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Schlegel, J., Porebski, B., Andronico, L., Hanke, L., Edwards, S., Brismar, H., . . . Sezgin, E. (2023). A Multiparametric and High-Throughput Platform for Host-Virus Binding Screens. Nano Letters, 23(9), 3701-3707
Open this publication in new window or tab >>A Multiparametric and High-Throughput Platform for Host-Virus Binding Screens
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2023 (English)In: Nano Letters, ISSN 1530-6984, E-ISSN 1530-6992, Vol. 23, no 9, p. 3701-3707Article in journal (Refereed) Published
Abstract [en]

Speed is key during infectious disease outbreaks. It is essential, for example, to identify critical host binding factors to pathogens as fast as possible. The complexity of host plasma membrane is often a limiting factor hindering fast and accurate determination of host binding factors as well as high-throughput screening for neutralizing antimicrobial drug targets. Here, we describe a multiparametric and high-throughput platform tackling this bottleneck and enabling fast screens for host binding factors as well as new antiviral drug targets. The sensitivity and robustness of our platform were validated by blocking SARS-CoV-2 particles with nanobodies and IgGs from human serum samples.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2023
Keywords
ACE2, flow cytometry, lipid bilayer, neuropilin-1, silica beads, virus binding
National Category
Infectious Medicine Biochemistry and Molecular Biology Cell and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-332980 (URN)10.1021/acs.nanolett.2c04884 (DOI)000948778700001 ()36892970 (PubMedID)2-s2.0-85149779176 (Scopus ID)
Note

QC 20230724

Available from: 2023-07-24 Created: 2023-07-24 Last updated: 2023-07-24Bibliographically approved
Jess, D. U., Ramdedovic, A., Butt, L., Plagmann, I., Hoehne, M., Hackl, A., . . . Benzing, T. (2023). Advanced optical imaging reveals preferred spatial orientation of podocyte processes along the axis of glomerular capillaries. Kidney International, 104(6), 1164-1169
Open this publication in new window or tab >>Advanced optical imaging reveals preferred spatial orientation of podocyte processes along the axis of glomerular capillaries
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2023 (English)In: Kidney International, ISSN 0085-2538, E-ISSN 1523-1755, Vol. 104, no 6, p. 1164-1169Article in journal (Refereed) Published
Abstract [en]

Mammalian kidneys filter enormous volumes of water and small solutes, a filtration driven by the hydrostatic pressure in glomerular capillaries, which is considerably higher than in most other tissues. Interdigitating cellular processes of podocytes form the slits for fluid filtration connected by the membrane-like slit diaphragm cell junction containing a mechanosensitive ion channel complex and allow filtration while counteracting hydrostatic pressure. Several previous publications speculated that podocyte processes may display a preferable orientation on glomerular capillaries instead of a random distribution. However, for decades, the controversy over spatially oriented filtration slits could not be resolved due to technical limitations of imaging technologies. Here, we used advanced high-resolution, three-dimensional microscopy with high data throughput to assess spatial orientation of podocyte processes and filtration slits quantitatively. Filtration-slit-generating secondary processes preferentially align along the capillaries' longitudinal axis while primary processes are preferably perpendicular to the longitudinal direction. This preferential orientation required maturation in development of the mice but was lost in mice with kidney disease due to treatment with nephrotoxic serum or with underlying heterologous mutations in the podocyte foot process protein podocin. Thus, the observation that podocytes maintain a preferred spatial orientation of their processes on glomerular capillaries goes well in line with the role of podocyte foot processes as mechanical buttresses to counteract mechanical forces resulting from pressurized capillaries. Future studies are needed to establish how podocytes establish and maintain their orientation and why orientation is lost under pathological conditions.

Place, publisher, year, edition, pages
Elsevier BV, 2023
Keywords
glomerulus, podocyte, ultrafiltration
National Category
Biophysics
Identifiers
urn:nbn:se:kth:diva-342067 (URN)10.1016/j.kint.2023.08.024 (DOI)001116585600001 ()37774923 (PubMedID)2-s2.0-85174456707 (Scopus ID)
Note

QC 20240110

Available from: 2024-01-10 Created: 2024-01-10 Last updated: 2024-01-10Bibliographically approved
Zysk, M., Beretta, C., Naia, L., Dakhel, A., Pavenius, L., Brismar, H., . . . Erlandsson, A. (2023). Amyloid-beta accumulation in human astrocytes induces mitochondrial disruption and changed energy metabolism. Journal of Neuroinflammation, 20(1), Article ID 43.
Open this publication in new window or tab >>Amyloid-beta accumulation in human astrocytes induces mitochondrial disruption and changed energy metabolism
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2023 (English)In: Journal of Neuroinflammation, ISSN 1742-2094, E-ISSN 1742-2094, Vol. 20, no 1, article id 43Article in journal (Refereed) Published
Abstract [en]

BackgroundAstrocytes play a central role in maintaining brain energy metabolism, but are also tightly connected to the pathogenesis of Alzheimer's disease (AD). Our previous studies demonstrate that inflammatory astrocytes accumulate large amounts of aggregated amyloid-beta (A beta). However, in which way these A beta deposits influence their energy production remain unclear.MethodsThe aim of the present study was to investigate how A beta pathology in astrocytes affects their mitochondria functionality and overall energy metabolism. For this purpose, human induced pluripotent cell (hiPSC)-derived astrocytes were exposed to sonicated A beta(42) fibrils for 7 days and analyzed over time using different experimental approaches.ResultsOur results show that to maintain stable energy production, the astrocytes initially increased their mitochondrial fusion, but eventually the A beta-mediated stress led to abnormal mitochondrial swelling and excessive fission. Moreover, we detected increased levels of phosphorylated DRP-1 in the A beta-exposed astrocytes, which co-localized with lipid droplets. Analysis of ATP levels, when blocking certain stages of the energy pathways, indicated a metabolic shift to peroxisomal-based fatty acid beta-oxidation and glycolysis.ConclusionsTaken together, our data conclude that A beta pathology profoundly affects human astrocytes and changes their entire energy metabolism, which could result in disturbed brain homeostasis and aggravated disease progression.

Place, publisher, year, edition, pages
Springer Nature, 2023
Keywords
Alzheimer's disease, Glia, Lipid droplets, Mitochondria dynamics, DRP-1
National Category
Neurosciences
Identifiers
urn:nbn:se:kth:diva-324909 (URN)10.1186/s12974-023-02722-z (DOI)000935963900001 ()36803838 (PubMedID)2-s2.0-85148401898 (Scopus ID)
Note

QC 20230321

Available from: 2023-03-21 Created: 2023-03-21 Last updated: 2023-03-21Bibliographically approved
Kouznetsova, A., Liu, J. G., Valentiniene, S., Brismar, H. & Höög, C. (2022). Age-dependent aneuploidy in mammalian oocytes instigated at the second meiotic division. Aging Cell, 21(7), Article ID e13649.
Open this publication in new window or tab >>Age-dependent aneuploidy in mammalian oocytes instigated at the second meiotic division
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2022 (English)In: Aging Cell, ISSN 1474-9718, E-ISSN 1474-9726, Vol. 21, no 7, article id e13649Article in journal (Refereed) Published
Abstract [en]

Ageing severely affects the chromosome segregation process in human oocytes resulting in aneuploidy, infertility and developmental disorders. A considerable amount of segregation errors in humans are introduced at the second meiotic division. We have here compared the chromosome segregation process in young adult and aged female mice during the second meiotic division. More than half of the oocytes in aged mice displayed chromosome segregation irregularities at anaphase II, resulting in dramatically increased level of aneuploidy in haploid gametes, from 4% in young adult mice to 30% in aged mice. We find that the post-metaphase II process that efficiently corrects aberrant kinetochore-microtubule attachments in oocytes in young adult mice is approximately 10-fold less efficient in aged mice, in particular affecting chromosomes that show small inter-centromere distances at the metaphase II stage in aged mice. Our results reveal that post-metaphase II processes have critical impact on age-dependent aneuploidy in mammalian eggs. 

Place, publisher, year, edition, pages
Wiley, 2022
Keywords
Aged, Aneuploidy, Animals, Chromosome Segregation, Female, Humans, Mammals, Meiosis, Mice, Oocytes, Spindle Apparatus, anaphase, animal cell, animal experiment, animal model, Article, cell aging, centromere, controlled study, developmental disorder, gamete, infertility, kinetochore, mammal, meiotic spindle, metaphase, microtubule, mouse, nonhuman, oocyte, time lapse imaging, animal, genetics, human, age-dependent aneuploidy, chromosome, second meiotic division, segregation
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-324362 (URN)10.1111/acel.13649 (DOI)000805561500001 ()35665589 (PubMedID)2-s2.0-85131214754 (Scopus ID)
Note

QC 20230228

Available from: 2023-02-28 Created: 2023-02-28 Last updated: 2023-02-28Bibliographically approved
Nordahl, L., Wennmalm, S., Jönsson, J., Elsässer, S., Akkuratov, E. E. & Brismar, H. (2022). Direct Observation of Na,K‐ATPase Oligomers in The Plasma Membrane of Living Cells by FRET‐FCS. The FASEB Journal, 36(S1)
Open this publication in new window or tab >>Direct Observation of Na,K‐ATPase Oligomers in The Plasma Membrane of Living Cells by FRET‐FCS
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2022 (English)In: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 36, no S1Article in journal (Refereed) Published
Abstract [en]

The sodium pump, Na,K-ATPase, is an integral plasma membrane protein, expressed in all eukaryotic cells. Na,K-ATPase transforms chemical energy from ATP into a gradient of Na+ and K+ over the plasma membrane by actively exporting three Na+ ions and importing two K+ ions for each hydrolyzed ATP. It is responsible for maintenance of the transmembrane Na+ gradient and is the major determinant of the membrane potential. It provides the driving force for all Na+-coupled transport processes, thereby controlling essential functions in the cell. Na,K-ATPase is formed by three subunits alpha/beta/FXYD, where alpha is the catalytic ion-transporting subunit, beta is a regulatory subunit and FXYD is accessory.

Self-interaction and oligomerization of the Na,K-ATPase alpha/beta heterodimer in cell membranes has been proposed and discussed for a long time but is still an open question.

Here we have used a combination of FRET and Fluorescence Correlation Spectroscopy, FRET-FCS, in order to detect oligomers of Na,K-ATPase. Compared to conventional cross-correlation FCCS, FRET-FCS is one to two orders of magnitude more sensitive when detecting oligomers. Moreover, FRET-FCS is inherently insensitive to unbalanced labeling, which is a great advantage during live cell measurements.

We hypothesized that Na,K-ATPase can exist in a higher order oligomeric state and demonstrate the use of FRET-FCS to test this hypothesis

We have introduced fluorescent labels by using expression of non-canonical amino acid modified beta subunits. The FRET pair Alexa488 and Alexa647 was directly conjugated to the beta subunits using selective click chemistry. Conventional FCS measurements of labeled cells revealed the absolute density of labeled and unlabeled Na,K-ATPase. With FRET-FCS we could observe FRET signals and FCS curves demonstrating the existence of oligomers. Positive controls for the FRET-FCS measurements were constructed by labeling alpha subunits with Alexa488 and beta subunits with Alexa647.

Furthermore, we performed Monte Carlo simulations of Na,K-ATPase, as monomer and as oligomer of increasing order, together with its ligands in a picket and fence diffusion model of the plasma membrane. The simulations suggest that oligomerization can have an impact on the net efficiency of the Na,K-ATPase measured as ATP turnover.

In conclusion we find that Na,K-ATPase can be found in the plasma membrane as oligomers. Further we discuss the consequences of oligomerization and propose that it can have a regulatory effect for the Na,K-ATPase net efficiency.

Place, publisher, year, edition, pages
Wiley, 2022
National Category
Biochemistry and Molecular Biology Biophysics
Identifiers
urn:nbn:se:kth:diva-312484 (URN)10.1096/fasebj.2022.36.s1.r4657 (DOI)000878430605101 ()35553550 (PubMedID)
Note

QC 20220520

Available from: 2022-05-19 Created: 2022-05-19 Last updated: 2023-09-21Bibliographically approved
Stenudd, M., Sabelstrom, H., Llorens-Bobadilla, E., Zamboni, M., Blom, H., Brismar, H., . . . Frisen, J. (2022). Identification of a discrete subpopulation of spinal cord ependymal cells with neural stem cell properties. Cell Reports, 38(9), Article ID 110440.
Open this publication in new window or tab >>Identification of a discrete subpopulation of spinal cord ependymal cells with neural stem cell properties
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2022 (English)In: Cell Reports, E-ISSN 2211-1247, Vol. 38, no 9, article id 110440Article in journal (Refereed) Published
Abstract [en]

Spinal cord ependymal cells display neural stem cell properties in vitro and generate scar-forming astrocytes and remyelinating oligodendrocytes after injury. We report that ependymal cells are functionally heterogeneous and identify a small subpopulation (8% of ependymal cells and 0.1% of all cells in a spinal cord segment), which we denote ependymal A (EpA) cells, that accounts for the in vitro stem cell potential in the adult spinal cord. After spinal cord injury, EpA cells undergo self-renewing cell division as they give rise to differentiated progeny. Single-cell transcriptome analysis revealed a loss of ependymal cell gene expression programs as EpA cells gained signaling entropy and dedifferentiated to a stem-cell-like transcriptional state after an injury. We conclude that EpA cells are highly differentiated cells that can revert to a stem cell state and constitute a therapeutic target for spinal cord repair.

Place, publisher, year, edition, pages
CELL PRESS, 2022
National Category
Neurosciences
Identifiers
urn:nbn:se:kth:diva-310212 (URN)10.1016/j.celrep.2022.110440 (DOI)000764832100005 ()35235796 (PubMedID)2-s2.0-85125224331 (Scopus ID)
Note

QC 20220329

Available from: 2022-03-29 Created: 2022-03-29 Last updated: 2024-01-17Bibliographically approved
Unnersjö Jess, D., Butt, L., Höhne, M., Witasp, A., Kühne, L., Hoyer, P. F., . . . Blom, H. (2021). A fast and simple clearing and swelling protocol for 3D in-situ imaging of the kidney across scales. Kidney International, 99(4), 1010-1020
Open this publication in new window or tab >>A fast and simple clearing and swelling protocol for 3D in-situ imaging of the kidney across scales
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2021 (English)In: Kidney International, ISSN 0085-2538, E-ISSN 1523-1755, Vol. 99, no 4, p. 1010-1020Article in journal (Refereed) Published
Abstract [en]

In recent years, many light-microscopy protocols have been published for visualization of nanoscale structures in the kidney. These protocols present researchers with new tools to evaluate both foot process anatomy and effacement, as well as protein distributions in foot processes, the slit diaphragm and in the glomerular basement membrane. However, these protocols either involve the application of different complicated super resolution microscopes or lengthy sample preparation protocols. Here, we present a fast and simple, five-hour long procedure for three-dimensional visualization of kidney morphology on all length scales. The protocol combines optical clearing and tissue expansion concepts to produce a mild swelling, sufficient for resolving nanoscale structures using a conventional confocal microscope. We show that the protocol can be applied to visualize a wide variety of pathologic features in both mouse and human kidneys. Thus, our fast and simple protocol can be beneficial for conventional microscopic evaluation of kidney tissue integrity both in research and possibly in future clinical routines.

Place, publisher, year, edition, pages
Elsevier BV, 2021
National Category
Biophysics Cell and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-312489 (URN)10.1016/j.kint.2020.10.039 (DOI)000740817900006 ()33285146 (PubMedID)2-s2.0-85101519750 (Scopus ID)
Funder
Swedish Foundation for Strategic ResearchSwedish Research Council
Note

QC 20220520

Available from: 2022-05-19 Created: 2022-05-19 Last updated: 2022-06-25Bibliographically approved
Ygberg, S., Akkuratov, E. E., Howard, R. J., Taylan, F., Jans, D. C., Mahato, D. R., . . . Aperia, A. (2021). A missense mutation converts the Na+,K+-ATPase into an ion channel and causes therapy-resistant epilepsy. Journal of Biological Chemistry, 297(6), Article ID 101355.
Open this publication in new window or tab >>A missense mutation converts the Na+,K+-ATPase into an ion channel and causes therapy-resistant epilepsy
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2021 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 297, no 6, article id 101355Article in journal (Refereed) Published
Abstract [en]

The ion pump Na+,K+-ATPase is a critical determinant of neuronal excitability; however, its role in the etiology of diseases of the central nervous system (CNS) is largely unknown. We describe here the molecular phenotype of a Trp931Arg mutation of the Na+,K+-ATPase catalytic alpha 1 subunit in an infant diagnosed with therapy-resistant lethal epilepsy. In addition to the pathological CNS phenotype, we also detected renal wasting of Mg2+. We found that membrane expression of the mutant alpha 1 protein was low, and ion pumping activity was lost. Arginine insertion into membrane proteins can generate water-filled pores in the plasma membrane, and our molecular dynamic (MD) simulations of the principle states of Na+,K+-ATPase transport demonstrated massive water inflow into mutant alpha 1 and destabilization of the ion-binding sites. MD simulations also indicated that a water pathway was created between the mutant arginine residue and the cytoplasm, and analysis of oocytes expressing mutant alpha 1 detected a nonspecific cation current. Finally, neurons expressing mutant alpha 1 were observed to be depolarized compared with neurons expressing wild-type protein, compatible with a lowered threshold for epileptic seizures. The results imply that Na+,K+-ATPase should be considered a neuronal locus minoris resistentia in diseases associated with epilepsy and with loss of plasma membrane integrity.

Place, publisher, year, edition, pages
Elsevier BV, 2021
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-308660 (URN)10.1016/j.jbc.2021.101355 (DOI)000748379600006 ()34717959 (PubMedID)2-s2.0-85120083717 (Scopus ID)
Note

QC 20220215

Available from: 2022-02-15 Created: 2022-02-15 Last updated: 2022-06-25Bibliographically approved
Butt, L., Unnersjö-Jess, D., Höhne, M., Hahnfeldt, R., Reilly, D., Rinschen, M. M., . . . Benzing, T. (2021). Super-Resolution Imaging of the Filtration Barrier Suggests a Role for Podocin R229Q in Genetic Predisposition to Glomerular Disease. Journal of the American Society of Nephrology, 33(1), 138-154
Open this publication in new window or tab >>Super-Resolution Imaging of the Filtration Barrier Suggests a Role for Podocin R229Q in Genetic Predisposition to Glomerular Disease
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2021 (English)In: Journal of the American Society of Nephrology, ISSN 1046-6673, E-ISSN 1533-3450, Vol. 33, no 1, p. 138-154Article in journal (Refereed) Published
Abstract [en]

Significance Statement 

Podocin R229Q results from the most frequent missense variant in NPHS2, and its association with FSGS when podocin R229Q is transassociated with a second mutation in NPHS2 is well recognized. However, because results from observational studies are ambiguous and appropriate animal studies are lacking, its isolated pathogenic potency is not entirely clear. In this study, the authors introduced this genetic alteration in mice and assessed the phenotype using super-resolution microscopy and albuminuria measurements. They demonstrated a deleterious effect of the variant on podocyte morphology and on the integrity of the glomerular filtration barrier under basal conditions and after external glomerular injury. Because this finding suggests that this mutation confers a genetic predisposition to glomerular disease, it has implications for a large number of carriers worldwide.

Background 

Diseases of the kidney’s glomerular filtration barrier are a leading cause of end stage renal failure. Despite a growing understanding of genes involved in glomerular disorders in children, the vast majority of adult patients lack a clear genetic diagnosis. The protein podocin p.R229Q, which results from the most common missense variant in NPHS2, is enriched in cohorts of patients with FSGS. However, p.R229Q has been proposed to cause disease only when transassociated with specific additional genetic alterations, and population-based epidemiologic studies on its association with albuminuria yielded ambiguous results.

Methods 

To test whether podocin p.R229Q may also predispose to the complex disease pathogenesis in adults, we introduced the exact genetic alteration in mice using CRISPR/Cas9-based genome editing (PodR231Q). We assessed the phenotype using super-resolution microscopy and albuminuria measurements and evaluated the stability of the mutant protein in cell culture experiments.

Results 

Heterozygous PodR231Q/wild-type mice did not present any overt kidney disease or proteinuria. However, homozygous PodR231Q/R231Q mice developed increased levels of albuminuria with age, and super-resolution microscopy revealed preceding ultrastructural morphologic alterations that were recently linked to disease predisposition. When injected with nephrotoxic serum to induce glomerular injury, heterozygous PodR231Q/wild-type mice showed a more severe course of disease compared with Podwild-type/wild-type mice. Podocin protein levels were decreased in PodR231Q/wild-type and PodR231Q/R231Q mice as well as in human cultured podocytes expressing the podocinR231Q variant. Our in vitro experiments indicate an underlying increased proteasomal degradation.

Conclusions 

Our findings demonstrate that podocin R231Q exerts a pathogenic effect on its own, supporting the concept of podocin R229Q contributing to genetic predisposition in adult patients.

Place, publisher, year, edition, pages
Wolters Kluwer, 2021
National Category
Biophysics
Identifiers
urn:nbn:se:kth:diva-312487 (URN)10.1681/asn.2020060858 (DOI)000731508200001 ()34853150 (PubMedID)2-s2.0-85123228347 (Scopus ID)
Funder
German Research Foundation (DFG), BR4917/3German Research Foundation (DFG), FOR2743German Research Foundation (DFG), KFO329
Note

QC 20220601

Available from: 2022-05-19 Created: 2022-05-19 Last updated: 2023-07-24Bibliographically approved
Bernhem, K., Fontana, J. M., Svensson, D., Zhang, L., Nilsson, L., Scott, L., . . . Aperia, A. (2021). Super-resolution microscopy reveals that Na+/K+-ATPase signaling protects against glucose-induced apoptosis by deactivating Bad. Cell Death and Disease, 12(8), Article ID 739.
Open this publication in new window or tab >>Super-resolution microscopy reveals that Na+/K+-ATPase signaling protects against glucose-induced apoptosis by deactivating Bad
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2021 (English)In: Cell Death and Disease, ISSN 2041-4889, E-ISSN 2041-4889, Vol. 12, no 8, article id 739Article in journal (Refereed) Published
Abstract [en]

Activation of the apoptotic pathway is a major cause of progressive loss of function in chronic diseases such as neurodegenerative and diabetic kidney diseases. There is an unmet need for an anti-apoptotic drug that acts in the early stage of the apoptotic process. The multifunctional protein Na+,K+-ATPase has, in addition to its role as a transporter, a signaling function that is activated by its ligand, the cardiotonic steroid ouabain. Several lines of evidence suggest that sub-saturating concentrations of ouabain protect against apoptosis of renal epithelial cells, a common complication and major cause of death in diabetic patients. Here, we induced apoptosis in primary rat renal epithelial cells by exposing them to an elevated glucose concentration (20mM) and visualized the early steps in the apoptotic process using super-resolution microscopy. Treatment with 10nM ouabain interfered with the onset of the apoptotic process by inhibiting the activation of the BH3-only protein Bad and its translocation to mitochondria. This occurred before the pro-apoptotic protein Bax had been recruited to mitochondria. Two ouabain regulated and Akt activating Ca2+/calmodulin-dependent kinases were found to play an essential role in the ouabain anti-apoptotic effect. Our results set the stage for further exploration of ouabain as an anti-apoptotic drug in diabetic kidney disease as well as in other chronic diseases associated with excessive apoptosis.

Place, publisher, year, edition, pages
Springer Nature, 2021
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-299672 (URN)10.1038/s41419-021-04025-8 (DOI)000679952600001 ()34315852 (PubMedID)2-s2.0-85111586846 (Scopus ID)
Note

QC 20210819

Available from: 2021-08-19 Created: 2021-08-19 Last updated: 2022-12-12Bibliographically approved
Organisations
Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0003-0578-4003

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