Timing of Chromosome DNA Integration throughout the Yeast Cell Cycle.

The dynamic mechanism of cell uptake and genomic integration of exogenous linear DNA still has to be completely clarified, especially within each phase of the cell cycle. We present a study of integration events of double-stranded linear DNA molecules harboring at their ends sequence homologies to the host's genome, all throughout the cell cycle of the model organism Saccharomyces cerevisiae, comparing the efficiency of chromosomal integration of two types of DNA cassettes tailored for site-specific integration and bridge-induced translocation. Transformability increases in S phase regardless of the sequence homologies, while the efficiency of chromosomal integration during a specific cycle phase depends upon the genomic targets. Moreover, the frequency of a specific translocation between chromosomes XV and VIII strongly increased during DNA synthesis under the control of Pol32 polymerase. Finally, in the null POL32 double mutant, different pathways drove the integration in the various phases of the cell cycle and bridge-induced translocation was possible outside the S phase even without Pol32. The discovery of this cell-cycle dependent regulation of specific pathways of DNA integration, associated with an increase of ROS levels following translocation events, is a further demonstration of a sensing ability of the yeast cell in determining a cell-cycle-related choice of DNA repair pathways under stress.

Acute Budd-Chiari Syndrome with Complete Portal Vein Thrombosis Complicated by Hepato-Renal Syndrome Treated Successfully by Emergent TIPS with Rheolytic Thrombectomy.

We described a case of a 73-year-old female admitted to the emergency department with acute hepatic and renal failure (hepato-renal syndrome, HRS) due to acute Budd-Chiari syndrome associated with complete portal vein thrombosis (BCS-PVT) for an unknown cause. Despite the initial therapy with anticoagulants, a sudden impairment of the renal function requiring hemodialysis was observed. The hepatic transplant was excluded for patient age and clinical conditions. Thus, the patient was successfully treated by emergent transjugular intrahepatic portosystemic shunt (TIPS) previous rheolytic thrombectomy of the PVT with AngioJet Ultra PE Thrombectomy System (Boston Scientific, Marlborough, MA, USA). After the procedure, the sudden resolution of the HRS was observed, and the patient is alive 13 months after hospital dismission with no TIPS dysfunction. In conclusion, emergent extended TIPS with the usage of rheolytic thrombectomy device in patient with acute BCS-PVT complicated by HRS is feasible by experienced operators and provide resolution of the HRS.

Computed Tomography Texture Analysis of Carotid Plaque as Predictor of Unfavorable Outcome after Carotid Artery Stenting: A Preliminary Study.

Novel biomarkers are advocated to manage carotid plaques. Therefore, we aimed to test the association between textural features of carotid plaque at computed tomography angiography (CTA) and unfavorable outcome after carotid artery stenting (CAS). Between January 2010 and January 2021, were selected 172 patients (median age, 77 years; 112/172, 65% men) who underwent CAS with CTA of the supra-aortic vessels performed within prior 6 months. Standard descriptors of the density histogram were derived by open-source software automated analysis obtained by CTA plaque segmentation. Multiple logistic regression analysis, receiver operating characteristic (ROC) curve analysis and the area under the ROC (AUC) were used to identify potential prognostic variables and to assess the model performance for predicting unfavorable outcome (periprocedural death or myocardial infarction and any ipsilateral acute neurological event). Unfavorable outcome occurred in 17/172 (10%) patients (median age, 79 years; 12/17, 70% men). Kurtosis was an independent predictor of unfavorable outcome (odds ratio, 0.79; confidence interval, 0.65-0.97; p = 0.029). The predictive model for unfavorable outcome including CTA textural features outperformed the model without textural features (AUC 0.789 vs. 0.695, p = 0.004). In patients with stenotic carotid plaque, kurtosis derived by CTA density histogram analysis is an independent predictor of unfavorable outcome after CAS.

Diverse human VH antibody fragments with bio-therapeutic properties from the Crescendo Mouse.

We describe the 'Crescendo Mouse', a human VH transgenic platform combining an engineered heavy chain locus with diverse human heavy chain V, D and J genes, a modified mouse Cγ1 gene and complete 3' regulatory region, in a triple knock-out (TKO) mouse background devoid of endogenous immunoglobulin expression. The addition of the engineered heavy chain locus to the TKO mouse restored B cell development, giving rise to functional B cells that responded to immunization with a diverse response that comprised entirely 'heavy chain only' antibodies. Heavy chain variable (VH) domain libraries were rapidly mined using phage display technology, yielding diverse high-affinity human VH that had undergone somatic hypermutation, lacked aggregation and showed enhanced expression in E. coli. The Crescendo Mouse produces human VH fragments, or Humabody® VH, with excellent bio-therapeutic potential, as exemplified here by the generation of antagonistic Humabody® VH specific for human IL17A and IL17RA.

Extracranial Visceral Artery Aneurysms/Pseudoaneurysms Repaired with Flow Diverter Device Developed for Cerebral Aneurysms: Preliminary Results.

BACKGROUND: Recently, there has been a shift toward elective endovascular repair of visceral artery aneurysms (VAAs). Transcatheter embolization (TE) and covered stenting (CS) represent the 2 most used endovascular techniques; however, TE carries the potential risk of end-organ ischemia, while CS is challenging when the parent arteries are tortuous. Flow diverter devices (FDDs) developed for cerebral aneurysms maintain distal flow and are characterized by high navigability in tortuous arteries. This report describes our initial experience in using FDD developed for cerebral aneurysms to treat extracranial VAAs/pseudoaneurysm (VAP). METHODS: The study was conducted on patients affected by VAP, who underwent endovascular repair using FDD, between January 2015 and April 2017. All patients underwent preinterventional computed tomography angiography (CTA) for diagnosis and procedural planning. VAP features (type, location, size) and the diameter of both the proximal and distal parent arteries were recorded. Since TE or CS was contraindicated or failed in the previous attempt, VAPs were repaired through an elective endovascular procedure with FDD (Surpass; Stryker Neurovascular, Fremont, CA). Follow-up CTAs were performed within 6 months and at 24 months after the endovascular repair, evaluating patency and proper position of the FDD, the maximum diameter of the VAP, any perfusion of the sac, and adequacy of end-organ perfusion. RESULTS: Four VAPs were repaired by FDD in 4 patients (2 females; median age: 72 years, range: 64-80 years). One patient suffered from cervical arterial anastomotic pseudoaneurysm, whereas the remaining VAPs were 2 splenic artery aneurysms and 1 common hepatic aneurysm. VAPs median size was 20 mm (range: 13-26 mm) with median parent artery caliber of 5 mm (range: 3-5 mm). The correct deployment of the device was obtained in all cases; 2/4 VAPs showed endoleak at the end of the procedure. At follow-up CTAs performed after the procedure in a median time of 25 months (range: 4-28 months), all devices were patent and not migrated. All VAPs showed shrinkage of the sac without endoleak or signs of end-organ ischemia. CONCLUSIONS: When high tortuosity and small caliber of the parent arteries prevent CS and the necessity to maintain vessel patency contraindicates TE, FDD could represent an option for the treatment of VAP; however, high costs and the off-label use in extracranial vessels demand an accurate selection of the patients suitable for the VAP treatment with FDD.

Interaction of Yna1 and Yna2 Is Required for Nuclear Accumulation and Transcriptional Activation of the Nitrate Assimilation Pathway in the Yeast Hansenula polymorpha.

A few yeasts, including Hansenula polymorpha are able to assimilate nitrate and use it as nitrogen source. The genes necessary for nitrate assimilation are organised in this organism as a cluster comprising those encoding nitrate reductase (YNR1), nitrite reductase (YNI1), a high affinity transporter (YNT1), as well as the two pathway specific Zn(II)2Cys2 transcriptional activators (YNA1, YNA2). Yna1p and Yna2p mediate induction of the system and here we show that their functions are interdependent. Yna1p activates YNA2 as well as its own (YNA1) transcription thus forming a nitrate-dependent autoactivation loop. Using a split-YFP approach we demonstrate here that Yna1p and Yna2p form a heterodimer independently of the inducer and despite both Yna1p and Yna2p can occupy the target promoter as mono- or homodimer individually, these proteins are transcriptionally incompetent. Subsequently, the transcription factors target genes containing a conserved DNA motif (termed nitrate-UAS) determined in this work by in vitro and in vivo protein-DNA interaction studies. These events lead to a rearrangement of the chromatin landscape on the target promoters and are associated with the onset of transcription of these target genes. In contrast to other fungi and plants, in which nuclear accumulation of the pathway-specific transcription factors only occur in the presence of nitrate, Yna1p and Yna2p are constitutively nuclear in H. polymorpha. Yna2p is needed for this nuclear accumulation and Yna1p is incapable of strictly positioning in the nucleus without Yna2p. In vivo DNA footprinting and ChIP analyses revealed that the permanently nuclear Yna1p/Yna2p heterodimer only binds to the nitrate-UAS when the inducer is present. The nitrate-dependent up-regulation of one partner protein in the heterodimeric complex is functionally similar to the nitrate-dependent activation of nuclear accumulation in other systems.

Different aneuploidies arise from the same bridge-induced chromosomal translocation event in Saccharomyces cerevisiae.

Chromosome translocations are gross chromosomal rearrangements that have often been associated with cancer development in mammalian cells. The feasibility of drastically reshaping the genome with a single translocation event also gives this molecular event a powerful capacity to drive evolution. Despite these implications and their role in genome instability, very little is known about the molecular mechanisms that promote and accompany these events. Here, at the molecular level, we describe 10 morphologically and physiologically different translocants ensuing from the induction of the same bridge-induced translocation (BIT) event in the budding yeast Saccharomyces cerevisiae. We have demonstrated that, despite their common origin from the integration of the same linear DNA construct, all 10 translocation mutant strains have different phenotypes and the ability to sporulate and regulate gene expression and morphology. We also provide insights into how heterogeneous phenotypic variations originate from the same initial genomic event. Here we show eight different ways in which yeast cells have dealt with a single initial event inducing translocation. Our results are in agreement with the formation of complex rearrangements and abnormal karyotypes described in many leukemia patients, thus confirming the modellistic value of the yeast BIT system for mammalian cells.

Comparison of the antibacterial efficacy and acceptability of an alcohol-based hand rinse with two alcohol-based hand gels during routine patient care.

The aims of this study were to compare the antibacterial efficacy of handrubbing with an alcoholic rinse (AHRR) and two different alcoholic gels (AHRG) in reducing hand contamination under practical use conditions. We wanted to assess the acceptability of the three products and to determine the effect of each product on overall hand hygiene compliance. A prospective alternating time-series clinical trial was performed in a medical intensive care unit. The study was divided into three six-week periods (P1, P2, P3). Handrubbing was achieved with Sterillium rinse (AHRR) during P1, sterillium gel(AHRG-1) during P2 and Manugel Plus (AHRG-2) during P3. Pre- and post-rubbing hand contaminations were assessed immediately after a direct contact with a patient, using the glove juice technique. Health care workers (HCWs) evaluated the acceptability of the products through a self-administered anonymous questionnaire. Compliance of HCWs with hand hygiene was assessed during the three periods. We studied 242 handrubbing opportunities. The mean reduction factor (expressed as the Log(10) CFU/mL) of the AHRR, AHRG-1 and AHRG-2 were 1.28+/-0.95, 1.29+/-0.84 and 0.51+/-0.73, respectively (p<0.001). Assessment of the three products by HCWs indicated that AHRR and AHRG-1 were significantly better accepted than AHRG-2. The overall compliance of HCWs to hand hygiene was better when gel was available. Under practical use conditions, AHRG-1 and AHRR were more effective than AHRG-2, although all were claimed to pass the European standard EN1500. In vivo trials are essential to compare the antimicrobial efficacy of products for handrubbing.

Hansenula polymorpha NMR2 and NMR4, two new loci involved in nitrogen metabolite repression.

In the yeast Hansenula polymorpha (Pichia angusta) nitrate assimilation is tightly regulated and subject to a dual control: nitrogen metabolite repression (NMR), triggered by reduced nitrogen compounds, and induction, elicited by nitrate itself. In a previous paper [Serrani, F., Rossi, B. and Berardi, E (2001) Nitrogen metabolite repression in Hansenula polymorpha: the nmrl-l mutation. Curr. Genet. 40, 243-250], we identified five loci (NMR1-NMR5) involved in NMR, and characterised one of them (NMR1), which likely identifies a regulatory factor. Here, we describe two more mutants, namely nmr2-1 and nmr4-1. The first one possibly identifies a regulatory factor involved in nitrogen metabolite repression by various nitrogen sources alternative to ammonium. The second one, apparently involved in ammonium assimilation, probably has sensor functions.