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European Journal of Endocrinology Feb 2024Pathogenic variants in the nicotinamide nucleotide transhydrogenase gene (NNT) are a rare cause of primary adrenal insufficiency (PAI), as well as functional impairment...
BACKGROUND
Pathogenic variants in the nicotinamide nucleotide transhydrogenase gene (NNT) are a rare cause of primary adrenal insufficiency (PAI), as well as functional impairment of the gonads.
OBJECTIVE
Despite the description of different homozygous and compound heterozygous NNT variants in PAI patients, the extent to which the function and expression of the mature protein are compromised remains to be clarified.
DESIGN
The activity and expression of mitochondrial NAD(P)+ transhydrogenase (NNT) were analyzed in blood samples obtained from patients diagnosed with PAI due to genetically confirmed variants of the NNT gene (n = 5), heterozygous carriers as their parents (n = 8), and healthy controls (n = 26).
METHODS
NNT activity was assessed by a reverse reaction assay standardized for digitonin-permeabilized peripheral blood mononuclear cells (PBMCs). The enzymatic assay was validated in PBMC samples from a mouse model of NNT absence. Additionally, the PBMC samples were evaluated for NNT expression by western blotting and reverse transcription quantitative polymerase chain reaction and for mitochondrial oxygen consumption.
RESULTS
NNT activity was undetectable (<4% of that of healthy controls) in PBMC samples from patients, independent of the pathogenic genetic variant. In patients' parents, NNT activity was approximately half that of the healthy controls. Mature NNT protein expression was lower in patients than in the control groups, while mRNA levels varied widely among genotypes. Moreover, pathogenic NNT variants did not impair mitochondrial bioenergetic function in PBMCs.
CONCLUSIONS
The manifestation of PAI in NNT-mutated patients is associated with a complete lack of NNT activity. Evaluation of NNT activity can be useful to characterize disease-causing NNT variants.
Topics: Animals; Humans; Mice; Addison Disease; Leukocytes, Mononuclear; Mitochondrial Proteins; NAD; NADP Transhydrogenase, AB-Specific; NADP Transhydrogenases
PubMed: 38261461
DOI: 10.1093/ejendo/lvae011 -
Discover Nano Jan 2024In this work, iron oxide (FeO) magnetic nanoparticles (MNPs) and graphene oxide (GO) nanosheets were prepared via the co-precipitation technique and the Modified Hummer...
In this work, iron oxide (FeO) magnetic nanoparticles (MNPs) and graphene oxide (GO) nanosheets were prepared via the co-precipitation technique and the Modified Hummer method. FeO MNPs and GO nanosheets were combined to prepare FeO/GO nanocomposite and subsequently conjugated with Digitonin (DIG) in order to obtain a dual-targeted delivery system based on DIG/FeO/GO nanocomposite. SEM images reveal the presence of FeO MNPs at a scale of 100 nm, exhibiting dispersion between the GO nanosheets. Aggregation of the DIG/FeO/GO nanocomposite was observed at various size scales. The XRD structural analysis confirms the crystal structure of the prepared samples. The FeO MNPs demonstrated the main XRD-diffracted peaks. Also, GO nanosheets exhibit crystalline characteristics on the (001) and (002) planes. The predominant peaks observed in the DIG/GO/FeO nanocomposite are attributed to the crystal phases of FeO MNPs. The FT-IR vibrational modes observed in the GO/DIG/FeO nanocomposite indicate the presence of crosslinking between GO nanosheet layers and the FeO MNPs. The antioxidant activity of the prepared samples was measured and the DIG/GO/FeO nanocomposite demonstrated a significantly high antioxidant activity in both 2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2-azino-bis-3-ethylbenzthiazoline-6-sulfonic acid (ABTS) tests.
PubMed: 38253925
DOI: 10.1186/s11671-024-03960-7 -
Science Bulletin Mar 2024Silk is one of the toughest fibrous materials known despite spun at ambient temperature and pressure with water as a solvent. It is a great challenge to reproduce...
Silk is one of the toughest fibrous materials known despite spun at ambient temperature and pressure with water as a solvent. It is a great challenge to reproduce high-performance artificial fibers comparable to natural silk by bionic for the incomplete understanding of silkworm spinning in vivo. Here, we found that amphipol and digitonin stabilized the structure of natural silk fibroin (NSF) by a large-scale screening in vitro, and then studied the close-to-native ultrastructure and hierarchical assembly of NSF in the silk gland lumen. Our study showed that NSF formed reversible flexible nanofibrils mainly composed of random coils with a sedimentation coefficient of 5.8 S and a diameter of about 4 nm, rather than a micellar or rod-like structure assembled by the aggregation of globular NSF molecules. Metal ions were required for NSF nanofibril formation. The successive pH decrease from posterior silk gland (PSG) to anterior silk gland (ASG) resulted in a gradual increase in NSF hydrophobicity, thus inducing the sol-gelation transition of NSF nanofibrils. NSF nanofibrils were randomly dispersed from PSG to ASG-1, and self-assembled into anisotropic herringbone patterns at ASG-2 near the spinneret ready for silkworm spinning. Our findings reveal the controlled self-assembly mechanism of the multi-scale hierarchical architecture of NSF from nanofibrils to herringbone patterns programmed by metal ions and pH gradient, which provides novel insights into the spinning mechanism of silk-secreting animals and bioinspired design of high-performance fibers.
Topics: Animals; Bombyx; Silk; Fibroins; Solvents; Metals; Hydrogen-Ion Concentration
PubMed: 38245448
DOI: 10.1016/j.scib.2023.12.050 -
Molecular Cell Dec 2023The cytoplasm is highly compartmentalized, but the extent and consequences of subcytoplasmic mRNA localization in non-polarized cells are largely unknown. We determined...
The cytoplasm is highly compartmentalized, but the extent and consequences of subcytoplasmic mRNA localization in non-polarized cells are largely unknown. We determined mRNA enrichment in TIS granules (TGs) and the rough endoplasmic reticulum (ER) through particle sorting and isolated cytosolic mRNAs by digitonin extraction. When focusing on genes that encode non-membrane proteins, we observed that 52% have transcripts enriched in specific compartments. Compartment enrichment correlates with a combinatorial code based on mRNA length, exon length, and 3' UTR-bound RNA-binding proteins. Compartment-biased mRNAs differ in the functional classes of their encoded proteins: TG-enriched mRNAs encode low-abundance proteins with strong enrichment of transcription factors, whereas ER-enriched mRNAs encode large and highly expressed proteins. Compartment localization is an important determinant of mRNA and protein abundance, which is supported by reporter experiments showing that redirecting cytosolic mRNAs to the ER increases their protein expression. In summary, the cytoplasm is functionally compartmentalized by local translation environments.
Topics: Endoplasmic Reticulum; Proteins; Cytosol; RNA, Messenger; Protein Transport; Protein Biosynthesis
PubMed: 38134885
DOI: 10.1016/j.molcel.2023.11.025 -
International Journal of Systematic and... Nov 2023Seal finger (sealer's finger, spekk finger), an extremely painful hand infection contracted by individuals handling seals, has previously been associated with . From...
Seal finger (sealer's finger, spekk finger), an extremely painful hand infection contracted by individuals handling seals, has previously been associated with . From 2000 to 2014, six independent strains of a novel species were isolated at Statens Serum Institut, Denmark, from Scandinavian patients with seal finger (M5725, M6447, M6620, M6642 and M6879) or septic arthritis (M6921). Prior to the onset of infection, all patients had reported contact with unspeciated seals. All isolates grew within 2-5 days in Friis' modified broth and metabolized glucose and arginine but not urea. Strains M5725, M6447, M6642 and M6921 also grew in Hayflick-type media. Colonies on agar media were large (0.5-1.0 mm) and had a typical 'fried egg' appearance, reduced tetrazolium, and were digitonin sensitive. Growth occurred at 32 °C but not at 42 °C. Strains were susceptible to doxycycline and moxifloxacin but resistant to azithromycin and erythromycin. The genomes of the six strains were sequenced and relatedness to all known species was inferred. Phylogenetic analyses using 16S rRNA gene sequences and core genome single nucleotide polymorphisms showed that the isolated strains were highly similar and phylogenetically distinct from all other species within the genus . The sizes of the genome sequences of the strains ranged from 744 321 to 772409 bp, with a G+C content of 25.0-25.2 mol%. Based on these analyses, we propose a novel species of the genus with the name sp. nov. with the first isolate M5725 (NCTC 14922=DSM 116188) as the proposed type strain and representative strains M6447, M6620, M6642, M6879 and M6921.
Topics: Humans; Animals; Phylogeny; RNA, Ribosomal, 16S; Base Composition; Sequence Analysis, DNA; DNA, Bacterial; Bacterial Typing Techniques; Fatty Acids; Seals, Earless; Arthritis, Infectious; Cellulitis
PubMed: 37966467
DOI: 10.1099/ijsem.0.006163 -
Drug Metabolism and Disposition: the... Nov 2023Our recent study revealed that SLC49A4, known as disrupted in renal carcinoma 2, is a H-coupled lysosomal exporter for pyridoxine (vitamin B6), a cationic compound, and...
Our recent study revealed that SLC49A4, known as disrupted in renal carcinoma 2, is a H-coupled lysosomal exporter for pyridoxine (vitamin B6), a cationic compound, and involved in the regulation of its lysosomal and cellular levels. We here examined a possibility that this transporter might also transport cationic amphiphilic drugs (CADs) that are known to undergo lysosomal trapping, using pyrilamine, an H-antagonist, as a model CAD and the COS-7 cell line as a model cell system for transient introduction of human SLC49A4 and a recombinant SLC49A4 protein (SLC49A4-AA), in which the N-terminal dileucine motif involved in lysosomal localization was removed by replacing with dialanine for redirected localization to the plasma membrane. The introduction of SLC49A4 into COS-7 cells induced a significant decrease in the accumulation of pyrilamine in the intracellular compartments in the cells treated with digitonin for permeabilization of plasma membranes, suggesting its operation for lysosomal pyrilamine export. Accordingly, functional analysis using the SLC49A4-AA mutant, which operates for cellular uptake at the plasma membrane, in transiently transfected COS-7 cells demonstrated its H-coupled operation for pyrilamine transport, which was saturable with a Michaelis constant of 132 μM at pH 5.5. In addition, many CADs that may potentially undergo lysosomal trapping, which include imipramine, propranolol, verapamil, and some others, were found to inhibit SLC49A4-AA-mediated pyrilamine transport, suggesting their affinity for SLC49A4. These results suggest that SLC49A4 is involved in the lysosomal trapping of pyrilamine, operating for its exit. The CADs that inhibited SLC49A4-AA-mediated pyrilamine transport could also be SLC49A4 substrate candidates. SLC49A4 mediates the transport of pyrilamine in a H-coupled manner at the lysosomal membrane. This could be a newly identified mechanism for lysosomal export involved in its lysosomal trapping.
PubMed: 37963658
DOI: 10.1124/dmd.123.001354 -
Nature Communications Oct 2023Hetero-pentameric Cys-loop receptors constitute a major type of neurotransmitter receptors that enable signal transmission and processing in the nervous system. Despite...
Hetero-pentameric Cys-loop receptors constitute a major type of neurotransmitter receptors that enable signal transmission and processing in the nervous system. Despite intense investigations into their working mechanism and pharmaceutical potentials, how neurotransmitters activate these receptors remains unclear due to the lack of high-resolution structural information in the activated open state. Here we report near-atomic resolution structures resolved in digitonin consistent with all principle functional states of the human α1β GlyR, which is a major Cys-loop receptor that mediates inhibitory neurotransmission in the central nervous system of adults. Glycine binding induces cooperative and symmetric structural rearrangements in the neurotransmitter-binding extracellular domain but asymmetrical pore dilation in the transmembrane domain. Symmetric response in the extracellular domain is consistent with electrophysiological data showing cooperative glycine activation and contribution from both α1 and β subunits. A set of functionally essential but differentially charged amino acid residues in the transmembrane domain of the α1 and β subunits explains asymmetric activation. These findings provide a foundation for understanding how the gating of the Cys-loop receptor family members diverges to accommodate specific physiological environments.
Topics: Humans; Receptors, Glycine; Ion Channel Gating; Cysteine Loop Ligand-Gated Ion Channel Receptors; Synaptic Transmission; Glycine
PubMed: 37821459
DOI: 10.1038/s41467-023-42051-6 -
Alternative direct-to-amplification cell lysis techniques for forensically relevant non-sperm cells.Journal of Forensic Sciences Nov 2023While efforts have been made to reduce the pervasive backlog of sexual assault evidence collection kits, the actual laboratory process remains very time-consuming due to...
While efforts have been made to reduce the pervasive backlog of sexual assault evidence collection kits, the actual laboratory process remains very time-consuming due to the requirement of a differential lysis step before DNA purification, as well as intricate mixture analysis towards the end of the DNA workflow. Recently, an alternative, direct-to-amplification sperm lysis method (using 1 M NaOH) was identified. However, a direct cell lysis method for non-sperm cells has not been identified yet. Thus, the primary objective of this work was to find an alternative method that is quick, inexpensive, and does not require multiple purification steps for the lysis of non-sperm cells in sexual assault samples. In this study, vaginal swab samples were lysed with the control method, prepGEM™, as well as six alternative reagents: alkaline buffer with 25-200 mM NaOH, high-salt stain extraction buffer, modified radioimmunoprecipitation assay (RIPA) buffer, mammalian protein extraction reagent (M-PER™), digitonin buffer, and urea/thiourea buffer. Quantification using Quantifiler® Trio of vaginal and semen lysates revealed that the alkaline (25 mM NaOH) and M-PER™ methods were efficient for the lysis of vaginal epithelial cells without substantial sperm cell lysis. Following quantification, analysis of STR profiles from vaginal lysates revealed that the M-PER™ method showed promising results across all metrics examined, including the percentage of detected STR alleles, mean peak heights, peak height ratio, and interlocus balance. Thus, this method was recommended as an alternative to the traditional differential lysis method for non-sperm cells given its ability to produce amplification-ready lysates without any DNA purification step.
Topics: Female; Male; Humans; Semen; DNA Fingerprinting; Sodium Hydroxide; Polymerase Chain Reaction; Spermatozoa; Indicators and Reagents; DNA; Microsatellite Repeats
PubMed: 37779342
DOI: 10.1111/1556-4029.15371 -
Journal of Visualized Experiments : JoVE Sep 2023An erratum was issued for: High-Resolution Respirometry to Assess Mitochondrial Function in Human Spermatozoa. The Protocol and Representative Result sections...
An erratum was issued for: High-Resolution Respirometry to Assess Mitochondrial Function in Human Spermatozoa. The Protocol and Representative Result sections were updated. Step 2.4.12 of the Protocol was updated from: Finally, inject 1 µL of 5 mM antimycin A (2.5 µM final concentration). This is a complex II inhibitor to discriminate between the mitochondrial and residual oxygen consumption (non-mitochondrial respiration). For the analysis of complex I, add 1 µL of 1 mM rotenone (0.5 µM final concentration), an inhibitor of this complex, instead of AA. Measure the oxygen consumption until the signal decreases and stabilizes. to: Finally, inject 1 µL of 5 mM antimycin A (2.5 µM final concentration). This is a complex III inhibitor to discriminate between the mitochondrial and residual oxygen consumption (non-mitochondrial respiration). For the analysis of complex I, add 1 µL of 1 mM rotenone (0.5 µM final concentration), an inhibitor of this complex, instead of AA. Measure the oxygen consumption until the signal decreases and stabilizes. Figure 3 in the Representative Results section was updated from: Figure 3: Determination of the optimal concentration of digitonin for the permeabilization of human sperm cells. The respiration rates were measured at 37 °C in MRM medium with glutamate, malate, and adenosine diphosphate. (A) Representative respiratory trace. The blue line is the O2 concentration, and the red line represents the O2 flow per volume correlated. (B) Mitochondria respiration rate means ± standard error, n = 4. The red arrow represents the optimal concentration. Abbreviation: dig = digitonin. Please click here to view a larger version of this figure. to: Figure 3: Determination of the optimal concentration of digitonin for the permeabilization of human sperm cells. The respiration rates were measured at 37 °C in MRM medium with glutamate, malate, and adenosine diphosphate. (A) Representative respiratory trace. The blue line is the O2 concentration, and the red line represents the O2 flow per volume correlated. (B) Mitochondria respiration rate means ± standard error, n = 4. The red arrow represents the optimal concentration. Abbreviation: dig = digitonin. Please click here to view a larger version of this figure.
PubMed: 37751599
DOI: 10.3791/6574 -
Journal of Virology Sep 2023Previously, we developed an infectious hepatitis E virus (HEV) harboring the nanoKAZ gene in the hypervariable region of the open reading frame 1 (ORF1) of the HEV3b...
Previously, we developed an infectious hepatitis E virus (HEV) harboring the nanoKAZ gene in the hypervariable region of the open reading frame 1 (ORF1) of the HEV3b (JE03-1760F/P10) genome and demonstrated the usefulness for screening anti-HEV drugs that inhibit the early infection process. In the present study, we constructed another reporter HEV (HEV3b-HiBiT) by placing a minimized HiBiT tag derived from NanoLuc luciferase at the 3'-end of the viral capsid (ORF2) coding sequence. It replicated efficiently in PLC/PRF/5 cells, produced membrane-associated particles identical to those of the parental virus, and was genetically stable and infectious. The HiBiT tag was fused to both secreted ORF2s (ORF2s-HiBiT) and ORF2c capsid protein (ORF2c-HiBiT). The ORF2c-HiBiT formed membrane-associated HEV particles (eHEV3b-HiBiT). By treating these particles with digitonin, we demonstrated that the HiBiT tag was expressed on the surface of capsid and was present inside the lipid membrane. To simplify the measurement of luciferase activity and provide a more convenient screening platform, we constructed an ORF2s-defective mutant (HEV3b-HiBiT/ΔORF2s) in which the secreted ORF2s are suppressed. We used this system to evaluate the effects of introducing small interfering RNAs and treatment with an inhibitor or accelerator of exosomal release on HEV egress and demonstrated that the effects on virus release can readily be analyzed. Therefore, HEV3b-HiBiT and HEV3b-HiBiT/ΔORF2s reporters may be useful for investigating the virus life cycle and can serve as a more convenient screening platform to search for candidate drugs targeting the late stage of HEV infection such as particle formation and release. IMPORTANCE The construction of recombinant infectious viruses harboring a stable luminescence reporter gene is essential for investigations of the viral life cycle, such as viral replication and pathogenesis, and the development of novel antiviral drugs. However, it is difficult to maintain the stability of a large foreign gene inserted into the viral genome. In the present study, we successfully generated a recombinant HEV harboring the 11-amino acid HiBiT tag in the ORF2 coding region and demonstrated the infectivity, efficient virus growth, particle morphology, and genetic stability, suggesting that this recombinant HEV is useful for assays. Furthermore, this system can serve as a more convenient screening platform for anti-HEV drugs. Thus, an infectious recombinant HEV is a powerful approach not only for elucidating the molecular mechanisms of the viral life cycle but also for the screening and development of novel antiviral agents.
PubMed: 37681960
DOI: 10.1128/jvi.00508-23