while stAPX - a , b and GR (from organelles) were the most abiotic stress related genes. Biotic and abiotic stress tolerant genotypes generally showed increased MDHAR , DHAR and/or GR mRNA levels compared to susceptible genotypes. Overall, these data clarified evolutionary events in leguminous plants and point to the functional specificity of duplicated genes of the AsA-GSH cycle in G. max. [ABSTRACT FROM AUTHOR]

The cellular localization of exogenous proteins expressed in transgenic crops not only determines their stability, but also their effects on crop growth and development, including under stressful conditions; however, the underlying molecular mechanisms remain unknown. Here, we determined the cellular distribution of exogenously expressed Cry1Ab/c protein in insect-resistant transgenic rice Huahui-1 (HH1) cells through subcellular localization, immunohistochemistry, immunofluorescence, and western blot analyses. Interaction between the Cry1Ab/c protein and the preliminarily screened endogenous plasma membrane protein Ca2+-ATPase was investigated through yeast two-hybrid, bimolecular fluorescence complementation (BIFC), and co-immunoprecipitation analyses. The potential interaction mechanism was analyzed by comparing the cellular localization and interaction sites between Cry1Ab/c and Ca2+-ATPase. Phenotypic indices and Ca2+-ATPase activity, which may be regulated by the Cry1Ab/c–Ca2+-ATPase interaction, were determined in transgenic HH1 and the parental line Minghui-63 under stress-free and salt-stress conditions. The results showed that Cry1Ab/c was not only distributed in the cytoplasm and nucleus but was also distributed on the plasma membrane, where it interacted with plasma membrane Ca2+-ATPase. This interaction partially retain plasma membrane protein Ca2+-ATPase in the nucleus by a BIFC experiment and thus may affect Ca2+-ATPase activity on the membrane by altering the cellular location of the protein. Consistently, our results confirmed that the presence of Cry1Ab/c in the transgenic HH1 resulted in a reduction in Ca2+-ATPase activity as well as causing detrimental effects on plant phenotype, including significantly reduced plant height and biomass, compared to parental MH63; and that these detrimental effects were more pronounced under salt stress conditions, impacting the salt resistance of the transgenic plants. We suggest that the Cry1Ab/c–Ca2+-ATPase interaction may explain the plasma membrane localization of Cry1Ab/c, which lacks a signal peptide and a transmembrane domain, and the adverse effects of Cry1Ab/c expression on the growth and development of transgenic HH1 plants under salt stress. This information may clarify the molecular mechanisms of these unintended effects and demonstrate the feasibility of evaluating the success and performance of genetic modification of commercially vital crops.

A rhodamine B (RhB)-based initiator for atom transfer radical polymerization (ATRP) was synthesized and applied for preparation of poly(2-trimethylammoniumethyl methacrylate) (PChMA), poly(2-hydroxyethyl methacrylate) (PHEMA) and poly(2-trimethylsilyloxyethyl methacrylate) (PHEMATMS). Polymer fluorescence was confirmed by determination of quantum yield by comparative method with piroxicam as the standard exhibiting dependency of emission intensity on the polymer chain hydrophilicity and the kind of solvent. The RhB functionalized polymers were used for biological tests in plant materials except for RhB-PHEMATMS because of weak fluorescence. These two polymers slightly differed in cellular localization. RhB-PChMA was mostly observed in cell walls of root tissues and cotyledon epidermis. It was also observed in cytoplasm and cell organelles of root cap cells and rhizodermis, in contrast with cytoplasm of cotyledon epidermis. RhB-PHEMA was also present in apoplast. A strong signal in protoxylem cell walls and a weak signal in cell walls of rhizodermis and cortex were visible. Moreover, it was also present in cell walls of cotyledon epidermis. However, RhB-PHEMA was mostly observed in cytoplasm and cell organelles of all root tissues and epidermis of cotyledons. Both RhB-polymers did not cause cell death which means that they can be used in living plant material.

TRIF, an important adaptor downstream of Toll-like receptor signaling, plays a critical role in the innate immune response. In this study, the full-length coding sequence of TRIF from common carp (Cyprinus carpio L.) was cloned and characterized. Bioinformatics analysis showed that common carp TRIF exhibited a conserved TIR domain and had the closest relationship with grass carp TRIF. Expression analysis revealed that TRIF was constitutively expressed in the examined tissues of common carp, with the highest expression in the spleen and the lowest expression in the head kidney, and could be upregulated under Aeromonas hydrophila and poly(I:C) stimulation in vivo and under poly(I:C), LPS, PGN, flagellin, and Pam3CSK4 stimulation in vitro. Laser confocal microscopy showed that common carp TRIF colocalized with the Golgi apparatus. A luciferase reporter assay showed that carp TRIF elicited the activity of ifn-1 and nf-κb through the C-terminal domain. Additionally, crystal violet staining and qPCR assays revealed that carp TRIF inhibited the replication of SVCV in epithelioma papulosum cyprini (EPC) cells. Then, the signaling downstream of carp TRIF was investigated. Coimmunoprecipitation and Western blotting analysis demonstrated that carp TRIF interacted with TBK1 and augmented the expression of TRAF6 and phosphorylation of TBK1. Overexpression of carp TRIF significantly enhanced the expression of interferon-stimulated genes and inflammatory cytokines. Furthermore, flow cytometric (FCM) analysis suggested that carp TRIF induced apoptosis through the activation of caspase-8. In summary, our study indicated that TRIF plays an essential role in the innate immune responses of common carp against bacterial and viral infection.

TRIF, an important adaptor downstream of Toll-like receptor signaling, plays a critical role in the innate immune response. In this study, the full-length coding sequence of TRIF from common carp (Cyprinus carpio L.) was cloned and characterized. Bioinformatics analysis showed that common carp TRIF exhibited a conserved TIR domain and had the closest relationship with grass carp TRIF. Expression analysis revealed that TRIF was constitutively expressed in the examined tissues of common carp, with the highest expression in the spleen and the lowest expression in the head kidney, and could be upregulated under Aeromonas hydrophila and poly(I:C) stimulation in vivo and under poly(I:C), LPS, PGN, flagellin, and Pam3CSK4 stimulation in vitro. Laser confocal microscopy showed that common carp TRIF colocalized with the Golgi apparatus. A luciferase reporter assay showed that carp TRIF elicited the activity of ifn-1 and nf-κb through the C-terminal domain. Additionally, crystal violet staining and qPCR assays revealed that carp TRIF inhibited the replication of SVCV in epithelioma papulosum cyprini (EPC) cells. Then, the signaling downstream of carp TRIF was investigated. Coimmunoprecipitation and Western blotting analysis demonstrated that carp TRIF interacted with TBK1 and augmented the expression of TRAF6 and phosphorylation of TBK1. Overexpression of carp TRIF significantly enhanced the expression of interferon-stimulated genes and inflammatory cytokines. Furthermore, flow cytometric (FCM) analysis suggested that carp TRIF induced apoptosis through the activation of caspase-8. In summary, our study indicated that TRIF plays an essential role in the innate immune responses of common carp against bacterial and viral infection. [ABSTRACT FROM AUTHOR]

Tumor necrosis factor receptor-associated factor 5 (TRAF5) is an intracellular protein that binds to the cytoplasmic portion of tumor necrosis factor receptors and mediates the activation of downstream nuclear factor-kappa B (NF-κB), interferon regulatory factor 3, and mitogen activated protein kinase signaling pathways. Compared with other TRAF proteins, TRAF5 is largely unknown in teleosts. In the present study, a TRAF5 homologue (HgTRAF5) from the hybrid grouper (Epinephelus fuscoguttatus♂ × Epinephelus lanceolatus ♀) was cloned and characterized. The open reading frame of HgTRAF5 consists of 1743 nucleotides encoding a 581 amino acid protein with a predicted molecular mass of 64.90 kDa. Similar to its mammalian counterpart, HgTRAF5 contains an N-terminal RING finger domain, a zinc finger domain, and a C-terminal TRAF domain, including a coiled-coil domain and a MATH domain. HgTRAF5 shares 99.83% identity with giant grouper (Epinephelus lanceolatus) TRAF5. Quantitative real-time PCR analysis indicated that HgTRAF5 mRNA was broadly expressed in all examined tissues. The expression of HgTRAF5 increased after Singapore grouper iridovirus (SGIV) infection in grouper spleen (GS) cells. Intracellular localization analysis demonstrated that the full-length HgTRAF5 protein mainly distributed in the cytoplasm. HgTRAF5 overexpression also promoted SGIV replication during viral infection in vitro. HgTRAF5 significantly promoted the activities of interferon-β, interferon-sensitive response element, and NF-κB. Taken together, these results are important for a better understanding of the function of TRAF5 in fish and reveal its involvement in the host response to immune challenge by SGIV. • HgTRAF5 mRNA was broadly expressed in all examined tissues. • HgTRAF5 were identified as a cytosolic protein. • HgTRAF5 enhanced SGIV replication during viral infection. [ABSTRACT FROM AUTHOR]

• The appearance of pepsin activity showed a certain lag behind the appearance of the gastric glands. • OpPGA1 was predominantly expressed in the carnivorous group, whereas the OpPGA2 gene was significantly higher in the phytophagous group than in the other groups. • Correlation between pepsinogen gene expression and pepsin activity and feeding time. • Pepsin activity was positively correlated with feeding time in different feeding groups. • In situ hybridization results revealed that mRNA of OpPGA1 and OpPGA 2 were expressed in the gastric gland cells. To solve the high mortality rate of early-stage larval feed conversion during aquaculture in Oplegnathus punctatus , the investigation of the structural and functional characteristics of the gastric tissue was conducted. Histological results showed that the gastric gland rudiment appeared at 17 dph. The basic structure of the stomach was fully developed between 26 and 35 dph. Two pepsinogen genes, named OpPGA1 and OpPGA2 , were identified in the spotted knifejaw genome. qPCR results of developmental period showed that the two genes were low in expression during early development (5 and 15 dph). At 20 dph, the two genes started to show trace expression, and at 30 dph the mRNA expression levels of OpPGA1 and OpPGA2 reached the highest levels. Results of pepsin activity detection during the development period showed lower activity was detected 22 dph, followed by a peak at 30 dph. Under different feeding inductions, OpPGA1 showed the highest expression in the basic diet group and hard-shell group, while the expression level in the phytophagous group remained consistently low. The mRNA expression level of OpPGA2 in the phytophagous group was significantly higher than in other groups. Enzyme activity determination under different feeding inductions showed slightly higher enzyme activity in the basic diet group and crustacean group. The results of in situ hybridization showed that the mRNA of both OpPGA1 and OpPGA2 genes was both expressed in gastric gland cells. These information can contribute to the development of practical feeding methods in terms of digestive physiology for the development of larvae. [ABSTRACT FROM AUTHOR]

Alzheimer’s disease is pathologically defined by accumulation of extracellular amyloid-β (Aβ). Approximately 25 mutations in β-amyloid precursor protein (APP) are pathogenic and cause autosomal dominant Alzheimer’s disease. To date, the mechanism underlying the effect of APP mutation on Aβ generation is unclear. Therefore, investigating the mechanism of APP mutation on Alzheimer’s disease may help understanding of disease pathogenesis. Thus, APP mutations (A673T, A673V, E682K, E693G, and E693Q) were transiently co-transfected into human embryonic kidney cells. Western blot assay was used to detect expression levels of APP, beta-secretase 1, and presenilin 1 in cells. Enzyme-linked immunosorbent assay was performed to determine Aβ1–40 and Aβ1–42 levels. Liquid chromatography-tandem mass chromatography was used to examine VVIAT, FLF, ITL, VIV, IAT, VIT, TVI, and VVIA peptide levels. Immunofluorescence staining was performed to measure APP and early endosome antigen 1 immunoreactivity. Our results show that the protective A673T mutation decreases Aβ42/Aβ40 rate by downregulating IAT and upregulating VVIA levels. Pathogenic A673V, E682K, and E693Q mutations promote Aβ42/Aβ40 rate by increasing levels of CTF99, Aβ42, Aβ40, and IAT, and decreasing VVIA levels. Pathogenic E693G mutation shows no significant change in Aβ42/Aβ40 ratio because of inhibition of γ-secretase activity. APP mutations can change location from the cell surface to early endosomes. Our findings confirm that certain APP mutations accelerate Aβ generation by affecting the long Aβ cleavage pathway and increasing Aβ42/40 rate, thereby resulting in Alzheimer’s disease.

Cancer is a disease caused by the accumulation of genetic and epigenetic changes in two types of genes: tumor suppressor genes (TSGs) and proto-oncogenes. Extensive research has been conducted over the last few decades to elucidate the role of TSGs in cancer development. In cancer, loss of TSG function occurs via the deletion or inactivation of two alleles, according to Knudson’s two-hit model hypothesis. It has become clear that mutations in TSGs are recessive at the level of an individual cell; therefore, a single mutation in a TSG is not sufficient to cause carcinogenesis. However, many studies have identified candidate TSGs that do not conform with this standard definition, including genes inactivated by epigenetic silencing rather than by deletion. In addition, proteasomal degradation by ubiquitination, abnormal cellular localization, and transcriptional regulation are also involved in the inactivation of TSGs. This review incorporates these novel additional mechanisms of TSG inactivation into the existing two-hit model and proposes a revised multiple-hit model that will enable the identification of novel TSGs that can be used as prognostic and predictive biomarkers of cancer.

Reduced growth habit is a desirable trait for ornamental potted plants and can successfully be obtained through Rhizobium rhizogenes transformation in a stable and heritable manner. Additionally, it can also be obtained by transformation with Agrobacterium tumefaciens harboring specific genes from R. rhizogenes. The bacterial T-DNA harbors four root oncogenic loci (rol) genes and 14 less known open reading frames (ORFs). The four rol genes, i.e., rolA, rolB, rolC, and rolD, are conceived as the common denominator for the compact phenotype and the other less characterized ORFs seem auxiliary but present a potential breeding target for less aberrant and/or more tailored phenotypes. In this study, Kalanchoë blossfeldiana ‘Molly’ was transformed with individual rol genes and selected ORFs in 35S overexpressing cassettes to comprehensively characterize growth traits, gene copy and expression, and ethylene tolerance of the flowers. An association of reduced growth habit, e.g. height and diameter, was observed for rolB2 and ORF14-2 when a transgene single copy and high gene expression were detected. Chlorophyll content was reduced in overexpressing lines compared to wild type (WT), except for one ΔORF13a (a truncated ORF13a, where SPXX DNA-binding motif is absent). The flower number severely decreased in the overexpressing lines compared to WT. The anthesis timing showed that WT opened the first flower at 68.9 ± 0.9 days and the overexpressing lines showed similar or up to 24 days delay in flowering. In general, a single or low relative gene copy insertion was correlated to higher gene expression, ca. 3 to 5-fold, in rolB and ΔORF13a lines, while in ORF14 such relation was not directly linked. The increased gene expression observed in rolB2 and ΔORF13a-2 contributed to reducing plant growth and a more compact habit. Tolerance of detached flowers to 0.5 μl L−1 ethylene was markedly higher for ORF14 with 66% less flower closure at day 3 compared to WT. The subcellular localization of rolC and ΔORF13a was investigated by transient expression in Nicotiana benthamiana and confocal images showed that rolC and ΔORF13a are soluble and localize in the cytoplasm being able to enter the nucleus.

Reduced growth habit is a desirable trait for ornamental potted plants and can successfully be obtained through Rhizobium rhizogenes transformation in a stable and heritable manner. Additionally, it can also be obtained by transformation with Agrobacterium tumefaciens harboring specific genes from R. rhizogenes. The bacterial T-DNA harbors four root oncogenic loci (rol) genes and 14 less known open reading frames (ORFs). The four rol genes, i.e., rol A, rol B, rol C, and rol D, are conceived as the common denominator for the compact phenotype and the other less characterized ORFs seem auxiliary but present a potential breeding target for less aberrant and/or more tailored phenotypes. In this study, Kalanchoë blossfeldiana 'Molly' was transformed with individual rol genes and selected ORFs in 35S overexpressing cassettes to comprehensively characterize growth traits, gene copy and expression, and ethylene tolerance of the flowers. An association of reduced growth habit, e.g. height and diameter, was observed for rol B2 and ORF14-2 when a transgene single copy and high gene expression were detected. Chlorophyll content was reduced in overexpressing lines compared to wild type (WT), except for one ΔORF13a (a truncated ORF13a, where SPXX DNA-binding motif is absent). The flower number severely decreased in the overexpressing lines compared to WT. The anthesis timing showed that WT opened the first flower at 68.9 ± 0.9 days and the overexpressing lines showed similar or up to 24 days delay in flowering. In general, a single or low relative gene copy insertion was correlated to higher gene expression, ca. 3 to 5-fold, in rol B and ΔORF13a lines, while in ORF14 such relation was not directly linked. The increased gene expression observed in rol B2 and ΔORF13a-2 contributed to reducing plant growth and a more compact habit. Tolerance of detached flowers to 0.5 μl L−1 ethylene was markedly higher for ORF14 with 66% less flower closure at day 3 compared to WT. The subcellular localization of rol C and ΔORF13a was investigated by transient expression in Nicotiana benthamiana and confocal images showed that rol C and ΔORF13a are soluble and localize in the cytoplasm being able to enter the nucleus. [ABSTRACT FROM AUTHOR]

Chemotaxis is essential for the competitiveness of motile bacteria in complex and harsh environments. The localization of chemotactic proteins in the cell is critical for coordinating a maximal response to chemotactic signals. One chemotaxis protein with a well-defined subcellular localization is the phosphatase CheZ. CheZ localizes to cell poles by binding with CheA in Escherichia coli and other enteric bacteria, or binding with a poorly understood protein called ChePep in epsilon-Proteobacteria. In alpha-Proteobacteria, CheZ lacks CheA-binding sites, and its cellular localization remains unknown. We therefore determined the localization of CheZ in the alpha-Proteobacteria Azorhizobium caulinodans ORS571. A. caulinodans CheZ, also termed as CheZAC, was found to be located to cell poles independently of CheA, and we suspect that either the N-terminal helix or the four-helix bundle of CheZAC is sufficient to locate to cell poles. We also found a novel motif, AXXFQ, which is adjacent to the phosphatase active motif DXXXQ, which effects the monopolar localization of CheZAC. This novel motif consisting of AXXFQ is conserved in CheZ and widely distributed among Proteobacteria. Finally, we found that the substitution of phosphatase active site affects the polar localization of CheZAC. In total, this work characterized the localization pattern of CheZ containing a novel motif, and we mapped the regions of CheZAC that are critical for its polar localization.

Preeclampsia, a disorder with a heterogeneous physiopathology, can be attributed to maternal, fetal, and/or placental factors. Long non-coding RNAs (lncRNAs) refer to a class of non-coding RNAs, the essential regulators of biological processes; their differential expression has been associated with the pathogenesis of multiple diseases. The study aimed to identify lncRNAs, expressed in the placentas and plasma of patients who presented with preeclampsia, as potential putative biomarkers of the disease. In silico analysis was performed to determine lncRNAs differentially expressed in the placentas of patients with preeclampsia, using a previously published RNA-Seq dataset. Seven placentas and maternal plasma samples collected at delivery from preterm preeclamptic patients (≤37 gestational weeks of gestation), and controls were used to validate the expression of lncRNAs by qRT-PCR. Six lncRNAs were validated and differentially expressed (p < 0.05) in the preeclampsia and control placentas: UCA1 and HCG4 were found upregulated, and LOC101927355, LINC00551, PART1, and NRAD1 downregulated. Two of these lncRNAs, HCG4 and LOC101927355, were also detected in maternal plasma, the latter showing a significant decrease (p = 0.03) in preeclamptic patients compared to the control group. In silico analyses showed the cytoplasmic location of LOC101927355, which suggests a role in post-transcriptional gene regulation. The detection of LOC101927355 in the placenta and plasma opens new possibilities for understanding the pathogenesis of preeclampsia and for its potential use as a biomarker.

The expression of prototype galectin‐14 (Gal‐14) in human placenta is higher than any other galectin, suggesting that it may play a role in fetal development and regulation of immune tolerance during pregnancy. Here, we solved the crystal structure of dimeric Gal‐14 and found that its global fold is significantly different from that of other galectins with two β‐strands (S5 and S6) extending from one monomer and contributing to the carbohydrate‐binding domain of the other. The hemagglutination assay showed that this lectin could induce agglutination of chicken erythrocytes, even though lactose could not inhibit Gal‐14‐induced agglutination activity. Calorimetry indicates that lactose does not interact with this lectin. Compared to galectin‐1, galectin‐3, and galectin‐8, Gal‐14 has two key amino acids (a histidine and an arginine) in the normally conserved, canonical sugar‐binding site, which are substituted by glutamine (Gln53) and histidine (His57), thus likely explaining why lactose binding to this lectin is very weak. Lactose was observed in the ligand‐binding site of one Gal‐14 structure, most likely because ligand binding is weak and crystals were allowed to grow over a long period of time in the presence of lactose. We also found that EGFP‐tagged Gal‐14 is primarily localized within the nucleus of different cell types. In addition, Gal‐14 colocalized with c‐Rel (a member of NF‐κB family) in HeLa cells. These findings indicate that Gal‐14 might regulate signal transduction pathways through NF‐κB hubs. Overall, the present study provides impetus for further research into the function of Gal‐14 in embryology. [ABSTRACT FROM AUTHOR]

Cotton leaf curl Multan betasatellite (CLCuMB) is a ubiquitous betasatellite commonly found along with cotton leaf curl disease (CLCuD) associated begomoviruses in the Old World. It has a promiscuous replicative nature and trans-replicated by a diverse range of geminiviruses. CLCuMB encodes a single ORF, βC1, in the complementary direction and has pathogenicity, symptoms determinant, suppressor of post-transcription and transcription gene silencing functions. After substituting the βC1 gene with the target gene, it has been used successfully as a gene delivery vector. In the present study, the βC1 gene of CLCuMB was substituted with the green fluorescent protein (GFP) gene, and the resulting construct utilized as a reporter vector to decipher in planta localization of geminiviruses. The recombinant CLCuMB expressing GFP (CLCuMB-GFP) was co-inoculated to Nicotiana benthamiana plants either with Cotton leaf curl Kokharan virus (CLCuKoV) alone or in a combination with the wild type CLCuMB to investigate the objectives of the study. Results showed that CLCuKoV successfully supported the replication and systemic movement of CLCuMB-GFP either alone or in the presence of wild type CLCuMB. The presence of CLCuMB-GFP was readily detected with PCR and Southern blot hybridization. The modified CLCuMB may serve as a tool useful for in planta localization of geminiviruses. [ABSTRACT FROM AUTHOR]