Institute

Complete genome sequence of Pediococcus pentosaceus 13.7 2A-1 isolated from a Holstein Friesian dairy cattle

2025-12-29T13:46:41Z

Complete genome sequence of Pediococcus pentosaceus 13.7 2A-1 isolated from a Holstein Friesian dairy cattle Mariani Corea, Nadine; Titgemeyer, Fritz; Wachtarczyk, Jennifer; Meyer, Frank; Fischer, Sebastian W. Pediococcus pentosaceus was isolated from a Holstein Friesian dairy cattle from a conventionally operated farm. The complete genome of strain P. pentosaceus 13.7 2A-1 comprises 1.84 Mbp and 1,746 protein-coding sequences. The decoded genome sequence will serve to further study the strain for its use in food fermentation and foodsafety.

Potent optogenetic regulation of gene expression in mammalian cells for bioproduction and basic research

2025-12-29T12:46:32Z

Potent optogenetic regulation of gene expression in mammalian cells for bioproduction and basic research Gebel, Jeannette; Ciglieri, Elisa; Stahl, Rainer; Duthie, Fraser; Frechen, Fabian; Möglich, Andreas; Müller-Hartmann, Herbert; Schmidt, Hanns-Martin; Wachten, Dagmar Precise temporal and spatial control of gene expression greatly benefits the study of specific cellular circuits and activities. Compared to chemical inducers, light-dependent control of gene expression by optogenetics achieves a higher spatial and temporal resolution. Beyond basic research, this could also prove decisive for manufacturing difficult-to-express proteins in pharmaceutical bioproduction. However, current optogenetic gene-expression systems limit this application in mammalian cells, as expression levels and the degree of induction upon light stimulation are insufficient. To overcome this limitation, we designed a photoswitch by fusing the blue light-activated light–oxygen–voltage receptor EL222 from Erythrobacter litoralis to the three transcriptional activator domains VP64, p65, and Rta in tandem. The result ant photoswitch, dubbed DEL-VPR, allows up to a 570-fold induction of target gene expression by blue light, thereby achieving expression levels of strong constitutive promoters. Here, we used DEL-VPR to enable light-induced expression of complex monoclonal and bispecific antibodies with reduced byproduct expression and increased yield of functional protein complexes. Our approach offers temporally controlled yet strong gene expression and applies to academic and industrial settings.

FVIII Trafficking Dynamics Across Subcellular Organelles Using CRISPR/Cas9 Specific Gene Knockouts

2025-12-29T11:31:33Z

FVIII Trafficking Dynamics Across Subcellular Organelles Using CRISPR/Cas9 Specific Gene Knockouts El Hazzouri, Salime; Al-Rifai, Rawya; Surges, Nicole; Rath, Melanie; Singer, Heike; Oldenburg, Johannes; El-Maarri, Osman Factor VIII (FVIII) interacts with Endoplasmic Reticulum (ER) chaperones Calnexin (CANX) and Calreticulin (CALR) and with ER-Golgi Intermediate Compartment (ERGIC) transporters, Lectin, mannose-binding 1 (LMAN1) and Multiple Coagulation Deficiency 2 (MCFD2). We previously reported that the Gamma-aminobutyric Acid Receptor-associated proteins (GABARAPs) also influence FVIII secretion. Here, we further investigated the intracellular dynamics of FVIII using single and double CRISPR/Cas9 Knockout (KO) models of the abovementioned chaperones as well as the GABARAP proteins in HEK293 cells expressing FVIII. Cellular pathways were manipulated by Brefeldin A (BFA), Chloroquine (CQ), a Rab7 inhibitor, and subjected to glucose starvation. The effect of each KO on FVIII secretion and organelle distribution was assessed by a two-stage chromogenic assay and immunofluorescence (IF) microscopy, prior and upon cell treatments. Using these approaches, we first observed distinct effects of each studied protein on FVIII trafficking. Notably, intracellular localization patterns revealed clustering of FVIII phenotypes in GABARAP^KO, CANX^KO, and CALR^KO cells together under both basal and treated conditions, an observation that was also reflected in their respective double KO combinations. Besides, a clear involvement of additional components of the endomembrane system was evident, specifically at the trans-Golgi space, as marked by FVIII colocalization with the Ras-like proteins in brain (Rab8 and Rab7) and with the Vesicle-Associated Membrane Protein (VAMP8), along with the observed impact of the selected cell treatments on FVIII phenotypes. These outcomes enhance our understanding of the molecular mechanisms regulating FVIII and pave the way for new perspectives, which could be further projected into FVIII replacement, cell and gene therapies.

Renal tissue-resident macrophages promote cystogenesis in early polycystic kidney disease

2025-12-29T11:16:35Z

Renal tissue-resident macrophages promote cystogenesis in early polycystic kidney disease Karl, Rudolfo; Ashraf, Arsila Palliyulla Kariat; Viola, Maria Francesca; Hopp, Katharina; Mass, Elvira; Wachten, Dagmar Autosomal-dominant polycystic kidney disease (ADPKD) is a ciliopathy characterized bymutations in PKD1 or PKD2, which drive cystogenesis in renal epithelial cells. Immune cells, particularly macrophages, contribute to disease progression, yet their role remains incompletely understood. Here, we performed an in-depth analysis of renal macrophage ontogeny and phenotype and investigated their function in an ADPKD mouse model (Pkd1^RC/RC) with adult onset and slow disease progression. We demonstrate that the numbers of tissue-resident macrophages were already increased before cyst formation. Using a flow cytometry screening panel, we further characterized the tissue-resident macrophage populations using surface markers and identified a novel marker that shows the potential to determine macrophage remodeling at different disease stages. To reveal the cellular interaction of tissue-resident macrophages and renal epithelial cells in further detail, we established a 3D co-culture system, demonstrating that tissue-resident macrophages from Pkd1^RC/RC mice, isolated at a stage before cysts were observed, already showed enhanced cystogenesis in vitro. These findings underscore the crucial role of tissue-resident macrophages in ADPKD and suggest targeting epithelial cell–macrophage interactions as a promising therapeutic avenue.