Transmembrane Transport Proteins Research Articles

Phytochemical modulation of P-Glycoprotein and its gene expression in an ivermectin-resistant Haemonchus contortus isolate in vitro

Haemonchus contortus is the most important gastrointestinal nematode in small ruminant systems worldwide and has developed resistance to several drugs, including ivermectin (IVM). IVM is not only a veterinary drug but also a safe, broad-spectrum, antiparasitic drug used in humans. One of the main IVM-resistance mechanisms in H. contortus involves P-glycoprotein (PgP), a trans-membrane transport protein that rids worm cells from toxic molecules. This study aimed to evaluate the anthelmintic activity of IVM, alone or combined with main terpenes of essential oils (alpha-terpinene, beta-citronellol, beta-pinene, citronellal, limonene, menthol, and terpinolene) and with phenolic compounds (epicatechin, epigallocatechin, gallocatechin, pentagalloylglucose, procyanidin, and quercetin). All compounds were tested, alone or combined with IVM, against susceptible (HcS) and resistant (HcR) isolates of H. contortus through the larval development test (LDT) and the adult motility assay (AMT) using verapamil (VP), a known PgP modulator, as a control. Results for the LDT determined that the lethal concentration required to kill 50% of nematodes (LC50) with IVM was 10 times greater (0.01 µg/mL) for HcR than for HcS (0.001 µg/mL). The combination IVM + VP inhibited the activity of PgP in HcR resulting in a LC50 = 0.002 ug.mL−1. Although limonene was the least effective and alpha-terpinene the most effective terpene when tested alone against HcR, the best combinations were IVM + limonene and IVM + quercetin both produced LC50 = 0.002 µg/mL (similar to IVM+VP) which were chosen for subsequent tests. Because adult parasites are the final target for anthelmintics, IVM was evaluated in HcS (LC50 = 0.067 µg/mL) and HcR (LC50 =164.94 µg/mL) through the AMT. Results obtained with IVM + VP (LC50 = 0.020 µg/mL) in HcR were similar to IVM + limonene (LC50 = 0.028 µg/mL) and outperformed IVM + quercetin (LC50 = 1.39 µg/mL). RNA extracts from HcR adult worms exposed to IVM, IVM+VP, and IVM + limonene were evaluated for PgP expression by RT-PCR. For most concentrations, PgP-9 was significantly more expressed in worms treated with IVM alone than in worms treated with IVM + VP or IVM + limonene. Our results suggest that limonene is involved in the modulation of the PgP-9 gene and that it can restore the activity of IVM in the HcR isolate down to levels seen in HcS. Limonene is one of the main compounds found in citrus peel and has the potential to be both safe and affordable if used in combination with IVM to restore its anthelmintic effects against multi-drug-resistant H. contortus isolates. Our results also suggest that we may be more successful by combining natural products with failing commercial anthelmintics than trying to find natural substitutes for them.

Metal Substrate Translocation and Transport Mechanism of the Sinorhizobium melilotiP1B‐5‐type ATPase Nia Revealed by In‐vitro Transport Assays in Proteoliposomes

P1B‐type ATPases are a class of transmembrane transporter proteins responsible for metal substrate translocation, and export across cellular membranes. These ion pumps are classified into seven sub‐families (P1B‐1‐P1B‐7‐type) based on conserved amino acid motifs in their transmembrane helices that appear to control each sub‐family’s substrate selectivity. The P1B‐5‐type sub‐family is widely found in nitrogen fixing bacteria, such as Sinorhizobium meliloti,which form a unique symbiotic relationship with legumes. This symbiotic relationship is highly dependent on the nutrition trafficking between the two species, including a tight control of transition metal homeostasis. The P1B‐5‐type ATPase Nia from S. Meliloti is recognized as a putative iron and nickel transporter, and besides possessing the typical P‐type ATPase domains and topology, it features of a C‐terminal hemerythrin‐like domain containing a di‐iron binding site involved in oxygen sensing, as proposed by in‐vivo studies. However, the substrate transport across lipid bi‐layers, the overall mechanism for cargo translocation and the transport kinetic parameters remain elusive due to lack of molecular tools to study putative substrate transport across membranes in real‐time.In this study, we coupled metal‐stimulated ATPase activity assays with an experimental platform based on multiple fluorescence sensor probes, to study substrate transport mechanism and translocation kinetics in real‐time with Nia reconstituted in a native‐like environment. We expressed and purified Nia in detergent micelles and subsequently incorporated it in artificial small unilamellar lipid bi‐layer vesicles (proteoliposomes) which allow to study the substrate specificity and transport mechanism in real‐time. The proteoliposomes were used as an in‐vitrotool to determine the kinetic parameters for metal transport by encapsulating fluorescent detector probes featuring turn‐on florescence signal upon substrate ion binding and translocation.The study revealed that Fe2+, Ni2+ and Co2+are actively translocated by Nia and the kinetic parameter analysis demonstrated that Fe2+ is the preferential Nia substrate. In parallel, by encapsulating in the proteoliposome lumen pH and membrane sensitive fluorescent probes we are also revealing the chemistry underlying the mechanism of transport and potential ions involved as counter‐transported cargos in P1B‐5‐type ATPase catalytic cycle, with the aim of revealing unique molecular features for this subclass of transmembrane metal pumps.

Relationship between Monocarboxylate Transporter-4 Expression and Breast Cancer Clinicopathology and Subtype in Sanglah General Hospital, Denpasar, Indonesia

BACKGROUND: Based on the global cancer observatory (GLOBOCAN) data, in 2018, there were 18.1 million new breast cancer cases. Breast cancer is the second-most common malignancy after lung cancer, which contributed to 11.6% of all new cancer cases in 2018. Breast cancer is the second-leading cause of death in women in developing countries. The activity of Warburg and reverse Warburg effects on breast cancer is reflected by the expression patterns of two molecules, namely caveolin-1 and monocarboxylate transporter-4 (MCT-4). MCT-4 is a transmembrane transport protein that functions in the transport of lactate from the cytoplasm to the intercellular fluid. METHODS: This is a cross-sectional analytical study to determine the relationship between MCT-4 expression and breast cancer clinicopathology and subtypes. The study was conducted between April and May of 2020 with 62 breast cancer patients as samples in Sanglah General Hospital, Denpasar. Analysis was done with SPSS 25. RESULTS: A logistic regression analysis was performed to analyze the relationship between the dependent variable (MCT-4) and covariates (stage, grade, and subtype). Of the three variables that were significantly associated with MCT-4 expression, only clinical stage and subtype (luminal and non-luminal) remained independently associated with MCT-4 expression. Analysis on the clinical stage and subtype variables showed an adjusted OR of 4.727 (p = 0.047; 95% CI: 1.109–21.922) and 17.850 (p = 0.009; 95% CI: 2.069–154.003), respectively. This suggests that MCT-4 has a significant association with subtype and clinical stage which increases the risk of progression of the cancer stage as well as the risk of developing a more malignant (non-luminal) subtype. CONCLUSION: High MCT-4 expression was significantly associated with malignant subtypes, high histological-grade cancer, and an advanced breast cancer.

A G-Protein-Coupled Receptor Modulates Gametogenesis via PKG-Mediated Signaling Cascade in Plasmodium berghei.

ABSTRACTGametogenesis is essential for malaria parasite transmission, but the molecular mechanism of this process remains to be refined. Here, we identified a G-protein-coupled receptor 180 (GPR180) that plays a critical role in signal transduction during gametogenesis in Plasmodium. The P. berghei GPR180 was predominantly expressed in gametocytes and ookinetes and associated with the plasma membrane in female gametes and ookinetes. Knockout of pbgpr180 (Δpbgpr180) had no noticeable effect on blood-stage development but impaired gamete formation and reduced transmission of the parasites to mosquitoes. Transcriptome analysis revealed that a large proportion of the dysregulated genes in the Δpbgpr180 gametocytes had assigned functions in cyclic nucleotide signal transduction. In the Δpbgpr180 gametocytes, the intracellular cGMP level was significantly reduced, and the cytosolic Ca2+ mobilization showed a delay and a reduction in the magnitude during gametocyte activation. These results suggest that PbGPR180 functions upstream of the cGMP-protein kinase G-Ca2+ signaling pathway. In line with this functional prediction, the PbGPR180 protein was found to interact with several transmembrane transporter proteins and the small GTPase Rab6 in activated gametocytes. Allele replacement of pbgpr180 with the P. vivax ortholog pvgpr180 showed equal competence of the transgenic parasite in sexual development, suggesting functional conservation of this gene in Plasmodium spp. Furthermore, an anti-PbGPR180 monoclonal antibody and the anti-PvGPR180 serum possessed robust transmission-blocking activities. These results indicate that GPR180 is involved in signal transduction during gametogenesis in malaria parasites and is a promising target for blocking parasite transmission.IMPORTANCE Environmental changes from humans to mosquitoes activate gametogenesis of the malaria parasite, an obligative process for parasite transmission, but how the signals are relayed remains poorly understood. Here, we show the identification of a Plasmodium G-protein-coupled receptor, GPR180, and the characterization of its function in gametogenesis. In P. berghei, GPR180 is dispensable for asexual development and gametocytogenesis, but its deletion impairs gametogenesis and reduces transmission to mosquitoes. GPR180 appears to function upstream of the cGMP-protein kinase G-Ca2+ signaling pathway and is required for the maximum activity of this pathway. Genetic complementation shows that the GPR180 ortholog from the human malaria parasite P. vivax was fully functional in P. berghei, indicating functional conservation of GPR180 in Plasmodium spp. With predominant expression and membrane association of GPR180 in sexual stages, GPR180 is a promising target for blocking transmission, and antibodies against GPR180 possess robust transmission-blocking activities.

RNA-Seq analysis reveals sex-dependent transcriptomic profiles of human subacromial bursa stratified by tear etiology.

Rotator cuff tendinopathy, a major cause of shoulder disability, occurs due to trauma or degeneration. Our molecular understanding of traumatic and degenerative tears remains elusive. Here, we probed transcript level differences between traumatic and degenerative tears. Subacromial bursa tissues were collected from patients with traumatic or degenerative tears during arthroscopy (N = 32). Transcripts differentially expressed by tear etiology were detected by RNA-seq. RNA-seq results were validated by real-time quantitative polymerase chain reaction. We identified 334 protein-coding transcripts differentially expressed between traumatic and degenerative tears in females and 167 in males at a fold-change greater than 2. In females, XIRP2, MYL1, MYBPC1, TNNT1, and LMOD2, were highly expressed in traumatic tears whereas TPSD1, CDSN, RCVRN, LTBP4, and PTGS1 were elevated in degen tears. Transcripts elevated in traumatic tears represented muscle cell differentiation and development, and muscle contraction whereas those elevated in degenerative tears represented cell activation and immune response. In males, AZGP1, CNTFR, COL9A1, ZNF98, and EREG were highly elevated in traumatic tears whereas MYL2, HOXD11, SLC6A7, CADM1, and MMP17 were highly expressed in degenerative tears. Transcripts elevated in traumatic tears represented metabolic/catabolic processes, and transmembrane protein transport while processes related to cell cycle were mainly enriched in degenerative tears. Numerous long noncoding RNAs were differentially expressed between traumatic and degenerative tears in both sexes. In summary, this study provides insights into molecular biology of bursa in patients with rotator cuff tendon disease based on tear acuity and novel sex-based transcript differences that could inform clinical decision making in treating patients with traumatic or degenerative shoulder injuries.

Relationship between Monocarboxylate Transporter 4 (MCT-4) Expression and Breast Cancer Clinicopathology and Subtype in Sanglah General Hospital, Denpasar, Indonesia

Background: Breast cancer is the second leading cause of death in women in developing countries. The activity of Warburg and Reverse-Warburg effects on breast cancer is reflected by the expression patterns of two molecules, namely caveolin-1 and Monocarboxylate Transporter-4 (MCT-4). MCT-4 is a transmembrane transport protein that transports lactate from the cytoplasm to the intercellular fluid.Method: This is a cross-sectional analytical study to determine the relationship between MCT-4 expression and breast cancer clinicopathology and subtypes. The study was conducted between April and May of 2020 with 62 breast cancer patients as samples in Sanglah General Hospital, Denpasar. Analysis was done with SPSS 25.Results: A logistic regression analysis was performed to analyze the relationship between the dependent variable (MCT-4) and the covariates (stage, grade, and subtype). Of the three variables significantly associated with MCT-4 expression, only clinical-stage and subtype (luminal and non-luminal) remained independently associated with MCT-4 expression. Analysis on the clinical stage and subtype variables showed an adjusted OR of 4.727 (p = 0.047; 95% CI: 1.109 - 21.922) and 17.850 (p = 0.009; 95% CI: 2.069 - 154.003) , respectively. This suggests that MCT-4 has a significant association with subtype and clinical-stage, increasing the risk of cancer stage progression and developing a more malignant (non-luminal) subtype.Conclusion: High MCT-4 expression was significantly associated with malignant subtypes, high histological-grade cancer and advanced breast cancer.

Cation/Proton Antiporter Genes in Tomato: Genomic Characterization, Expression Profiling, and Co-Localization with Salt Stress-Related QTLs

The cation/proton antiporter (CPA) family represents a class of transmembrane transporter proteins that play a crucial role in plants during high salinity stress by maintaining the cell’s ionic balance and pH homeostasis. So far, the CPA genes have not been systematically characterized in tomato (Solanum lycopersicum). In this study, we identified and analyzed 33 putative CPA genes in tomato. Phylogenetic analysis showed that tomato CPAs could be classified into three subgroups, i.e., CHX (18 genes), KEA (8 genes), and NHX (7 genes). CPA genes within each subgroup shared similar motifs, conserved catalytic domains and gene structure. Further analysis revealed that the CPA genes were unevenly distributed on the chromosomes and segmental duplication events played a major role in the expansion of the CPA gene family in tomato. Gene expression analysis exhibited that CPA genes were differentially expressed in different tissues, various stages of fruit development, and differentially regulated in response to abiotic stresses, especially salt stress. Further, co-localization of tomato CPA genes with quantitative trait loci (QTL) of salt stress-related phenotypes revealed their broader functions in salt stress tolerance. Finally, predicted protein–protein interactions of tomato CPAs, gene ontology analysis, and the presence of putative cis-elements in the promoter further support the diverse role of tomato CPAs in plant development and plant stress tolerance. In brief, this study highlights the potential role of tomato CPAs in plant development and abiotic stress tolerance, especially in salt stress, and provides comprehensive information to explore new candidate genes for salt tolerance in tomato.

Effects of Selenium as a Dietary Source on Performance, Inflammation, Cell Damage, and Reproduction of Livestock Induced by Heat Stress: A Review.

Heat stress as a result of global warming has harmful consequences for livestock and is thus becoming an urgent issue for animal husbandry worldwide. Ruminants, growing pigs, and poultry are very susceptible to heat stress because of their fast growth, rapid metabolism, high production levels, and sensitivity to temperature. Heat stress compromises the efficiency of animal husbandry by affecting performance, gastrointestinal health, reproductive physiology, and causing cell damage. Selenium (Se) is an essential nutritional trace element for livestock production, which acts as a structural component in at least 25 selenoproteins (SELs); it is involved in thyroid hormone synthesis, and plays a key role in the antioxidant defense system. Dietary Se supplementation has been confirmed to support gastrointestinal health, production performance, and reproductive physiology under conditions of heat stress. The underlying mechanisms include the regulation of nutrient digestibility influenced by gastrointestinal microorganisms, antioxidant status, and immunocompetence. Moreover, heat stress damage to the gastrointestinal and mammary barrier is closely related to cell physiological functions, such as the fluidity and stability of cellular membranes, and the inhibition of receptors as well as transmembrane transport protein function. Se also plays an important role in inhibiting cell apoptosis and reducing cell inflammatory response induced by heat stress. This review highlights the progress of research regarding the dietary supplementation of Se in the mitigation of heat stress, addressing its mechanism and explaining the effect of Se on cell damage caused by heat stress, in order to provide a theoretical reference for the use of Se to mitigate heat stress in livestock.

Prediction and Analysis of miRNA Targets in Poplar in Response to the Infection of <i>Lonsdalea quercina</i> subsp <i>Populi</i>

Euramerican poplar bacterial canker disease caused by  Lonsdalea quercina  subsp.  Populi  is one of the stem diseases and seriously harmful to the growth and survival of poplar. MiRNAs play crucial roles in the regulation of the stress response of plants. To identify target genes of miRNAs responsive to the infection of this pathogen and provide candidate genes to the molecular breeding of forest disease resistance, the mature sequences of poplar miRNAs in miRBase database were used as probes to in silico predict the differentially expressed targets in poplar ‘Zhonglin 46’ inoculated with bacterial canker by using psRNATarget in this investigation. A total of 566 target genes of 276 miRNAs belonging to 127 miRNA families were screened, and these targets were found to be involved in multiple pathways such as plant pathogen interaction, plant hormone signaling, and phenylpropanoid biosynthesis, suggesting that these miRNAs were involved in the regulation of the response to  L. quercina  infection. Combined with GO analysis, KEGG pathway enrichment and gene function annotation of target genes, it was showed that miRNAs including miR482, miR6459, miR7812 and miR7835 were predicted to target genes encoding RPS2, NBS-LRR, AUX1, CCR, and Ca 2+  transmembrane transport protein genes, which were involved in the resistance regulation of poplar to  L. quercina  infection. Six of the nine differentially expressed genes targeted by miR7835 encode the CCR enzyme in the phenylpropanol biosynthesis pathway, which might be involved in the defense response of poplars by affecting the synthesis of lignin. The miRNA:mRNA regulatory gene pairs found in this investigation enlarged the gene resources of molecular breeding of forest disease resistance, and provided new clues for the research of the molecular regulatory mechanism of disease resistance in trees.

Impact of nanodisc lipid composition on cell-free expression of proton-coupled folate transporter.

The Proton-Coupled Folate Transporter (PCFT) is a transmembrane transport protein that controls the absorption of dietary folates in the small intestine. PCFT also mediates uptake of chemotherapeutically used antifolates into tumor cells. PCFT has been identified within lipid rafts observed in phospholipid bilayers of plasma membranes, a micro environment that is altered in tumor cells. The present study aimed at investigating the impact of different lipids within Lipid-protein nanodiscs (LPNs), discoidal lipid structures stabilized by membrane scaffold proteins, to yield soluble PCFT expression in an E. coli lysate-based cell-free transcription/translation system. In the absence of detergents or lipids, we observed PCFT quantitatively as precipitate in this system. We then explored the ability of LPNs to support solubilized PCFT expression when present during in-vitro translation. LPNs consisted of either dimyristoyl phosphatidylcholine (DMPC), palmitoyl-oleoyl phosphatidylcholine (POPC), or dimyristoyl phosphatidylglycerol (DMPG). While POPC did not lead to soluble PCFT expression, both DMPG and DMPC supported PCFT translation directly into LPNs, the latter in a concentration dependent manner. The results obtained through this study provide insights into the lipid preferences of PCFT. Membrane-embedded or solubilized PCFT will enable further studies with diverse biophysical approaches to enhance the understanding of the structure and molecular mechanism of folate transport through PCFT.

Overexpression of ZNT1 and NRAMP4 from the Ni Hyperaccumulator Noccaea caerulescens Population Monte Prinzera in Arabidopsis thaliana Perturbs Fe, Mn, and Ni Accumulation.

Metalliferous soils are characterized by a high content of metal compounds that can hamper plant growth. The pseudometallophyte Noccaea caerulescens is able to grow on metalliferous substrates by implementing both tolerance and accumulation of usually toxic metal ions. Expression of particular transmembrane transporter proteins (e.g., members of the ZIP and NRAMP families) leads to metal tolerance and accumulation, and its comparison between hyperaccumulator N. caerulescens with non-accumulator relatives Arabidopsis thaliana and Thlaspi arvense has deepened our knowledge on mechanisms adopted by plants to survive in metalliferous soils. In this work, two transporters, ZNT1 and NRAMP4, expressed in a serpentinic population of N. caerulescens identified on the Monte Prinzera (Italy) are considered, and their expression has been induced in yeast and in A. thaliana. In the latter, single transgenic lines were crossed to test the effect of the combined over-expression of the two transporters. An enhanced iron and manganese translocation towards the shoot was induced by overexpression of NcZNT1. The combined overexpression of NcZNT1 and NcNRAMP4 did perturb the metal accumulation in plants.

Novel Candidate Genes Differentially Expressed in Glyphosate-Treated Horseweed (Conyza canadensis).

The evolution of herbicide-resistant weed species is a serious threat for weed control. Therefore, we need an improved understanding of how gene regulation confers herbicide resistance in order to slow the evolution of resistance. The present study analyzed differentially expressed genes after glyphosate treatment on a glyphosate-resistant Tennessee ecotype (TNR) of horseweed (Conyza canadensis), compared to a susceptible biotype (TNS). A read size of 100.2 M was sequenced on the Illumina platform and subjected to de novo assembly, resulting in 77,072 gene-level contigs, of which 32,493 were uniquely annotated by a BlastX alignment of protein sequence similarity. The most differentially expressed genes were enriched in the gene ontology (GO) term of the transmembrane transport protein. In addition, fifteen upregulated genes were identified in TNR after glyphosate treatment but were not detected in TNS. Ten of these upregulated genes were transmembrane transporter or kinase receptor proteins. Therefore, a combination of changes in gene expression among transmembrane receptor and kinase receptor proteins may be important for endowing non-target-site glyphosate-resistant C. canadensis.

Twitchy, the Drosophila orthologue of the ciliary gating protein FBF1/dyf-19, is required for coordinated locomotion and male fertility.

ABSTRACTPrimary cilia are compartmentalised from the rest of the cell by a ciliary gate comprising transition fibres and a transition zone. The ciliary gate allows the selective import and export of molecules such as transmembrane receptors and transport proteins. These are required for the assembly of the cilium, its function as a sensory and signalling centre and to maintain its distinctive composition. Certain motile cilia can also form within the cytosol as exemplified by human and Drosophila sperm. The role of transition fibre proteins has not been well described in the cytoplasmic cilia.Drosophila have both compartmentalised primary cilia, in sensory neurons, and sperm flagella that form within the cytosol. Here, we describe phenotypes for twitchy the Drosophila orthologue of a transition fibre protein, mammalian FBF1/C. elegans dyf-19. Loss-of-function mutants in twitchy are adult lethal and display a severely uncoordinated phenotype. Twitchy flies are too uncoordinated to mate but RNAi-mediated loss of twitchy specifically within the male germline results in coordinated but infertile adults. Examination of sperm from twitchy RNAi-knockdown flies shows that the flagellar axoneme forms, elongates and is post-translationally modified by polyglycylation but the production of motile sperm is impaired. These results indicate that twitchy is required for the function of both sensory cilia that are compartmentalised from the rest of the cell and sperm flagella that are formed within the cytosol of the cell. Twitchy is therefore likely to function as part of a molecular gate in sensory neurons but may have a distinct function in sperm cells.

Identifying the Phytotoxicity and Defense Mechanisms Associated with Graphene-Based Nanomaterials by Integrating Multiomics and Regular Analysis.

The application of graphene-based nanomaterials (GBNs) has attracted global attention in various fields, and understanding defense mechanisms against the phytotoxicity of GBNs is crucial for assessing their environmental risks and safe-by-design. However, the related information is lacking, especially for edible vegetable crops. In the present study, GBNs (0.25, 2.5, and 25 mg/kg plant fresh weight) were injected into the stems of pepper plants. The results showed that the plant defense was regulated by reducing the calcium content by 21.7-48.3%, intercellular CO2 concentration by 12.0-35.2%, transpiration rate by 8.7-40.2%, and stomatal conductance by 16.9-50.5%. The defense pathways of plants in response to stress were further verified by the downregulation of endocytosis and transmembrane transport proteins, leading to a decrease in the nanomaterial uptake. The phytohormone gibberellin and abscisic acid receptor PYL8 were upregulated, indicating the activation of defense systems. However, reduced graphene oxide and graphene oxide quantum dots trigger stronger oxidative stress (e.g., H2O2 and malondialdehyde) than graphene oxide in fruits due to the breakdown of antioxidant defense systems (e.g., cytochrome P450 86A22 and P450 77A1). Both nontargeted proteomics and metabolomics consistently demonstrated that the downregulation of carbohydrate and upregulation of amino acid metabolism were the main mechanisms underlying the phytotoxicity and defense mechanisms, respectively.

ActTRANS: Functional classification in active transport proteins based on transfer learning and contextual representations

MotivationPrimary and secondary active transport are two types of active transport that involve using energy to move the substances. Active transport mechanisms do use proteins to assist in transport and play essential roles to regulate the traffic of ions or small molecules across a cell membrane against the concentration gradient. In this study, the two main types of proteins involved in such transport are classified from transmembrane transport proteins. We propose a Support Vector Machine (SVM) with contextualized word embeddings from Bidirectional Encoder Representations from Transformers (BERT) to represent protein sequences. BERT is a powerful model in transfer learning, a deep learning language representation model developed by Google and one of the highest performing pre-trained model for Natural Language Processing (NLP) tasks. The idea of transfer learning with pre-trained model from BERT is applied to extract fixed feature vectors from the hidden layers and learn contextual relations between amino acids in the protein sequence. Therefore, the contextualized word representations of proteins are introduced to effectively model complex structures of amino acids in the sequence and the variations of these amino acids in the context. By generating context information, we capture multiple meanings for the same amino acid to reveal the importance of specific residues in the protein sequence. ResultsThe performance of the proposed method is evaluated using five-fold cross-validation and independent test. The proposed method achieves an accuracy of 85.44 %, 88.74 % and 92.84 % for Class-1, Class-2, and Class-3, respectively. Experimental results show that this approach can outperform from other feature extraction methods using context information, effectively classify two types of active transport and improve the overall performance.

Peppermint Essential Oil Suppresses Geotrichum citri-aurantii Growth by Destructing the Cell Structure, Internal Homeostasis, and Cell Cycle.

Peppermint essential oil (Peo) is an efficient antifungal agent, and 2.0 μL of Peo per milliliter culture medium can completely inhibit the mycelium growth and spore germination of Geotrichum citri-aurantii. In vitro experiments showed that the main functional component in Peo was l-menthol, which could lead to changes in sugar and protein contents, reduce the content of alkaline phosphatase (AKP), and destroy the spore membrane structure, with a significant increase in electrical conductivity. Meanwhile, the content of reactive oxygen (ROS) accumulated sharply, and the enzyme activity changed significantly with the change in the gene expression level. In addition, l-menthol could cause degradation in spore genetic material differently. Furthermore, a total of 1704 differentially expressed genes (DEGs) in G. citri-aurantii after 1.6 μL/mL l-menthol exposure for 2 h were obtained by the transcriptome sequencing. These DEGs were involved in transmembrane transport, carbohydrate transmembrane transport protein activity, and mitogen-activated protein kinase (MAPK) signaling pathway. The protein-protein interaction (PPI) analysis of DEGs yielded 10 highly cross-linked nodes, and these genes were associated with DNA replication and cell cycle. The expression level of the hub gene was confirmed by real-time quantitative PCR (RT-qPCR), with the most significant changes in POL 30 (5.9-fold). Molecular simulation was performed and it was found that the binding site between l-menthol and POL 30 was the 44th ARG residue in POL 30, and it was speculated that l-menthol and POL 30 may be combined by hydrogen bonding interaction. The results of flow cytometry assay showed that l-menthol blocked the replication process in the S-phase of G. citri-aurantii. This study provides new insights into the development and application of Peo in food safety.

Treatment with HIV-Protease Inhibitor Nelfinavir Identifies Membrane Lipid Composition and Fluidity as a Therapeutic Target in Advanced Multiple Myeloma.

The HIV-protease inhibitor nelfinavir has shown broad anticancer activity in various preclinical and clinical contexts. In patients with advanced, proteasome inhibitor (PI)-refractory multiple myeloma, nelfinavir-based therapy resulted in 65% partial response or better, suggesting that this may be a highly active chemotherapeutic option in this setting. The broad anticancer mechanism of action of nelfinavir implies that it interferes with fundamental aspects of cancer cell biology. We combined proteome-wide affinity-purification of nelfinavir-interacting proteins with genome-wide CRISPR/Cas9-based screening to identify protein partners that interact with nelfinavir in an activity-dependent manner alongside candidate genetic contributors affecting nelfinavir cytotoxicity. Nelfinavir had multiple activity-specific binding partners embedded in lipid bilayers of mitochondria and the endoplasmic reticulum. Nelfinavir affected the fluidity and composition of lipid-rich membranes, disrupted mitochondrial respiration, blocked vesicular transport, and affected the function of membrane-embedded drug efflux transporter ABCB1, triggering the integrated stress response. Sensitivity to nelfinavir was dependent on ADIPOR2, which maintains membrane fluidity by promoting fatty acid desaturation and incorporation into phospholipids. Supplementation with fatty acids prevented the nelfinavir-induced effect on mitochondrial metabolism, drug-efflux transporters, and stress-response activation. Conversely, depletion of fatty acids/cholesterol pools by the FDA-approved drug ezetimibe showed a synergistic anticancer activity with nelfinavir in vitro. These results identify the modification of lipid-rich membranes by nelfinavir as a novel mechanism of action to achieve broad anticancer activity, which may be suitable for the treatment of PI-refractory multiple myeloma. SIGNIFICANCE: Nelfinavir induces lipid bilayer stress in cellular organelles that disrupts mitochondrial respiration and transmembrane protein transport, resulting in broad anticancer activity via metabolic rewiring and activation of the unfolded protein response.

Rapid Single-Step Growth of MOF Exoskeleton on Mammalian Cells for Enhanced Cytoprotection.

Mammalian cells are promising agents for cell therapy, diagnostics, and drug delivery. For full utilization of the cells, development of an exoskeleton may be beneficial to protecting the cells against the environmental stresses and cytotoxins to which they are susceptible. We report here a rapid single-step method for growing metal-organic framework (MOF) exoskeletons on a mammalian cell surface under cytocompatible conditions. The MOF exoskeleton coating on the mammalian cells was developed via a one-pot biomimetic mineralization process. With the exoskeleton on, the individual cells were successfully protected against cell protease (i.e., Proteinase K), whereas smaller-sized nutrient transport across the exoskeleton was maintained. Moreover, vital cellular activities mediated by transmembrane GLUT transporter proteins were also unaffected by the MOF exoskeleton formation on the cell surfaces. Altogether, this ability to control the access of specific molecules to a single cell through the porous exoskeleton, along with the cytoprotection provided, should be valuable for biomedical applications of mammalian cells.

Zinc and the Zinc Transporter SLC39A10/ZIP10 Are Required for Heme Synthesis in Developing Erythroid Progenitors

ObjectivesZinc is an essential nutrient for diverse biological processes in the body. Cellular zinc homeostasis is established through differential expressions of the transmembrane zinc transporter proteins, ZnTs and ZIPs. The aims of the current studies were to elucidate the roles of cellular zinc in erythrocyte maturation, and to determine the zinc transporters essential to erythroid zinc homeostasis. MethodsG1E-ER4 mouse cells were employed as an in vitro study model of terminal erythroid differentiation. A cell-impermeable zinc chelator, diethylenetriamine pentaacetate (DTPA), was used to limit extracellular zinc availability. For gene silencing, gene-specific siRNAs were introduced to cells via Nucleofection. Functional impacts of zinc and gene deficiency were assessed via ICP-MS-based metal quantitation, heme assays, and gene expression assays using RNA-seq, qPCR, and western analyses. ResultsG1E-ER4 cells featured a 1.7-fold increase in total cellular zinc contents after 48 h of differentiation. Restriction of zinc import by 50 μM of DTPA led to less red coloration and lower increases in mean corpuscular hemoglobin contents by development. The heme deficiency by DTPA was fully restored by the addition of equimolar zinc, and was not due to changes in cellular iron contents. Zinc-deficient G1E-ER4 cells differentiated with normal Alas2 and Hbb-b1 transcript responses, but less Alad and alpha-globin expressions. Among the 24 zinc transporter genes, Zip10 produced the most prominent response to zinc restriction in differentiating erythroid cells. ZIP10-deficient G1E-ER4 cells were less efficient than control cells in hemoglobin production under zinc restriction. ZIP10 deficiency alone had no effects on the molecular indices of red cell hemoglobinization. ConclusionsOur studies characterize zinc as a nutrient essential to normal erythroid maturation and heme synthesis. Moreover, we have identified a compensatory role of ZIP10 for erythroid zinc homeostasis during zinc restriction. Thus, poor zinc status and ZIP10 mutations might serve as potential risk factors and thus new therapeutic targets for anemia and other erythrocyte-related disorders. Funding SourcesSupported by CFANS Graduate Fellowship to JK, and the Allen Foundation, Inc. and USDA NIFA Hatch Funds to M-SR.

Palmitoylation and Phosphorylation of the Na + ‐H + Exchanger Isoform 1 (NHE1) Regulate of Cell Proliferation and Stress Fiber Formation: PI3 Kinase and AKT Signaling

The Na+-H+ Exchanger Isoform 1 (NHE1) is a 12-pass transmembrane transport protein that exchanges one extracellular Na+ for one intracellular H+ with the primary function of intracellular pH regulation. Through the pH regulation NHE1 contributes to the regulation of cell proliferation and stress fiber formation. The regulation of NHE1 is extremely complex and involves protein and lipid binding sites including phosphorylation and palmitoylation sites. NHE1 transport activity is regulated by a series of signaling pathways which include kinases of the PI3 Kinase / AKT signaling pathways. Recently it was demonstrated that inhibition of either PI3 Kinase or AKT lead to an increase in the palmitoylation of NHE1. Here we assess the role of this interaction between palmitoylation and phosphorylation in the regulation of cell proliferation and stress fiber formation in PSN cells. It is hypothesized that inhibiting phosphorylation, palmitoylation, or both will differentially regulate cell proliferation and stress fiber formation in these cells. PSN cells are Chinese hamster lung fibroblast that stably express human NHE1. We evaluated cell proliferation and stress fiber formation in the presence and absence of combinations of inhibitors that alter NHE1 regulation and transport activity including, LY294002 (PI3 Kinase), MK 2206 (AKT), 2-Bromopalmitate (Palmitoylation), and Ethylisopropylamiloride (NHE1).