To the Heart of IFs Function: Do they Aggregate on Purpose?

Background: One of the molecular hallmarks in the development of heart failure (HF) is loss of ultrastructure within the cardiac myocyte. In addition, HF is increasingly recognized as a proteinopathy characterized by the accumulation of misfolded proteins similar to Alzheimer and Parkinson disease. However, despite its increasing prevalence and poor prognosis, the advances in the pharmacological treatment of HF have been limited, highlighting an urgent need for the discovery of new therapeutic targets. We reported a consistent accumulation of mono-phosporylated desmin in experimental and clinical models of HF. We also demonstrated how mono-phosphorylated desmin is more prone to cleavage and aggregation in isolated cardiac myocytes. Therefore, if on the one hand desmin cleavage could easily explain the loss of a cardiac myocyte\u2019s ultrastructure, its high abundance and propensity to aggregate make it an ideal candidate as the seed generating pre-amyloid-oligomers (PAOs) and amyloid fibrils in the heart. Methods: Using a combination of novel and established protein biochemistry techniques, we aimed at demonstrating desmin\u2019s identity as the seed starting the nucleation process which leads to the formation of cardiac PAOs and amyloid fibrils. Results: Desmin displayed common features shared by other established PAOs and fibrils (e.g. tinctorial properties) in experimental and clinical models of HF. Conclusions: The inherent propensity of intermediate filaments to aggregate, combined with the use of cardiac tissue as a model for repeated mechanical stretch, suggest that intermediate filaments aggregation could be used as a way to dissipate/scavange mechanical as well as chemical stress. We will therefore use the highly organized structure of cardiac myocytes to infer IFs function in mammalian cells

Nuclear Nox4-derived reactive oxygen species in myelodysplastic syndromes

A role for intracellular ROS production has been recently implicated in the pathogenesis and progression of a wide variety of neoplasias. ROS sources, such as NAD(P)H oxidase (Nox) complexes, are frequently activated in AML (acute myeloid leukemia) blasts and strongly contribute to their proliferation, survival, and drug resistance. Myelodysplastic syndromes (MDS) comprise a heterogeneous group of disorders characterized by ineffective hematopoiesis, with an increased propensity to develop AML. The molecular basis for MDS progression is unknown, but a key element in MDS disease progression is the genomic instability. NADPH oxidases are now recognized to have specific subcellular localizations, this targeting to specific compartments for localized ROS production. Local Nox-dependent ROS production in the nucleus may contribute to the regulation of redox-dependent cell growth, differentiation, senescence, DNA damage, and apoptosis. We observed that Nox1, 2, and 4 isoforms and p22phox and Rac1 subunits are expressed in MDS/AML cell lines and MDS samples, also in the nuclear fractions. Interestingly, Nox4 interacts with ERK and Akt1 within nuclear speckle domain, suggesting that Nox4 could be involved in regulating gene expression and splicing factor activity. These data contribute to the elucidation of the molecular mechanisms used by nuclear ROS to drive MDS evolution to AML

The open body: a “new” book

At the beginning of the ‘300, Mondino de’ Liuzzi, a physician from Bologna, was the first anatomist who started again the dissection of human body neglected from the III century. He hinted at the existence of the conflict between book and body, between “auctoritas” and the direct observation of the human body . The Mondino’s masterwork “Anothomia” remained the key book up to the middle of the sixth century, when Andrea Vesalio wrote “De Umani Corporis Fabrica,” in which the body (cadaver) eventually became the main player of the book . During the years, the technologic evolution led to the wrong conviction that dissection could be dismissed, albeit, still in our day, doctors in training feel the need to associate the direct experience on the cadaver with the very valuable digital means and the modern imaging technologies even in 3D. Thinking to Anatomy as an already fully well known discipline is a mistake. The most advanced methodologies for surgical access, namely the minimally invasive surgery, require the evolution of the traditional anatomical knowledge. The Human Anatomy Institute of the University of Bologna, among the first in Italy, has recognized this need. Thanks to the generosity of the people enrolled in the Body donation programme for research and teaching, our Institute allows medical students to practice dissection on cadavers, beginning as Freshman, then Sophomore, Junior and Senior. The sharing of Bologna’s experience could be the chance to think about the perspectives offered by the dissection of the corpse: a wide range of possibilities spanning from research projects to advanced training courses in collaboration with clinicians and surgeons belonging to different branches. Moreover the practice of corpse dissection is extremely important for the recruitment of young graduates in Medicine which, by means of the experience vested acting as “tutor of anatomy”, acquire interest in the field of research of morphological sciences, spanning from macroscopic up to the cellular and molecular level. Hic mors gaudet succurrere vitae: the motto, reported in dissection room of most of the Italian anatomical institutes, represents the synthesis of the experience of an ancient discipline which, nowadays , has the chance to rewrite a new chapter dedicated to modern frontiers of scientific research and medical education

The wide and growing range of lamin B‑related diseases: from laminopathies to cancer

B-type lamins are fundamental components of the nuclear lamina, a complex structure that acts as a scafold for organization and function of the nucleus. Lamin B1 and B2, the most represented isoforms, are encoded by LMNB1 and LMNB2 gene, respectively. All B-type lamins are synthesized as precursors and undergo sequential post-translational modifcations to generate the mature protein. B-type lamins are involved in a wide range of nuclear functions, including DNA replication and repair, regulation of chromatin and nuclear stifness. Moreover, lamins B1 and B2 regulate several cellular processes, such as tissue development, cell cycle, cellular proliferation, senescence, and DNA damage response. During embryogenesis, B-type lamins are essential for organogenesis, in particular for brain development. As expected from the numerous and pivotal functions of B-type lamins, mutations in their genes or fuctuations in their expression levels are critical for the onset of several diseases. Indeed, a growing range of human disorders have been linked to lamin B1 or B2, increasing the complexity of the group of diseases collectively known as laminopathies. This review highlights the recent fndings on the biological role of B-type lamins under physiological or pathological conditions, with a particular emphasis on brain disorders and cancer

Three-Dimensional Virtual Anatomy as a New Approach for Medical Student’s Learning

none8noMost medical and health science schools adopt innovative tools to implement the teaching of anatomy to their undergraduate students. The increase in technological resources for educational purposes allows the use of virtual systems in the field of medicine, which can be considered decisive for improving anatomical knowledge, a requisite for safe and competent medical practice. Among these virtual tools, the Anatomage Table 7.0 represents, to date, a pivotal anatomical device for student education and training medical professionals. This review focuses attention on the potential of the Anatomage Table in the anatomical learning process and clinical practice by discussing these topics based on recent publication findings and describing their trends during the COVID-19 pandemic period. The reports documented a great interest in and a positive impact of the use of this technological table by medical students for teaching gross anatomy. Anatomage allows to describe, with accuracy and at high resolution, organ structure, vascularization, and innervation, as well as enables to familiarize with radiological images of real patients by improving knowledge in the radiological and surgical fields. Furthermore, its use can be considered strategic in a pandemic period, since it ensures, through an online platform, the continuation of anatomical and surgical training on dissecting cadavers.openBartoletti-Stella, Anna; Gatta, Valentina; Mariani, Giulia Adalgisa; Gobbi, Pietro; Falconi, Mirella; Manzoli, Lucia; Faenza, Irene; Salucci, SaraBartoletti-Stella, Anna; Gatta, Valentina; Mariani, Giulia Adalgisa; Gobbi, Pietro; Falconi, Mirella; Manzoli, Lucia; Faenza, Irene; Salucci, Sar

IPMK and β-catenin take part in PLC-β1-dependent signaling pathway during myogenic differentiation

Phospholipase C (PLC)-β1 catalytic activity plays an essential role in the initiation of myogenic differentiation but the effectors involved in its signaling pathway are not well defined[1,2]. Here, we show that the overexpression of the Inositol Polyphosphate Multikinase (IPMK) promotes myogenic differentiation, and that IPMK targets the same cyclin D3 promoter region activated by PLC-β1. Moreover, cyclin D3 promoter activation relies upon c-jun binding to the promoter, both in response to PLC-β1 and to IPMK overexpression. Furthermore, both IPMK and PLC-β1 overexpression determines an increase in β-catenin translocation and accumulation to the nuclei of differentiating myoblasts resulting in higher MyoD activation. Therefore, our data show that PLC-β1, IPMK and β-catenin are mediators of the same signaling pathway that regulates cyclin D3 and myosin heavy chain (MYH) induction during myogenic differentiation

A Role for Nuclear Phospholipase Cβ1 in Cell Cycle Control

Phosphoinositide signaling resides in the nucleus, and among the enzymes of the cycle, phospholipase C (PLC) appears as the key element both in Saccharomyces cerevisiae and in mammalian cells. The yeast PLC pathway produces multiple inositol polyphosphates that modulate distinct nuclear processes. The mammalian PLCbeta(1), which localizes in the nucleus, is activated in insulin-like growth factor 1-mediated mitogenesis and undergoes down-regulation during murine erythroleukemia differentiation. PLCbeta(1) exists as two polypeptides of 150 and 140 kDa generated from a single gene by alternative RNA splicing, both of them containing in the COOH-terminal tail a cluster of lysine residues responsible for nuclear localization. These clues prompted us to try to establish the critical nuclear target(s) of PLCbeta(1) subtypes in the control of cell cycle progression. The results reveal that the two subtypes of PLCbeta(1) that localize in the nucleus induce cell cycle progression in Friend erythroleukemia cells. In fact when they are overexpressed in the nucleus, cyclin D3, along with its kinase (cdk4) but not cyclin E is overexpressed even though cells are serum-starved. As a consequence of this enforced expression, retinoblastoma protein is phosphorylated and E2F-1 transcription factor is activated as well. On the whole the results reveal a direct effect of nuclear PLCbeta(1) signaling in G(1) progression by means of a specific target, i.e. cyclin D3/cdk4

Morphological and immunoistochemical evaluation of cadaveric gingival specimens to estimate the post-mortem Interval

The estimation of the post-mortem interval (PMI) is a critical step in forensic medicine and remains one of the most challenging variables to determine. In general, the numerous methods currently used in estimating post-mortem interval yield to large post-mortem windows, are influenced by several factors and sometime contradict each other.  With the aim to obtain a much more accurate determination of the post-mortem interval we combined morphological ultrastructural and immunoistochemical analyses to reach a more detailed knowledge on tissue organization and degradation after death. Samples of gingival tissues obtained from 20 cadavers at different post-mortem intervals were processed for transmission electron microscopy to evaluate ultrastructural modifications in the epithelium and connective tissue. Gingival cells and the extracellular matrix of gingival tissues have been observed by a transmission electron microscopy (TEM) in combination with the evaluation of potential post-mortem biochemical markers, with the final goal to find a tight correlation between the ultrastructural modifications, the biomarker expression and the time of death. All the samples were also immunostained with anti-hypoxia-induced factor 1-α (HIF1-α) antibody, a transcription factor expressed in response to hypoxia, in order to evaluate the expression of HIF1-α, and to establish a correlation between the protein presence and the time of death. Results showed nuclear chromatin changes and cytoplasmic vacuolization in both epithelial and connective tissues and a different pattern of expression of HIF1alpha protein that correlate to the time of death. In conclusion, our preliminary findings suggest that ultrastructural investigations in combination with immunohistochemistry techniques in gingival specimens may represent a new tool to accurately and systematically estimate post-mortem interval

Nuclear DGKα regulates cell cycle progression in K562 cells

The existence of an independent nuclear inositide pathway distinct from the cytoplasmic one has been demonstrated in different physiological systems and in diseases (1). Phosphatidylinositols (PIs) play an important role in nuclear function regulation and behave differently from their counterparts in the cytoplasm. The autonomous nuclear PI cycle in eukaryotic cells is involved in different regulation processes, from cell proliferation to differentiation and many others (2). At nuclear level an array of kinases and phosphatases can modulate PIs. Among these, Diacylglycerol Kinases (DGKs) are a class of phosphotransferases that phosphorylate diacylglycerol (DAG) and induce the synthesis of phosphatidic acid. We Investigated DGKα localization and function in human erythroleukemia cell line K562. Synchronization experiments at different cell cycle checkpoints showed an important expression of DGKα in the nuclear fraction of this cell model, slightly peaking at G2/M. This suggested that DGKα might have a function in nuclear signaling. In particular, nuclear DGKα expression can modulate cell cycle progression, leading to changes in the phosphorylated status of the Retinoblastoma protein (pRb), thus, regulating G1/S transition: DGKα silencing or downregulation leads to impaired G1/S transition and its overexpression leads to S phase progression. The molecular mechanism by which nuclear DGKα controls pRb phosphorylation and therefore cell cycle regulation in K562 cell line are still unclear. Further studies are needed to better understand the role of DGKα in relation to other pivotal PIs involved in cell cycle regulation in the hematopoietic system

Multiple roles of phosphoinositide-specific phospholipase C isozymes

Phosphoinositide-specific phospholipase C is an effector molecule in the signal transduction process. It generates two second messengers, inositol-1,4,5-trisphosphate and diacylglycerol from phosphatidylinositol 4,5-bisphosphate. Currently, thirteen mammal PLC isozymes have been identified, and they are divided into six groups: PLC-beta, -gamma, -delta, -epsilon, -zeta and -eta. Sequence analysis studies demonstrated that each isozyme has more than one alternative splicing variant. PLC isozymes contain the X and Y domains that are responsible for catalytic activity. Several other domains including the PH domain, the C2 domain and EF hand motifs are involved in various biological functions of PLC isozymes as signaling proteins. The distribution of PLC isozymes is tissue and organ specific. Recent studies on isolated cells and knockout mice depleted of PLC isozymes have revealed their distinct phenotypes. Given the specificity in distribution and cellular localization, it is clear that each PLC isozyme bears a unique function in the modulation of physiological responses. In this review, we discuss the structural organization, enzymatic properties and molecular diversity of PLC splicing variants and study functional and physiological roles of each isozyme.open19320