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ENaC channels
(2023)
The epithelial sodium channel (ENaC) is a heterotrimeric ion channel that plays a key role in sodium and water homeostasis in tetrapod vertebrates. In the aldosterone-sensitive distal nephron, hormonally controlled ENaC expression matches dietary sodium intake to its excretion. Furthermore, ENaC mediates sodium absorption across the epithelia of the colon, sweat ducts, reproductive tract, and lung. ENaC is a constitutively active ion channel and its expression, membrane abundance, and open probability (PO) are controlled by multiple intracellular and extracellular mediators and mechanisms [9]. Aberrant ENaC regulation is associated with severe human diseases, including hypertension, cystic fibrosis, pulmonary edema, pseudohypoaldosteronism type 1, and nephrotic syndrome [9].
Once aberrantly activated, the Wnt/βcatenin pathway may result in uncontrolled proliferation and eventually cancer. Efforts to counter and inhibit this pathway are mainly directed against βcatenin, as it serves a role on the cytoplasm and the nucleus. In addition, speciallygenerated lymphocytes are recruited for the purpose of treating liver cancer. Peripheral blood mononuclear lymphocytes are expanded by the timely addition of interferon γ, interleukin (IL)1β, IL2 and anticluster of differentiation 3 antibody. The resulting cells are called cytokineinduced killer (CIK) cells. The present study utilised these cells and combine them with drugs inhibiting the Wnt pathway in order to examine whether this resulted in an improvement in the killing ability of CIK cells against liver cancer cells. Drugs including ethacrynic acid (EA) and ciclopirox olamine (CPX) were determined to be suitable candidates, as determined by previous studies. Drugs were administered on their own and combined with CIK cells and then a cell viability assay was performed. These results suggest that EAtreated cells demonstrated apoptosis and were significantly affected compared with untreated cells. Unlike EA, CPX killed normal and cancerous cells even at low concentrations. Subsequent to combining EA with CIK cells, the potency of killing was increased and a greater number of cells died, which proves a synergistic action. In summary, EA may be used as an antihepatocellular carcinoma drug, while CPX possesses a high toxicity to cancerous as well as to normal cells. It was proposed that EA should be integrated into present therapeutic methods for cancer.
SLC6A14 (ATB0,+) is unique among SLC proteins in its ability to transport 18 of the 20 proteinogenic (dipolar and cationic) amino acids and naturally occurring and synthetic analogues (including anti-viral prodrugs and nitric oxide synthase (NOS) inhibitors). SLC6A14 mediates amino acid uptake in multiple cell types where increased expression is associated with pathophysiological conditions including some cancers. Here, we investigated how a key position within the core LeuT-fold structure of SLC6A14 influences substrate specificity. Homology modelling and sequence analysis identified the transmembrane domain 3 residue V128 as equivalent to a position known to influence substrate specificity in distantly related SLC36 and SLC38 amino acid transporters. SLC6A14, with and without V128 mutations, was heterologously expressed and function determined by radiotracer solute uptake and electrophysiological measurement of transporter-associated current. Substituting the amino acid residue occupying the SLC6A14 128 position modified the binding pocket environment and selectively disrupted transport of cationic (but not dipolar) amino acids and related NOS inhibitors. By understanding the molecular basis of amino acid transporter substrate specificity we can improve knowledge of how this multi-functional transporter can be targeted and how the LeuT-fold facilitates such diversity in function among the SLC6 family and other SLC amino acid transporters.
Among the celestial bodies in the Solar System, Mars currently represents the main target for the search for life beyond Earth. However, its surface is constantly exposed to high doses of cosmic rays (CRs) that may pose a threat to any biological system. For this reason, investigations into the limits of resistance of life to space relevant radiation is fundamental to speculate on the chance of finding extraterrestrial organisms on Mars. In the present work, as part of the STARLIFE project, the responses of dried colonies of the black fungus Cryomyces antarcticus Culture Collection of Fungi from Extreme Environments (CCFEE) 515 to the exposure to accelerated iron (LET: 200 keV/μm) ions, which mimic part of CRs spectrum, were investigated. Samples were exposed to the iron ions up to 1000 Gy in the presence of Martian regolith analogues. Our results showed an extraordinary resistance of the fungus in terms of survival, recovery of metabolic activity and DNA integrity. These experiments give new insights into the survival probability of possible terrestrial-like life forms on the present or past Martian surface and shallow subsurface environments.
Dried serum spots that are well prepared can be attractive alternatives to frozen serum samples for shelving specimens in a medical or research center's biobank and mailing freshly prepared serum to specialized laboratories. During the pre-analytical phase, complications can arise which are often challenging to identify or are entirely overlooked. These complications can lead to reproducibility issues, which can be avoided in serum protein analysis by implementing optimized storage and transfer procedures. With a method that ensures accurate loading of filter paper discs with donor or patient serum, a gap in dried serum spot preparation and subsequent serum analysis shall be filled. Pre-punched filter paper discs with a 3 mm diameter are loaded within seconds in a highly reproducible fashion (approximately 10% standard deviation) when fully submerged in 10 μl of serum, named the "Submerge and Dry" protocol. Such prepared dried serum spots can store several hundred micrograms of proteins and other serum components. Serum-borne antigens and antibodies are reproducibly released in 20 μl elution buffer in high yields (approximately 90%). Dried serum spot-stored and eluted antigens kept their epitopes and antibodies their antigen binding abilities as was assessed by SDS-PAGE, 2D gel electrophoresis-based proteomics, and Western blot analysis, suggesting pre-punched filter paper discs as handy solution for serological tests.
Amaç: Keton cisim oluşumu (ketogenez) bozuklukları; mitokondriyel 3-hidroksi-3metil glutaril CoA sentaz (Mhs) ve 3-hidroksi-3-metil glutaril CoA liyaz (HL) enzim eksiklikleri sonucu oluşur. Keton cisim yıkımı (ketoliz) bozuklukları ise suksinil CoA: 3 oksoasit CoA transferaz (SCOT) ve asetoasetil CoA thiolaz-beta ketotiolaz (MAT) enzim eksiklikleri sonucu oluşmaktadır. Keton metabolizma bozukluğu tanısıyla izlenen hastaların klinik ve laboratuvar bulguları ile değerlendirilmesi amaçlandı.
Yöntem: Keton metabolizması bozukluğu tanısıyla izlenen hasta verileri retrospektif olarak incelendi.
Bulgular: Dört hastada HL eksikliği, 3 hastada MAT eksikliği ve 2 hastada SCOT eksikliği tanısı mevcuttu. Hastaların ortanca yaşı 5 yıl (6 ay-15,5 yıl), ilk metabolik dekompanzasyon atak yaşı ortalama 7,7 ay (22 gün-19 ay) idi. MAT eksikliği olan bir hasta, kardeş taraması ile asemptomatik dönemde tanı aldı. İki hastada spastik tetraparezi gibi ağır nörolojik defisit gelişti. Dekompanzasyon ataklarının beslenememe, kusma ve gastroenterit gibi infeksiyon sonrası geliştiği görüldü.
Sonuç: Açıklanamayan metabolik asidoz atakları durumunda keton metabolizma bozuklukları akılda tutulmalıdır. Akut dekompanzasyon değişik yaşlarda ortaya çıkabilir, klinik şiddeti değişken olabilir. Erken tanı ve uygun tedavi mortalite ve morbidite açısından çok önemlidir.
A firm link between endoplasmic reticulum (ER) stress and tumors has been wildly reported. Endoplasmic reticulum oxidoreductase 1 alpha (ERO1α), an ER-resident thiol oxidoreductase, is confirmed to be highly upregulated in various cancer types and associated with a significantly worse prognosis. Of importance, under ER stress, the functional interplay of ERO1α/PDI axis plays a pivotal role to orchestrate proper protein folding and other key processes. Multiple lines of evidence propose ERO1α as an attractive potential target for cancer treatment. However, the unavailability of specific inhibitor for ERO1α, its molecular inter-relatedness with closely related paralog ERO1β and the tightly regulated processes with other members of flavoenzyme family of enzymes, raises several concerns about its clinical translation. Herein, we have provided a detailed description of ERO1α in human cancers and its vulnerability towards the aforementioned concerns. Besides, we have discussed a few key considerations that may improve our understanding about ERO1α in tumors.
PURPOSE
Cervical cancer (CC) is caused by a persistent high-risk human papillomavirus (hrHPV) infection. The cervico-vaginal microbiome may influence the development of (pre)cancer lesions. Aim of the study was (i) to evaluate the new CC screening program in Germany for the detection of high-grade CC precursor lesions, and (ii) to elucidate the role of the cervico-vaginal microbiome and its potential impact on cervical dysplasia.
METHODS
The microbiome of 310 patients referred to colposcopy was determined by amplicon sequencing and correlated with clinicopathological parameters.
RESULTS
Most patients were referred for colposcopy due to a positive hrHPV result in two consecutive years combined with a normal PAP smear. In 2.1% of these cases, a CIN III lesion was detected. There was a significant positive association between the PAP stage and Lactobacillus vaginalis colonization and between the severity of CC precursor lesions and Ureaplasma parvum.
CONCLUSION
In our cohort, the new cervical cancer screening program resulted in a low rate of additional CIN III detected. It is questionable whether these cases were only identified earlier with additional HPV testing before the appearance of cytological abnormalities, or the new screening program will truly increase the detection rate of CIN III in the long run. Colonization with U. parvum was associated with histological dysplastic lesions. Whether targeted therapy of this pathogen or optimization of the microbiome prevents dysplasia remains speculative.
It has become increasingly clear that caspases, far from being merely cell death effectors, have a much wider range of functions within the cell. These functions are as diverse as signal transduction and cytoskeletal remodeling, and caspases are now known to have an essential role in cell proliferation, migration, and differentiation. There is also evidence that apoptotic cells themselves can direct the behavior of nearby cells through the caspase-dependent secretion of paracrine signaling factors. In some processes, including the differentiation of skeletal muscle myoblasts, both caspase activation in differentiating cells as well as signaling from apoptotic cells has been reported. Here, we review the non-apoptotic outcomes of caspase activity in a range of different model systems and attempt to integrate this knowledge.
Intact Transition Epitope Mapping - Targeted High-Energy Rupture of Extracted Epitopes (ITEM-THREE)
(2019)
Epitope mapping, which is the identification of antigenic determinants, is essential for the design of novel antibody-based therapeutics and diagnostic tools. ITEM-THREE is a mass spectrometry-based epitope mapping method that can identify epitopes on antigens upon generating an immune complex in electrospray-compatible solutions by adding an antibody of interest to a mixture of peptides from which at least one holds the antibody's epitope. This mixture is nano-electrosprayed without purification. Identification of the epitope peptide is performed within a mass spectrometer that provides an ion mobility cell sandwiched in-between two collision cells and where this ion manipulation setup is flanked by a quadrupole mass analyzer on one side and a time-of-flight mass analyzer on the other side. In a stepwise fashion, immune-complex ions are separated from unbound peptide ions and dissociated to release epitope peptide ions. Immune complex-released peptide ions are separated from antibody ions and fragmented by collision induced dissociation. Epitope-containing peptide fragment ions are recorded, and mass lists are submitted to unsupervised data base search thereby retrieving both, the amino acid sequence of the epitope peptide and the originating antigen. ITEM-THREE was developed with antiTRIM21 and antiRA33 antibodies for which the epitopes were known, subjecting them to mixtures of synthetic peptides of which one contained the respective epitope. ITEM-THREE was then successfully tested with an enzymatic digest of His-tagged recombinant human β-actin and an antiHis-tag antibody, as well as with an enzymatic digest of recombinant human TNFα and an antiTNFα antibody whose epitope was previously unknown.