PaperPlayer biorxiv molecular biology podcast

The Macerprep: a minimalist kit- and enzyme-free high-yield miniprep utilising alkaline lysis and alkaline hydrolysis principles

0:00
0:00
Spola tillbaka 15 sekunder
Spola framåt 15 sekunder
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.08.13.249607v1?rss=1 Authors: Hu, I. Abstract: The commercialisation of miniprep kits supplanted the original alkaline lysis method for plasmid DNA preparation, and had remained relatively unchanged for almost two decades. The Miraprep substantially improved the yields of miniprep kits. However, the method still relies on commercial kits, which can be a burden financially to certain projects. Additionally, Pronobis et al. also identified loss of RNAse activities in miniprep kits over time. The present novel plasmid DNA isolation protocol addresses the two issues mentioned above utilising alkaline lysis and alkaline hydrolysis principles. With a largely identical workflow and operation time, the Macerprep will significantly reduce costs of establishing new laboratories as well as maintenance of running molecular biology laboratories. Copy rights belong to original authors. Visit the link for more info

Fler avsnitt från "PaperPlayer biorxiv molecular biology"

  • PaperPlayer biorxiv molecular biology podcast

    Assembly and symmetry of the fungal E3BP-containing core of the Pyruvate Dehydrogenase Complex

    Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.08.13.232140v1?rss=1 Authors: Forsberg, B. O., Aibara, S., Howard, R. J., Mortezaei, N., Lindahl, E. Abstract: The pyruvate dehydrogenase complex (PDC) is a central component of all aerobic respiration, connecting glycolysis to mitochondrial oxidation of pyruvate. Despite its central metabolic role, its precise composition and means of regulation re- main unknown. To explain the variation in stoichiometry reported for the E3-recruiting protein X (PX) in the fungal PDC, we established cryo-EM reconstructions of the native and recombinant PDC from the filamentous fungus and model organism Neurospora crassa. We find that the PX C-terminal domain localizes interior to the E2 core. Critically, we show that two distinct arrangements of a trimeric oligomer exists, which both result in strict tetrahedral symmetry of the PDC core interior. Both oligomerization and volume occlusion of the PDC interior by PX appears to limit its binding stoichiometry, which explains the variety of stoichiometries found previously for S. cerevisiae. This also suggests that the PX oligomer stability and size are potential mechanisms to dynamically adjust PDC compostion in response to external cues. Moreover, we find that the site where PX binds is conserved within fungi but not mammals, suggesting that it could be therapeutically targeted. To this end, we also show that a PX knockout results in loss of activity through dysfunctional E3 recruitment, leading to severely impaired N. crassa growth on sucrose. The fungal PDC is thus shown to be fundamentally similar to the mammalian PDC in function but subject to other conditions of possible regulation, conditioned by a steric restrictions imposed by the symmetry of the PDC and its components. Copy rights belong to original authors. Visit the link for more info
  • PaperPlayer biorxiv molecular biology podcast

    A Strategy to Assess the Cellular Activity of E3 Ligases against Neo-Substrates using Electrophilic Probes

    Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.08.13.249482v1?rss=1 Authors: Pinch, B. J., Buckley, D. L., Gleim, S., Brittain, S. M., Tandeske, L., D'Alessandro, P. L., Harvey, E. P., Hauseman, Z. J., Schirle, M., Sprague, E. R., Forrester, W. C., Dovala, D., McGregor, L. M., Thoma, C. R. Abstract: Targeted protein degradation is a rapidly developing therapeutic modality that promises lower dosing and enhanced selectivity as compared to traditional occupancy-driven inhibitors, and the potential to modulate historically intractable targets. While the well-characterized E3 ligases CRBN and VHL have been successfully redirected to degrade numerous proteins, there are approximately 600 predicted additional E3 family members that may offer improved activity, substrate selectivity, and/or tissue distribution; however, characterizing the potential applications of these many ligases for targeted protein degradation has proven challenging. Here, we report the development of an approach to evaluate the ability of recombinant E3 ligase components to support neo-substrate degradation. Bypassing the need for hit finding to identify specific E3 ligase binders, this approach makes use of simple chemistry for Covalent Functionalization Followed by E3 Electroporation into live cells (COFFEE). We demonstrate this method by electroporating recombinant VHL, covalently functionalized with JQ1 or dasatinib, to induce degradation of BRD4 or kinase targets, respectively. Furthermore, by applying COFFEE to SPSB2, a SOCS box and SPRY-domain E3 ligase that has not previously been redirected for targeted protein degradation, we validate this method as a powerful approach to define the activity of previously uncharacterized ubiquitin ligases against neo-substrates. Copy rights belong to original authors. Visit the link for more info
  • PaperPlayer biorxiv molecular biology podcast

    Missa inte ett avsnitt av PaperPlayer biorxiv molecular biology och prenumerera på det i GetPodcast-appen.

    iOS buttonAndroid button
  • PaperPlayer biorxiv molecular biology podcast

    The RNA-binding protein Puf5 buffers mRNA levels against chromatin-mediated changes in nascent transcription

    Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.08.13.249912v1?rss=1 Authors: Kochan, D. Z., Mawer, J. S. P., Tishinov, K., Parekh, S., Maassen, J., Graef, M., Spang, A., Tessarz, P. Abstract: Gene expression is a dynamic process regulated at all stages, starting with opening of chromatin, transcription, and continuing with mRNA export, translation and, finally, degradation. While there are feedback mechanisms within the system, it is not clear whether these extend to crosstalk between chromatin architecture and mRNA decay. Here, we show that changes in nascent transcription, mediated by mutating H3K56 to alanine, are post-transcriptionally buffered by the Pumilio protein Puf5, which stabilizes transcripts in a context-dependent manner. Depleting Puf5 in an H3K56A background leads to synthetic lethality. This genetic interaction can be explained by a decrease in translation due to downregulation of its direct mRNA targets, largely consisting of ribosomal protein genes. Importantly, we show that this post-transcriptional buffering is not only linked to H3K56A, but may be a more widespread phenomenon that also buffers against an increase in nascent RNA transcription in order to maintain physiological mRNA levels and cellular homeostasis. Copy rights belong to original authors. Visit the link for more info
  • PaperPlayer biorxiv molecular biology podcast

    The Macerprep: a minimalist kit- and enzyme-free high-yield miniprep utilising alkaline lysis and alkaline hydrolysis principles

    Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.08.13.249607v1?rss=1 Authors: Hu, I. Abstract: The commercialisation of miniprep kits supplanted the original alkaline lysis method for plasmid DNA preparation, and had remained relatively unchanged for almost two decades. The Miraprep substantially improved the yields of miniprep kits. However, the method still relies on commercial kits, which can be a burden financially to certain projects. Additionally, Pronobis et al. also identified loss of RNAse activities in miniprep kits over time. The present novel plasmid DNA isolation protocol addresses the two issues mentioned above utilising alkaline lysis and alkaline hydrolysis principles. With a largely identical workflow and operation time, the Macerprep will significantly reduce costs of establishing new laboratories as well as maintenance of running molecular biology laboratories. Copy rights belong to original authors. Visit the link for more info
  • PaperPlayer biorxiv molecular biology podcast

    Maternal high fat diet alters lactation-specific miRNA expression and programs the DNA methylome in the amygdala of female offspring

    Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.08.13.249300v1?rss=1 Authors: Abuaish, S., Wijenayake, S., de Vega, W. C., Lum, C. M. W., Sasaki, A., McGowan, P. Abstract: Adverse maternal diets high in saturated fats are associated with impaired neurodevelopment and epigenetic modifications in offspring. Maternal milk, the primary source of early life nutrition in mammals, contains lactation-specific microRNAs (miRNAs). Lactation-specific miRNAs have been found in various offspring tissues in early life, including the brain. We examined the effects of maternal high saturated fat diet (mHFD) on lactation-specific miRNAs that inhibit DNA methyltransferases (DNMTs), enzymes that catalyze DNA methylation modifications, in the amygdala of female offspring during early life and adulthood. Offspring exposed to mHFD showed reduced miR-148/152 and miR-21 transcripts in stomach milk and amygdala in the first week of life. This was associated with increased DNMT1 expression, DNMT activity, and global DNA methylation in the amygdala. In addition, persistent DNA methylation modifications from early life to adulthood were observed in pathways involved in neurodevelopment as well as genes regulating the DNMT machinery and protein function in mHFD offspring. The findings indicate a novel link between exogenous, lactation-specific miRNAs and developmental programming of the neural DNA methylome in offspring. Copy rights belong to original authors. Visit the link for more info
  • PaperPlayer biorxiv molecular biology podcast

    Homologous recombination defects in Shwachman-Diamond syndrome and Diamond-Blackfan anemia

    Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.08.13.250068v1?rss=1 Authors: Asik, E., Chatterjee, N., Bertuch, A. A. Abstract: Shwachman-Diamond syndrome (SDS) and Diamond-Blackfan anemia (DBA) are ribosomopathies characterized by impaired hematopoiesis and cancer predisposition. The mechanisms underlying cancer predisposition in these disorders are not well understood. We found that LCLs derived from patients with SDS or DBA had a prolonged DNA damage response and hypersensitivity to ionizing radiation, suggesting impaired DNA double strand break (DSB) repair. Consistent with this, depletion of SBDS and RPS19, the most common etiologic factors in SDS and DBA, respectively, resulted in reduced homologous recombination (HR) in HCT116 and U2OS cells. Surprisingly, depletion of EFL1, which functions with SBDS in ribosome biogenesis, did not impair HR and depletion of eIF6, which restores ribosome joining in SBDS-depleted cells, did not rescue the HR defect associated with SBDS depletion. Instead, we found SBDS and RPS19 recruitment to sites of DSBs suggesting that SBDS and RPS19 have more proximate roles in regulating HR, independent of their ribosomal functions. We propose that reduced HR shifts DSB repair toward error-prone NHEJ and this may contribute to oncogenesis in SDS and DBA. Additionally, we found SBDS and RPS19 depleted cells were hypersensitive to PARP inhibition, potentially uncovering a therapeutic target for SDS- and DBA-associated malignancies. Copy rights belong to original authors. Visit the link for more info
  • PaperPlayer biorxiv molecular biology podcast

    UPRmt scales mitochondrial network expansion with protein synthesis via mitochondrial import

    Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.08.12.248161v1?rss=1 Authors: Shpilka, T., Du, Y., Yung, Q., Melber, A., Uma Naresh, N., Lavelle, J., Liu, P., Weidberg, H., Li, R., Yu, J., Zhu, L. J., Strittmatter, L., Haynes, C. M. Abstract: As organisms develop, individual cells generate mitochondria to fulfill physiologic requirements. However, it remains unknown how mitochondrial network expansion is scaled to cell growth and impacted by environmental cues. The mitochondrial unfolded protein response (UPRmt) is a signaling pathway mediated by the transcription factor ATFS-1 which harbors a mitochondrial targeting sequence (MTS). Here, we demonstrate that ATFS-1 mediates an adaptable mitochondrial expansion program that is active throughout normal development. Developmental mitochondrial network expansion required the relatively inefficient MTS in ATFS-1, which allowed the transcription factor to be responsive to parameters that impact protein import capacity of the entire mitochondrial network. Increasing the strength of the ATFS-1 MTS impaired UPRmt activity throughout development due to increased accumulation within mitochondria. The insulin-like signaling-TORC1 and AMPK pathways affected UPRmt activation in a manner that correlated with protein synthesis. Manipulation to increase protein synthesis caused UPRmt activation. Alternatively, S6 kinase inhibition had the opposite effect due to increased mitochondrial accumulation of ATFS-1. However, ATFS-1 with a dysfunctional MTS constitutively increased UPRmt activity independent of TORC1 function. Lastly, expression of a single protein with a strong MTS, was sufficient to expand the muscle cell mitochondrial network in an ATFS-1-dependent manner. We propose that mitochondrial network expansion during development is an emergent property of the synthesis of highly expressed mitochondrial proteins that exclude ATFS-1 from mitochondrial import, causing UPRmt activation. Mitochondrial network expansion is attenuated once ATFS-1 can be imported. Copy rights belong to original authors. Visit the link for more info
  • PaperPlayer biorxiv molecular biology podcast

    Structure Basis of Cav1.1 Modulation by Dihydropyridine Compounds

    Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.08.13.250340v1?rss=1 Authors: Gao, S., Yan, N. Abstract: 1,4-Dihydropyridines (DHP), the most commonly used antihypertensives, function by inhibiting the L-type voltage-gated Ca2+ (Cav) channels. DHP compounds exhibit chirality-specific antagonistic or agonistic effects. Recent structural elucidation of rabbit Cav1.1 bound to an achiral drug nifedipine reveals the general binding mode for DHP drugs, but the molecular basis for chiral specificity remains elusive. Here, we report five cryo-EM structures of nanodisc-embedded Cav1.1 in the presence of the bestselling drug amlodipine, a DHP antagonist (R)-(+)-Bay K8644, and a titration of its agonistic enantiomer (S)-(-)-Bay K8644 at resolutions of 2.9-3.4 [A]. The amlodipine-bound structure reveals the molecular basis for the high efficacy of the drug. All structures with the addition of the Bay K8644 enantiomers exhibit similar inactivated conformations, suggesting that the agonistic effect of (S)-(-)-Bay K8644 might be transient. The similarity of these structures to that obtained in detergent micelles alleviates the concerns about potential structural perturbation by detergents. Copy rights belong to original authors. Visit the link for more info
  • PaperPlayer biorxiv molecular biology podcast

    Creb5 establishes the competence for Prg4 expression in articular cartilage

    Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.08.12.248609v1?rss=1 Authors: Zhang, C.-H., Gao, Y., Jadhav, U., Hung, H.-H., Holton, K. M., Grodzinsky, A. J., Shivdasani, R. A., Lassar, A. B. Abstract: A hallmark of cells comprising the superficial zone of articular cartilage is their expression of lubricin, encoded by the Prg4 gene, that lubricates the joint and protects against the development of arthritis. Here, we identify Creb5 as a transcription factor that is specifically expressed in superficial zone articular chondrocytes and is required for TGF-beta and EGFR signaling to induce Prg4 expression. Notably, forced expression of Creb5 in chondrocytes derived from the deep zone of the articular cartilage confers the competence for TGF-beta and EGFR signals to induce Prg4 expression. Chromatin-IP and ATAC-Seq analyses have revealed that Creb5 directly binds to two Prg4 promoter-proximal regulatory elements, that display an open chromatin conformation specifically in superficial zone articular chondrocytes; and which work in combination with a more distal regulatory element to drive induction of Prg4 by TGF-beta. Our results indicate that Creb5 is a critical regulator of Prg4/lubricin expression in the articular cartilage. Copy rights belong to original authors. Visit the link for more info
  • PaperPlayer biorxiv molecular biology podcast

    KIAA1841, a novel SANT and BTB domain-containing protein, inhibits class switch recombination

    Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.08.12.247809v1?rss=1 Authors: Zheng, S., Matthews, A. J., Rahman, N., Herrick-Reynolds, K., Choi, J. E., Sible, E., Ng, Y. K., Rhodes, D., Elledge, S. J., Vuong, B. Q. Abstract: Class switch recombination (CSR) enables B cells to produce different immunoglobulin isotypes and mount an effective immune response against pathogens. Timely resolution of CSR prevents damage due to an uncontrolled and prolonged immune response. While many positive regulators of CSR have been described, negative regulators of CSR are relatively unknown. Using a shRNA library screen in a mouse B cell line, we have identified the novel protein KIAA1841 (NM_027860) as a negative regulator of CSR. KIAA1841 is an uncharacterized protein of 82kD containing SANT and BTB domains. The BTB domain of KIAA1841 exhibited characteristic properties such as self-dimerization and interaction with co-repressor proteins. Overexpression of KIAA1841 inhibited CSR in primary mouse splenic B cells, and inhibition of CSR is dependent on the BTB domain while the SANT domain is largely dispensable. Thus, we have identified a new member of the BTB family that serves as a negative regulator of CSR. Copy rights belong to original authors. Visit the link for more info

Kom åt alla poddsändningar i gratisappen GetPodcast.

Prenumerera på dina favoritpoddar, lyssna på avsnitt offline och få spännande rekommendationer.

iOS buttonAndroid button