Initiation of stem cell differentiation involves cell cycle-dependent regulation of developmental genes by Cyclin D.

Coordination of differentiation and cell cycle progression represents an essential process for embryonic development and adult tissue homeostasis. These mechanisms ultimately determine the quantities of specific cell types that are generated. Despite their importance, the precise molecular interplays between cell cycle machinery and master regulators of cell fate choice remain to be fully uncovered. Here, we demonstrate that cell cycle regulators Cyclin D1-3 control cell fate decisions in human pluripotent stem cells by recruiting transcriptional corepressors and coactivator complexes onto neuroectoderm, mesoderm, and endoderm genes. This activity results in blocking the core transcriptional network necessary for endoderm specification while promoting neuroectoderm factors. The genomic location of Cyclin Ds is determined by their interactions with the transcription factors SP1 and E2Fs, which result in the assembly of cell cycle-controlled transcriptional complexes. These results reveal how the cell cycle orchestrates transcriptional networks and epigenetic modifiers to instruct cell fate decisions.This work was supported by the European Research Council grant Relieve IMDs and the Cambridge Hospitals National Institute for Health Research Biomedical Research Center (L.V.). A.B. was funded by the British Heart Foundation Ph.D. Studentship. S.P. was funded by a Federation of European Biochemical Societies long-term fellowship and a InnovaLiv EuFP7 grant. S.P. and L.V. conceived the research and wrote the manuscript. S.P. and A.B. performed the experiments. P.M. performed bioinformatic analyses.This is the final version of the article. It first appeared from Cold Spring Harbor Laboratory Press via http://dx.doi.org/10.1101/gad.271452.11

Overproduction and Characterization of the Bacillus subtilis Anti-sigma Factor FlgM

FlgM is an anti-sigma factor of the flagellar-specific sigma (sigma) subunit of RNA polymerase in Bacillus subtilis, and it is responsible of the coupling of late flagellar gene expression to the completion of the hook-basal body structure. We have overproduced the protein in soluble form and characterized it. FlgM forms dimers as shown by gel exclusion chromatography and native polyacrylamide gel electrophoresis and interacts in vitro with the cognate sigmaD factor. The FlgM.sigmaD complex is a stable heterodimer as demonstrated by gel exclusion chromatography, chemical cross-linking, native polyacrylamide gel electrophoresis, and isoelectric focusing. sigmaD belongs to the group of sigma factors able to bind to the promoter sequence even in the absence of core RNA polymerase. The FlgM.sigmaD complex gave a shift in a DNA mobility shift assay with a probe containing a sigmaD-dependent promoter sequence. Limited proteolysis studies indicate the presence of two structural motifs, corresponding to the N- and C-terminal regions, respectively

The Importance of Being “That” Colorectal pT1: A Combined Clinico-Pathological Predictive Score to Improve Nodal Risk Stratification

The management of endoscopically resected pT1 colorectal cancer (CRC) relies on nodal metastasis risk estimation based on the assessment of specific histopathological features. Avoiding the overtreatment of metastasis-free patients represents a crucial unmet clinical need. By analyzing a consecutive series of 207 pT1 CRCs treated with colectomy and lymphadenectomy, this study aimed to develop a novel clinicopathological score to improve pT1 CRC metastasis prediction. First, we established the clinicopathological profile of metastatic cases: lymphovascular invasion (OR: 23.8; CI: 5.12–110.9) and high-grade tumor budding (OR: 5.21; CI: 1.60–16.8) correlated with an increased risk of nodal metastasis, while age at diagnosis >65 years (OR: 0.26; CI: 0.09–0.71) and high tumor-infiltrating lymphocytes (OR: 0.19; CI: 0.06–0.59) showed a protective effect. Combining these features, we built a five-tier risk score that, applied to our series, identified cases with a higher risk (score ≥ 2) of nodal metastasis (OR: 7.7; CI: 2.4–24.4). Notably, a score of 0 was only assigned to cases with no metastases (13/13 cases) and all the score 4 samples (2/2 cases) showed nodal metastases. In conclusion, we developed an effectively combined score to assess pT1 CRC nodal metastasis risk. We believe that its adoption within a multidisciplinary pT1 unit could improve patients' clinical management and limit surgical overtreatment

Homozygous loss of autism-risk gene CNTNAP2 results in reduced local and long-range prefrontal functional connectivity

Functional connectivity aberrancies, as measured with resting-state fMRI (rsfMRI), have been consistently observed in the brain of autism spectrum disorders (ASD) patients. However, the genetic and neurobiological underpinnings of these findings remain unclear. Homozygous mutations in Contactin Associated Protein-like 2 (CNTNAP2), a neurexin-related cell-adhesion protein, are strongly linked to autism and epilepsy. Here we used rsfMRI to show that homozygous mice lacking Cntnap2 exhibit reduced long-range and local functional connectivity in prefrontal and midline brain “connectivity hubs”. Long-range rsfMRI connectivity impairments affected heteromodal cortical regions and were prominent between frontoposterior components of the mouse default-mode network (DMN), an effect that was associated with reduced social investigation, a core “autism trait” in mice. Notably, viral tracing revealed reduced frequency of prefrontal-projecting neural clusters in the cingulate cortex of Cntnap2-/- mutants, suggesting a possible contribution of defective mesoscale axonal wiring to the observed functional impairments. Macroscale cortico-cortical white matter organization appeared to be otherwise preserved in these animals. These findings reveal a key contribution of ASD-associated gene CNTNAP2 in modulating macroscale functional connectivity, and suggest that homozygous loss-of-function mutations in this gene may predispose to neurodevelopmental disorders and autism through a selective dysregulation of connectivity in integrative prefrontal areas

DNAJB9 Is a Reliable Immunohistochemical Marker of Fibrillary Glomerulonephritis: Evaluation of Diagnostic Efficacy in a Large Series of Kidney Biopsies

Fibrillary glomerulonephritis (FGN) is a rare glomerular disease characterized by a challenging diagnostic workup requiring ultrastructural identification of 20 nm-thick randomly oriented fibrillar deposits. However, the recent introduction of DNAJB9 as a putative diagnostic marker of FGN could thoroughly improve this diagnostic scenario. This study aims to assess the DNAJB9 immunohistochemical expression in a large series of FGN cases and to eventually confirm its role as a diagnostic marker of FGN. We evaluated the immunohistochemical expression of DNAJB9 (Rabbit Polyclonal, ThermoFisher) in a series of 77 FGN and 128 non-FGN cases diagnosed between January 1992 and June 2022 at the Pathology Unit of the AOU Città della Salute e della Scienza Hospital. DNAJB9 was expressed in 73 of the 74 evaluable FGN cases, mostly showing a strong glomerular positivity (68 cases). Additionally, DNAJB9 resulted positive in all challenging scenarios [early-stage (6), congophilic (4), combined (4), and uncertain (4) cases of FGN)]. DNAJB9 was negative in all non-FGN cases, eventually resulting in a specificity of 100% and sensitivity of 99%. In conclusion, we confirmed the role of DNAJB9 as a diagnostic marker of FGN. Its adoption in the clinical routine will allow a faster, more feasible, and more accurate FGN diagnosis

Deletion of autism risk gene Shank3 disrupts prefrontal connectivity

Mutations in the synaptic scaffolding protein Shank3 are a major cause of autism, and are associated with prominent intellectual and language deficits. However, the neural mechanisms whereby SHANK3 deficiency affects higher order socio-communicative functions remain unclear. Using high-resolution functional and structural MRI in adult male mice, here we show that loss of Shank3 (Shank3B-/-) results in disrupted local and long-range prefrontal and fronto-striatal functional connectivity. We document that prefrontal hypo-connectivity is associated with reduced short-range cortical projections density, and reduced gray matter volume. Finally, we show that prefrontal disconnectivity is predictive of social communication deficits, as assessed with ultrasound vocalization recordings. Collectively, our results reveal a critical role of SHANK3 in the development of prefrontal anatomy and function, and suggest that SHANK3 deficiency may predispose to intellectual disability and socio-communicative impairments via dysregulation of higher-order cortical connectivity