Saccharomyces cerevisiae Gle2/Rae1 is involved in septin organization, essential for cell cycle progression.

Gle2/Rae1 is highly conserved from yeast to humans and has been described as an mRNA export factor. Additionally, it is implicated in the anaphase-promoting complex-mediated cell cycle regulation in higher eukaryotes. Here we identify an involvement for Saccharomyces cerevisiae Gle2 in septin organization, which is crucial for cell cycle progression and cell division. Gle2 genetically and physically interacts with components of the septin ring. Importantly, deletion of GLE2 leads to elongated buds, severe defects in septin-assembly and their cellular mislocalization. Septin-ring formation is triggered by the septin-regulating GTPase Cdc42, which establishes and maintains cell polarity. Additionally, activity of the master cell cycle regulator Cdc28 (Cdk1) is needed, which is, besides other functions, also required for G2 /M-transition, and in yeast particularly responsible for initiating the apical-isotropic switch. We show genetic and physical interactions of Gle2 with both Cdc42 and Cdc28. Most importantly, we find that gle2∆ severely mislocalizes Cdc42, leading to defects in septin-complex formation and cell division. Thus, our findings suggest that Gle2 participates in the efficient organization of the septin assembly network, where it might act as a scaffold protein. © 2017 The Authors. Yeast published by John Wiley & Sons, Ltd.

A Robust and Versatile Method of Combinatorial Chemical Synthesis of Gene Libraries via Hierarchical Assembly of Partially Randomized Modules.

A major challenge in gene library generation is to guarantee a large functional size and diversity that significantly increases the chances of selecting different functional protein variants. The use of trinucleotides mixtures for controlled randomization results in superior library diversity and offers the ability to specify the type and distribution of the amino acids at each position. Here we describe the generation of a high diversity gene library using tHisF of the hyperthermophile Thermotoga maritima as a scaffold. Combining various rational criteria with contingency, we targeted 26 selected codons of the thisF gene sequence for randomization at a controlled level. We have developed a novel method of creating full-length gene libraries by combinatorial assembly of smaller sub-libraries. Full-length libraries of high diversity can easily be assembled on demand from smaller and much less diverse sub-libraries, which circumvent the notoriously troublesome long-term archivation and repeated proliferation of high diversity ensembles of phages or plasmids. We developed a generally applicable software tool for sequence analysis of mutated gene sequences that provides efficient assistance for analysis of library diversity. Finally, practical utility of the library was demonstrated in principle by assessment of the conformational stability of library members and isolating protein variants with HisF activity from it. Our approach integrates a number of features of nucleic acids synthetic chemistry, biochemistry and molecular genetics to a coherent, flexible and robust method of combinatorial gene synthesis.

Budding yeast Mph1 promotes sister chromatid interactions by a mechanism involving strand invasion.

Stalling of replication forks at lesions is a serious threat to genomic integrity and cell viability. Cells have developed a variety of pathways that allow continuation of synthesis, including translesion synthesis, postreplication repair and homologous recombination. We have devised a sensitive genetic system for detection of sister chromatid interactions in Saccharomyces cerevisiae. A 266bp sequence duplication in the KanMX4 module was generated and reversions were scored via G418 resistant colonies. Both 4-NQO induced and spontaneous reversions are strictly dependent on RAD52. Damage-induced reversions are also largely dependent on RAD51. Thus, most damage-induced events require a strand invasion step. Induced reversions were not affected in rev3 mutants and partially reduced in rad30 mutants indicating an involvement of Pol η. In cells lacking Mph1, a member of the FANCM family of DNA helicases, that has been implicated in a pathway for fork reactivation involving homologous recombination, damage-induced events are significantly reduced. Together with the spontaneous mutator phenotype of mph1 mutants this data strongly suggest that Mph1 has an additional function in recombination besides its previously described ability to disrupt D-loops. We propose that Mph1 promotes D-loop formation.

Helix-hairpin-helix protein MJ1434 from Methanocaldococcus jannaschii and EndoIV homologue TTC0482 from Thermus thermophilus HB27 do not process DNA uracil residues.

The mutagenic threat of hydrolytic DNA cytosine deamination is met mostly by uracil DNA glycosylases (UDG) initiating base excision repair. However, several sequenced genomes of archaeal organisms are devoid of genes coding for homologues of the otherwise ubiquitous UDG superfamily of proteins. Previously, two possible solutions to this problem were offered by (i) a report of a newly discovered family of uracil DNA glycosylases exemplified by MJ1434, a protein found in the hyperthermophilic archaeon Methanocaldococcus jannaschii, and (ii) the description of TTC0482, an EndoIV homologue from the hyperthermophilic bacterium Thermus thermophilus HB27, as being able to excise uracil from DNA. Sequence homologues of both proteins can be found throughout the archaeal domain of life. Three proteins orthologous to MJ1434 and the family founder itself were tested for but failed to exhibit DNA uracil glycosylase activity when produced in an Ung-deficient Escherichia coli host. Likewise, no DNA uracil processing activity could be detected to be associated with TTC0482, while the protein was fully active as an AP endonuclease. We propose that the uracil processing activities formerly found were due to contaminations with Ung enzyme. Use of Deltaung-strains as hosts for production of putatively DNA-U processing enzymes provides a simple safeguard.

Archaeal DNA uracil repair via direct strand incision: A minimal system reconstituted from purified components.

Hydrolytic deamination of DNA cytosine residues results in U/G mispairs, pre-mutagenic lesions threatening long-term genetic stability. Hence, DNA uracil repair is ubiquitous throughout all extant life forms and base excision repair, triggered by a uracil DNA glycosylase (UDG), is the mechanistic paradigm adopted, as it seems, by all bacteria and eukaryotes and a large fraction of archaea. However, members of the UDG superfamily of enzymes are absent from the extremely thermophilic archaeon Methanothermobacter thermautotrophicus DeltaH. This organism, as a hitherto unique case, initiates repair by direct strand incision next to the DNA-U residue, a reaction catalyzed by the DNA uridine endonuclease Mth212, an ExoIII homologue. To elucidate the detailed mechanism, in particular to identify the molecular partners contributing to this repair process, we reconstituted DNA uracil repair in vitro from only four purified enzymes of M. thermautotrophicus DeltaH. After incision at the 5'-side of a 2'-d-uridine residue by Mth212 DNA polymerase B (mthPolB) is able to take over the 3'-OH terminus and carry out repair synthesis generating a 5'-flap structure that is resolved by mthFEN, a 5'-flap endonuclease. Finally, DNA ligase seals the resulting nick. This defines mechanism and minimal enzymatic requirements of DNA-U repair in this organism.

Genetic evidence for a role of Saccharomyces cerevisiae Mph1 in recombinational DNA repair under replicative stress.

In yeast as in human, DNA helicases play critical roles in assisting replication fork progression. The Saccharomyces cerevisiae MPH1 gene, homologue of human FANCM, has been involved in homologous recombination and DNA repair. We describe a synthetic growth defect of an mph1 deletion if combined with an srs2 deletion that can result-depending on the genetic background-in synthetic lethality. The lethality is suppressed by mutations in homologous recombination (rad51, rad52, rad55, rad57) and in the DNA damage checkpoint (rad9, rad24, rad17). Importantly, rad54 and mph1, epistatic for damage sensitivity, are subadditive for spontaneous mutator phenotype. Therefore, Mph1 could be placed at the Rad51-mediated strand invasion process, with a function distinct from Rad54. Moreover, siz1 mutation is viable with mph1 and additive for DNA damage sensitivity. mph1 srs2 double mutants, isolated in a background where they are viable, are synergistically sensitive to DNA damage. Moderate overexpression of SGS1 partially suppresses this sensitivity. Finally, we observe an epistatic relationship in terms of sensitivity to camptothecin of mms4 or mus81 to mph1. Overall, our results support a role of Mph1 in assisting replication progression. We propose two models for the resumption of DNA synthesis under replicative stress where Mph1 is placed at the sister chromatid interaction step.

A randomized comparison of transradial versus transfemoral approach for coronary angiography and angioplasty.

OBJECTIVES: The aim of the study was to evaluate the safety, feasibility, and procedural variables by the transradial approach compared with the transfemoral access in a standard population of patients undergoing coronary catheterization. BACKGROUND: Coronary catheterization is usually performed via the transfemoral approach. Transradial access may offer some advantages in comparison with transfemoral access especially under conditions of aggressive anticoagulation and antiplatelet treatment. METHODS: Between July 2006 and January 2008, a total of 1,024 patients undergoing coronary catheterization were randomly assigned to the transradial or transfemoral approach. Patients with an abnormal Allen's test, history of coronary artery bypass surgery, simultaneous right heart catheterization, chronic renal insufficiency, or known difficulties with the radial or femoral access were excluded. RESULTS: Successful catheterization was achieved in 494 of 512 patients (96.5%) in the transradial and in 511 of 512 patients (99.8%) in the transfemoral group (p < 0.0001). Median procedural duration (37.0 min, interquartile range [IQR] 19.6 to 49.1 min vs. 40.2 min, IQR 24.3 to 50.8 min; p = 0.046) and median dose area product (38.2 Gycm(2), IQR 20.4 to 48.5 Gycm(2) vs. 41.9 Gycm(2), IQR 22.6 to 52.2 Gycm(2); p = 0.034) were significantly lower in the transfemoral group compared with the transradial access group. A median amount of contrast agent was similar among both groups. Vascular access site complications were higher in the transfemoral group (3.71%) than in the transradial group (0.58%; p = 0.0008) CONCLUSIONS: The findings of the present study show that transradial coronary angiography and angioplasty are safe, feasible, and effective with similar results to those of the transfemoral approach. However, procedural duration and radiation exposure are higher using the transradial access. In contrast to the transfemoral route, the rate of major vascular complications was negligible using the transradial approach.

DNA uracil repair initiated by the archaeal ExoIII homologue Mth212 via direct strand incision.

No genes for any of the known uracil DNA glycosylases of the UDG superfamily are present in the genome of Methanothermobacter thermautotrophicus DeltaH, making it difficult to imagine how DNA-U repair might be initiated in this organism. Recently, Mth212, the ExoIII homologue of M. thermautotrophicus DeltaH has been characterized as a DNA uridine endonuclease, which suggested the possibility of a novel endonucleolytic entry mechanism for DNA uracil repair. With no system of genetic experimentation available, the problem was approached biochemically. Assays of DNA uracil repair in vitro, promoted by crude cellular extracts, provide unequivocal confirmation that this mechanism does indeed operate in M. thermautotrophicus DeltaH.

Yeast Mph1 helicase dissociates Rad51-made D-loops: implications for crossover control in mitotic recombination.

Eukaryotes possess mechanisms to limit crossing over during homologous recombination, thus avoiding possible chromosomal rearrangements. We show here that budding yeast Mph1, an ortholog of human FancM helicase, utilizes its helicase activity to suppress spontaneous unequal sister chromatid exchanges and DNA double-strand break-induced chromosome crossovers. Since the efficiency and kinetics of break repair are unaffected, Mph1 appears to channel repair intermediates into a noncrossover pathway. Importantly, Mph1 works independently of two other helicases-Srs2 and Sgs1-that also attenuate crossing over. By chromatin immunoprecipitation, we find targeting of Mph1 to double-strand breaks in cells. Purified Mph1 binds D-loop structures and is particularly adept at unwinding these structures. Importantly, Mph1, but not a helicase-defective variant, dissociates Rad51-made D-loops. Overall, the results from our analyses suggest a new role of Mph1 in promoting the noncrossover repair of DNA double-strand breaks.

[Superior vena cava syndrome by cardiac tumor]. / Sekundäres kardiales Non-Hodgkin-Lymphom als Differentialdiagnose des Vena-cava-superior-Syndroms.

CASE REPORT: A 59-year-old man with a 4-week history of dyspnea, coughing, and chest discomfort was referred to hospital for further evaluation. Moreover, he reported fever and fatigue. There were neither cardiovascular risk factors nor drug medication. 6 months earlier, a deep vein thrombosis of his left lower limb was diagnosed followed by an anticoagulation for 4 weeks. Physical examination revealed a dilatation of the neck veins with a present Kussmaul sign and a diastolic murmur at the left lower sternal border. The findings on the rest of his physical examination were unremarkable. Electrocardiography showed normal sinus rhythm, low voltage, and anterolateral T wave inversion. Initial laboratory results were remarkable for elevated lactate dehydrogenase level. Transthoracic echocardiography revealed a small pericardial effusion with a large intracardiac mass adjacent to the right atrium. Thoracic computed tomography confirmed the tumor mass and showed enlargement of mediastinal lymph nodes. The patient underwent transesophageal echocardiography-guided transvenous biopsy of the tumor. The immunohistology of the specimen revealed non-Hodgkin's lymphoma. The patient subsequently received a chemotherapy with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisolone. His clinical response after the first cycle was remarkable with total regression of the superior vena cava syndrome. After the third cycle of therapy, both tumor and pericardial effusion had disappeared. CONCLUSION: A cardiac tumor is a rare cause of a superior vena cava syndrome. Tissue biopsy is warranted to guide diagnosis and therapy. Transvenous biopsy is generally safe when guided by echocardiography.

Incidence of coronary artery disease and necessity of revascularization in symptomatic patients requiring permanent pacemaker implantation.

BACKGROUND AND PURPOSE: The cause of severe cardiac conduction disturbances or sinus node dysfunction is often unknown. The objective of this study was to assess the incidence of coronary artery disease (CAD) and the necessity of revascularization in patients with symptomatic bradycardia requiring permanent pacemaker implantation and to try to find a causal association between the need for a pacemaker and the presence of CAD. PATIENTS AND METHODS: From January 2002 to December 2005, 507 pacemakers were implanted due to symptomatic bradycardia. In the presence of at least one atherosclerotic risk factor, patients were recommended to undergo coronary angiography. Each patient underwent exercise or dobutamine echocardiography to detect myocardial ischemia. RESULTS: 212 of the 507 patients (42%) with permanent pacemaker implantation (141 men, mean age 70 +/- 9 years) underwent coronary angiography within 2 months before or after pacemaker implantation. Twelve patients (6%) had a normal angiogram. No significant CAD was found in 37 patients (17%), and significant narrowing of the coronary arteries in 150 (71%). Conservative treatment was recommended in 128 patients (60%), 40 patients (19%) were treated with percutaneous coronary intervention, and 44 patients (21%) underwent coronary artery bypass grafting. CONCLUSION: The data indicate that patients with severe conduction disturbances or sinus node dysfunction requiring permanent pacemaker implantation are more likely to have CAD with subsequent myocardial revascularization in the presence of at least one atherosclerotic risk factor. A causal association between the need for pacemaker and CAD could not be established from the results.

[Coincidence of coronary artery disease and takotsubo cardiomyopathy in a 72-year-old female patient]. / Koinzidenz von koronarer Herzkrankheit und Takotsubo-Kardiomyopathie bei einer 72-jährigen Patientin.

BACKGROUND: Takotsubo cardiomyopathy is characterized by transient left ventricular dysfunction in patients with normal findings on coronary angiography. The simultaneous incidence of coronary vessel disease and takotsubo cardiomyopathy is described. CASE REPORT: A 72-year-old, previously healthy female patient reported about chest pain. Acute emotional and physical stress were denied. Cardiac enzymes and electrocardiogram presented typical findings of myocardial infarction. Coronary angiography revealed a coronary one-vessel disease with occlusion of the second branch of the circumflex branch of the left coronary artery which did not explain the severe reduction of left ventricular function. After 4 months, left ventricular function and electrocardiogram had returned to normal. CONCLUSION: Coronary artery disease does not rule out takotsubo cardiomyopathy.

Aortic valve stenosis due to alkaptonuria.

Cardiovascular disease is a less-well appreciated aspect of alkaptonuria. A 69-year-old man presented with shortness of breath and exertional chest pain. He had a previous diagnosis of alkaptonuria (endogenous ochronosis), confirmed on the basis of urine coloration, skin pigmentation and ochronotic arthropathy in the knees. Echocardiography and coronary angiography revealed severe aortic valve stenosis and concomitant coronary artery disease. The patient underwent biological aortic valve replacement (AVR) and coronary artery bypass grafting (CABG). Operative findings included ochronosis of a severely calcified aortic valve and the aortic intima, and bioprosthetic AVR and CABG were successfully performed.

The late open infarct-related artery hypothesis: evidence-based medicine or not?

Randomized clinical trials have clearly shown that early reperfusion of coronary arteries is the established treatment of myocardial infarction preserving left ventricular function and reducing mortality. However, late patency of the infarct-related artery is an independent predictor of survival leading to the late open-artery hypothesis. This concept implies restoration of antegrade blood flow of the infarct-related artery in patients with myocardial infarction to improve survival by mechanisms less time-dependent or even time-independent. Possible explanations for this benefit include improved left ventricular function and electrical stability by perfusion of hibernating myocardium, accelerated infarct healing and limitation of ventricular remodeling. This review focuses on the evidence of late recanalization of occluded infarct-related arteries in patients with coronary artery disease.

Inferior vena cava approach to permanent pacemaker implantation.

A 89-year-old woman required permanent pacemaker implantation because of symptomatic bradyarrhythmia with multiple falls and repeated fractures. Because of the obstruction of the thoracic veins and infection of both groins, an alternative approach via directly punctured inferior vena cava was performed. At follow-up, the patient remained well with an excellent symptomatic response to pacing. The method seems simple to perform and is an alternative when the usual pectoral implantation site is inaccessible.

Antiplatelet therapy early after bioprosthetic aortic valve replacement is unnecessary in patients without thromboembolic risk factors.

BACKGROUND: The use of antithrombotic therapy during the postoperative period after biological aortic valve replacement (AVR) in patients without thromboembolic risk factors remains controversial. Treatment with warfarin is recommended for the first 3 months after biological AVR. The use of antiplatelet therapy - mainly aspirin (ASA) - is suggested as an alternative treatment but its efficacy is still unsettled. Due to the increased risk of bleeding complications even no use of any antithrombotic or antiplatelet therapy was advocated. Given this ongoing dispute, the aim of this retrospective double-institutional study was to evaluate the necessity of antiplatelet treatment by ASA with no postoperative antiplatelet therapy in terms of survival, major bleedings and cerebral thromboembolism of patients undergoing biological AVR without thromboembolic risk factors. METHODS: From January 2001 to December 2003, 288 consecutive patients (72.8+/-7.5 years, 134 males) with sinus rhythm and no other thromboembolic risk factors underwent single biological AVR with porcine or bovine pericardial valves without concurrent coronary artery bypass graft surgery. By surgeons preference, 100 mg ASA was given to 132 patients, and 156 patients received no antiplatelet therapy. Patients were followed for cerebral ischemic events, major bleedings, need for repeat operation, NYHA class and survival at three time intervals postoperatively (30 days, 3 and 12 months). RESULTS: None of all patients died during the operation. Mortality within 30 days was 3.8% in the ASA and 3.9% in the no ASA group (p=0.777). There were no statistically significant differences for cerebral ischemia within 3 months after AVR (ASA 0.8% vs no ASA 1.3%: p=0.884) and 3 to 12 months after AVR (ASA 0.8% vs no ASA 0%; p=0.933). Major bleedings occurred in two ASA-treated patients and in one patient without antiplatelet therapy (p=0.884). The incidence of NYHA class III-IV after 3 months (1.5% vs 1.9%; p=0.850) and 12 months (9.0% vs 5.1%; p=0.278) were similar, as were the need for repeat operative AVR after 12 months (0.8% vs 0.6%; p=0.553). Survival rates at 12-month follow-up were 95.5% for ASA treatment and 94.9% for no ASA treatment (p=0.963). CONCLUSIONS: In patients without thromboembolic risk factors undergoing biological AVR administration of ASA confers no advantage compared to no antiplatelet therapy. Functional status, thromboembolic events and survival were not adversely affected by withholding any antiplatelet therapy. Guidelines need to be reviewed for the antithrombotic therapy of patients without risk factors undergoing bioprosthetic AVR.

Hybrid treatment for complex aortic problems combining surgery and stenting in the integrated operating theater.

OBJECTIVES: Conventional surgical treatment of complex aortic pathologies involving several thoracoabdominal aortic segments necessitates extended incisions or subsequent surgeries, resulting in significant mortality and morbidity rates. The combination of surgery and simultaneous stenting in the operating theater may reduce the surgical trauma. METHODS: A total of nine patients (62 +/- 10 years, range 44-70) underwent a combined surgical and endovascular treatment of thoracic or thoracoabdominal aortic aneurysms or chronic dissection. Five patients were treated with viscero-renal artery translocation followed by transfemoral stenting of the entire thoracoabdominal aorta. Two patients underwent debranching of the supraaortic vessels followed by immediate transfemoral stenting of the aortic arch, and two patients with a history of an ascending aortic aneurysm repair were treated with open surgical debranching of the supraaortic trunks and repair of the ascending aorta and aortic arch with elephant trunk technique. Preoperatively, magnetic resonance imaging was used to check supraaortic and intracranial vessels as well as the completeness of the Circle of Willisi prior to arch stenting and/or supraaortic vessel surgery. Cerebrospinal fluid drainage and induced mild hypertension have been used for one-step thoracoabdominal aortic stenting. RESULTS: Thirty-day mortality rate and incidence of paraplegia was 0%. There was a single reversible perioperative stroke after aortic arch stenting. One patient required temporary renal replacement therapy using continuous arterio-venous hemofiltration. There was one early reoperation at the superior mesenteric artery after viscero-renal translocation. Four type I endoleaks occurred in three patients requiring two interventions. All patients have been discharged to home. CONCLUSION: The innovative combination of simultaneous conventional surgery and stenting reduces the operative burden for patients with complex aortic pathologies involving several segments of the thoracic and thoracoabdominal aorta. Arch debranching and viscero-renal artery translocation may avoid the use of thoracoabdominal incisions, cardiopulmonary bypass techniques, deep hypothermia, and circulatory arrest.

The Methanothermobacter thermautotrophicus ExoIII homologue Mth212 is a DNA uridine endonuclease.

The genome of Methanothermobacter thermautotrophicus, as a hitherto unique case, is apparently devoid of genes coding for general uracil DNA glycosylases, the universal mediators of base excision repair following hydrolytic deamination of DNA cytosine residues. We have now identified protein Mth212, a member of the ExoIII family of nucleases, as a possible initiator of DNA uracil repair in this organism. This enzyme, in addition to bearing all the enzymological hallmarks of an ExoIII homologue, is a DNA uridine endonuclease (U-endo) that nicks double-stranded DNA at the 5'-side of a 2'-d-uridine residue, irrespective of the nature of the opposing nucleotide. This type of activity has not been described before; it is absent from the ExoIII homologues of Escherichia coli, Homo sapiens and Methanosarcina mazei, all of which are equipped with uracil DNA repair glycosylases. The U-endo activity of Mth212 is served by the same catalytic center as its AP-endo activity.