A Haspin-ARHGAP11A axis regulates epithelial morphogenesis through Rho-ROCK dependent modulation of LIMK1-Cofilin.

Throughout mitosis, a plethora of processes must be efficiently concerted to ensure cell proliferation and tissue functionality. The mitotic spindle does not only mediate chromosome segregation, but also defines the axis of cellular division, thus determining tissue morphology. Functional spindle orientation relies on precise actin dynamics, shaped in mitosis by the LIMK1-Cofilin axis. The kinase Haspin acts as a guardian of faithful chromosome segregation that ensures amphitelic chromosome attachment and prevents unscheduled cohesin cleavage. Here, we report an unprecedented role for Haspin in the determination of spindle orientation in mitosis. We show that, during mitosis, Haspin regulates Rho-ROCK activity through ARHGAP11A, a poorly characterized GAP, and that ROCK is in turn responsible for the mitotic activation of LIMK1 and stabilization of the actin cytoskeleton, thus supporting a functional spindle orientation. By exploiting 3D cell cultures, we show that this pathway is pivotal for the establishment of a morphologically functional tissue.

Blood purification therapy with a hemodiafilter featuring enhanced adsorptive properties for cytokine removal in patients presenting COVID-19: a pilot study.

BACKGROUND: Systemic inflammation in COVID-19 often leads to multiple organ failure, including acute kidney injury (AKI). Renal replacement therapy (RRT) in combination with sequential extracorporeal blood purification therapies (EBP) might support renal function, attenuate systemic inflammation, and prevent or mitigate multiple organ dysfunctions in COVID-19. AIM: Describe overtime variations of clinical and biochemical features of critically ill patients with COVID-19 treated with EBP with a hemodiafilter characterized by enhanced cytokine adsorption properties. METHODS: An observational prospective study assessing the outcome of patients with COVID-19 admitted to the ICU (February to April 2020) treated with EBP according to local practice. Main endpoints included overtime variation of IL-6 and multiorgan function-scores, mortality, and occurrence of technical complications or adverse events. RESULTS: The study evaluated 37 patients. Median baseline IL-6 was 1230 pg/ml (IQR 895) and decreased overtime (p < 0.001 Kruskal-Wallis test) during the first 72 h of the treatment, with the most significant decrease in the first 24 h (p = 0.001). The reduction in serum IL-6 concentrations correlated with the improvement in organ function, as measured in the decrease of SOFA score (rho = 0.48, p = 0.0003). Median baseline SOFA was 13 (IQR 6) and decreased significantly overtime (p < 0.001 at Kruskal-Wallis test) during the first 72 h of the treatment, with the most significant decrease in the first 48 h (median 8 IQR 5, p = 0.001). Compared to the expected mortality rates, as calculated by APACHE IV, the mean observed rates were 8.3% lower after treatment. The best improvement in mortality rate was observed in patients receiving EBP early on during the ICU stay. Premature clotting (running < 24 h) occurred in patients (18.9% of total) which featured higher effluent dose (median 33.6 ml/kg/h, IQR 9) and higher filtration fraction (median 31%, IQR 7.4). No electrolyte disorders, catheter displacement, circuit disconnection, unexpected bleeding, air, or thromboembolisms due to venous cannulation of EBP were recorded during the treatment. In one case, infection of vascular access occurred during RRT, requiring replacement. CONCLUSIONS: EBP with heparin-coated hemodiafilter featuring cytokine adsorption properties administered to patients with COVID-19 showed to be feasible and with no adverse events. During the treatment, patients experienced serum IL-6 level reduction, attenuation of systemic inflammation, multiorgan dysfunction improvement, and reduction in expected ICU mortality rate.

Haspin regulates Ras localization to promote Cdc24-driven mitotic depolarization.

Cell polarization is of paramount importance for proliferation, differentiation, development, and it is altered during carcinogenesis. Polarization is a reversible process controlled by positive and negative feedback loops. How polarized factors are redistributed is not fully understood and is the focus of this work. In Saccharomyces cerevisiae, mutants defective in haspin kinase exhibit stably polarized landmarks and are sensitive to mitotic delays. Here, we report a new critical role for haspin in polarisome dispersion; failure to redistribute polarity factors, in turn, leads to nuclear segregation defects and cell lethality. We identified a mitotic role for GTP-Ras in regulating the local activation of the Cdc42 GTPase, resulting in its dispersal from the bud tip to a homogeneous distribution over the plasma membrane. GTP-Ras2 physically interacts with Cdc24 regulateing its mitotic distribution. Haspin is shown to promote a mitotic shift from a bud tip-favored to a homogenous PM fusion of Ras-containing vesicles. In absence of haspin, active Ras is not redistributed from the bud tip; Cdc24 remains hyperpolarized promoting the activity of Cdc42 at the bud tip, and the polarisome fails to disperse leading to erroneously positioned mitotic spindle, defective nuclear segregation, and cell death after mitotic delays. These findings describe new functions for key factors that modulate cell polarization and mitotic events, critical processes involved in development and tumorigenesis.

Effects of liver ischemia-reperfusion injury on respiratory mechanics and driving pressure during orthotopic liver transplantation.

BACKGROUND: During orthotopic liver transplantation (OLT), liver graft ischemia-reperfusion injury (IRI) triggers a cytokine-mediated systemic inflammatory response, which impairs graft function and disrupts distal organ homeostasis. The objective of this prospective, observational trial was to assess the effects of IRI on lung and chest wall mechanics in the intraoperative period of patients undergoing OLT. METHODS: In 26 patients undergoing OLT, we measured elastance of the respiratory system (ERS), partitioned into lung (EL) and chest wall (ECW), hemodynamics, and fluid and blood product intake before laparotomy (T1), after portal/caval surgical clamp (T2), and immediately (T3) and, at 90 and 180 minutes post-reperfusion (T4 and T5, respectively). Interleukin-6 (IL-6), monocyte chemotactic protein-1 (MCP-1), IL-1ß and tumor necrosis factor-α plasma concentrations were assessed at T1, T4 and T5. RESULTS: EL significantly decreased from T1 to T2 (13.5±4.4 vs 9.7±4.8 cmH2O/L, P<0.05), remained stable at T3, while at T4 (12.3±4.4 cmH2O/L, P<0.05) was well above levels recorded at T2, reaching its highest value at T5 (15±3.9 cmH2O/L, P<0.05). Variations in ERS, EL, driving pressure (∆P) and trans-pulmonary pressure (∆PL) significantly correlated with changes in IL-6 and MCP-1 plasma concentrations, but not with changes in wedge pressure, fluid amounts, and red blood cells and platelets administered. No correlation was found between changes in cytokine concentrations and ECW. CONCLUSIONS: We found that EL, ECW, ∆P and ∆PL underwent significant variations during the OLT procedure. Further, we documented a significant association between the respiratory mechanics changes and the inflammatory response following liver graft reperfusion.

Intratracheal Administration of Small Interfering RNA Targeting Fas Reduces Lung Ischemia-Reperfusion Injury.

OBJECTIVES: Lung ischemia-reperfusion injury is the main cause of primary graft dysfunction after lung transplantation and results in increased morbidity and mortality. Fas-mediated apoptosis is one of the pathologic mechanisms involved in the development of ischemia-reperfusion injury. We hypothesized that the inhibition of Fas gene expression in lungs by intratracheal administration of small interfering RNA could reduce lung ischemia-reperfusion injury in an ex vivo model reproducing the procedural sequence of lung transplantation. DESIGN: Prospective, randomized, controlled experimental study. SETTING: University research laboratory. SUBJECTS: C57/BL6 mice weighing 28-30 g. INTERVENTIONS: Ischemia-reperfusion injury was induced in lungs isolated from mice, 48 hours after treatment with intratracheal small interfering RNA targeting Fas, control small interfering RNA, or vehicle. Isolated lungs were exposed to 6 hours of cold ischemia (4°C), followed by 2 hours of warm (37°C) reperfusion with a solution containing 10% of fresh whole blood and mechanical ventilation with constant low driving pressure. MEASUREMENTS AND MAIN RESULTS: Fas gene expression was significantly silenced at the level of messenger RNA and protein after ischemia-reperfusion in lungs treated with small interfering RNA targeting Fas compared with lungs treated with control small interfering RNA or vehicle. Silencing of Fas gene expression resulted in reduced edema formation (bronchoalveolar lavage protein concentration and lung histology) and improvement in lung compliance. These effects were associated with a significant reduction of pulmonary cell apoptosis of lungs treated with small interfering RNA targeting Fas, which did not affect cytokine release and neutrophil infiltration. CONCLUSIONS: Fas expression silencing in the lung by small interfering RNA is effective against ischemia-reperfusion injury. This approach represents a potential innovative strategy of organ preservation before lung transplantation.

Posttraumatic stress symptoms, dissociation, and alexithymia in an Italian sample of flood victims.

BACKGROUND: Several studies have demonstrated a significant association between dissociation and posttraumatic symptoms. A dissociative reaction during a traumatic event may seem to predict the later development of posttraumatic stress symptoms. Moreover, several researchers also observed an alexithymic condition in a variety of traumatized samples. METHODS: A total of 287 flood victims (men =159, 55.4%; women =128, 44.6%) with an age range of 17-21 years (mean =18.33; standard deviation =0.68) completed the following: Impact of Event Scale-Revised, Dissociative Experiences Scale II, Twenty-Item Toronto Alexithymia Scale, and Peritraumatic Dissociative Experiences Questionnaire. RESULTS: We found significant correlations among all variables. Linear regression showed that peritraumatic dissociation plays a mediator role between alexithymia, dissociation, and post-traumatic stress symptoms. CONCLUSION: Our results seem to confirm the significant roles of both dissociation and alexithymia for the development of posttraumatic symptoms.

The Impact of Event Scale - Revised: psychometric properties of the Italian version in a sample of flood victims.

BACKGROUND: This study aims to verify the main psychometric properties of the Italian version of the Impact of Event Scale - Revised (IES-R) in a sample of flood victims. METHODS: The sample was composed of 262 subjects involved in the natural disaster of 2009 in the city of Messina (Italy). All participants completed the IES-R and the Dissociative Experiences Scale-II (DES-II) in order to verify some aspects of convergent validity. RESULTS: THE EXPLORATORY AND CONFIRMATORY FACTOR ANALYSIS, USED TO VERIFY THE CONSTRUCT VALIDITY OF THE MEASURE, SHOWED A CLEAR FACTOR STRUCTURE WITH THREE INDEPENDENT DIMENSIONS: intrusion, avoidance, and hyper-arousal. The goodness-of-fit indices (non-normed fit index [NNFI] = 0.99; comparative fit index [CFI] = 0.99; standardized root mean square residual [SRMR] = 0.04; and root mean square error of approximation [RMSEA] = 0.02) indicated a good adaptation of the model to the data. The IES-R scales showed satisfactory values of internal consistency (intrusion, α = 0.78; avoidance, α = 0.72; hyper-arousal, α = 0.83) and acceptable values of correlation with the DES-II. CONCLUSION: These results suggest that this self-reported and easily administered instrument for assessing the dimensions of trauma has good psychometric properties and can be adopted usefully, both for research and for practice in Italy.

Yeast haspin kinase regulates polarity cues necessary for mitotic spindle positioning and is required to tolerate mitotic arrest.

Haspin is an atypical protein kinase that in several organisms phosphorylates histone H3Thr3 and is involved in chromosome segregation. In Saccharomyces cerevisiae, H3Thr3 phosphorylation has never been observed and the function of haspin is unknown. We show that deletion of ALK1 and ALK2 haspin paralogs causes the mislocalization of polarisome components. Following a transient mitotic arrest, this leads to an overly polarized actin distribution in the bud where the mitotic spindle is pulled. Here it elongates, generating anucleated mothers and binucleated daughters. Reducing the intensity of the bud-directed pulling forces partially restores proper cell division. We propose that haspin controls the localization of polarity cues to preserve the coordination between polarization and the cell cycle and to tolerate transient mitotic arrests. The evolutionary conservation of haspin and of the polarization mechanisms suggests that this function of haspin is likely shared with other eukaryotes, in which haspin may regulate asymmetric cell division.

Expression plasmids for use in Candida glabrata.

We describe a series of CEN/ARS episomal plasmids containing different Candida glabrata promoters, allowing for a range of constitutive or regulated expression of proteins in C. glabrata. The set of promoters includes three constitutive promoters (EGD2pr, HHT2pr, PDC1pr), two macrophage/phagocytosis-induced promoters (ACO2pr, LYS21pr), and one nutritionally regulated promoter (MET3pr). Each promoter was cloned into two plasmid backbones that differ in their selectable marker, URA3, or the dominant-selectable NAT1 gene, which confers resistance to the drug nourseothricin. Expression from the 12 resulting plasmids was assessed using GFP as a reporter and flow cytometry or quantitative reverse-transcription polymerase chain reaction to assess expression levels. Together this set of plasmids expands the toolkit of expression vectors available for use with C. glabrata.

Non-canonical CRL4A/4B(CDT2) interacts with RAD18 to modulate post replication repair and cell survival.

The Cullin-4(CDT2) E3 ubiquitin ligase plays an essential role in DNA replication origin licensing directing degradation of several licensing factors at the G1/S transition in order to prevent DNA re-replication. Recently a RAD18-independent role of Cullin-4(CDT2) in PCNA monoubiquitylation has been proposed. In an effort to better understand the function of Cullin-4(CDT2) E3 ubiquitin ligase in mammalian Post-Replication Repair during an unperturbed S-phase, we show that down-regulation of Cullin-4(CDT2) leads to two distinguishable independent phenotypes in human cells that unveil at least two independent roles of Cullin-4(CDT2) in S-phase. Apart from the re-replication preventing activity, we identified a non-canonical Cullin-4(CDT2) complex, containing both CUL4A and CUL4B, associated to the COP9 signalosome, that controls a RAD18-dependent damage avoidance pathway essential during an unperturbed S-phase. Indeed, we show that the non-canonical Cullin-4A/4B(CDT2) complex binds to RAD18 and it is required to modulate RAD18 protein levels onto chromatin and the consequent dynamics of PCNA monoubiquitylation during a normal S-phase. This function prevents replication stress, ATR hyper-signaling and, ultimately, apoptosis. A very similar PRR regulatory mechanism has been recently described for Spartan. Our findings uncover a finely regulated process in mammalian cells involving Post-Replication Repair factors, COP9 signalosome and a non-canonical Cullin4-based E3 ligase which is essential to tolerate spontaneous damage and for cell survival during physiological DNA replication.

The ocular albinism type 1 protein, an intracellular G protein-coupled receptor, regulates melanosome transport in pigment cells.

The protein product of the ocular albinism type 1 gene, named OA1, is a pigment cell-specific G protein-coupled receptor exclusively localized to intracellular organelles, namely lysosomes and melanosomes. Loss of OA1 function leads to the formation of macromelanosomes, suggesting that this receptor is implicated in organelle biogenesis, however the mechanism involved in the pathogenesis of the disease remains obscure. We report here the identification of an unexpected abnormality in melanosome distribution both in retinal pigment epithelium (RPE) and skin melanocytes of Oa1-knock-out (KO) mice, consisting in a displacement of the organelles from the central cytoplasm towards the cell periphery. Despite their depletion from the microtubule (MT)-enriched perinuclear region, Oa1-KO melanosomes were able to aggregate at the centrosome upon disruption of the actin cytoskeleton or expression of a dominant-negative construct of myosin Va. Consistently, quantification of organelle transport in living cells revealed that Oa1-KO melanosomes displayed a severe reduction in MT-based motility; however, this defect was rescued to normal following inhibition of actin-dependent capture at the cell periphery. Together, these data point to a defective regulation of organelle transport in the absence of OA1 and imply that the cytoskeleton might represent a downstream effector of this receptor. Furthermore, our results enlighten a novel function for OA1 in pigment cells and suggest that ocular albinism type 1 might result from a different pathogenetic mechanism than previously thought, based on an organelle-autonomous signalling pathway implicated in the regulation of both membrane traffic and transport.

The Shq1p.Naf1p complex is required for box H/ACA small nucleolar ribonucleoprotein particle biogenesis.

Small nucleolar ribonucleoprotein particles (snoRNPs) are essential cofactors in ribosomal RNA metabolism. Although snoRNP composition has been thoroughly characterized, the biogenesis process of these particles is poorly understood. We have identified two proteins from the yeast Saccharomyces cerevisiae, Yil104c/Shq1p and Ynl124w/Naf1p, which are essential and required for the stability of box H/ACA snoRNPs. Depletion of either Shq1p or Naf1p leads to a dramatic and specific decrease in box H/ACA snoRNA levels in vivo. A severe concomitant defect in ribosomal RNA processing is observed, consistent with the depletion of this family of snoRNAs. Shq1p and Naf1p show nuclear localization and interact with Nhp2p and Cbf5p, two core proteins of mature box H/ACA snoRNPs. Shq1p and Naf1p form a complex, but they are not strongly associated with box H/ACA snoRNPs. We propose that Shq1p and Naf1p are involved in the early biogenesis steps of box H/ACA snoRNP assembly.