Sen1 architecture: RNA-DNA hybrid resolution, autoregulation, and insights into SETX inactivation in AOA2.

The senataxin (SETX, Sen1 in yeasts) RNA-DNA hybrid resolving helicase regulates multiple nuclear transactions, including DNA replication, transcription, and DNA repair, but the molecular basis for Sen1 activities is ill defined. Here, Sen1 cryoelectron microscopy (cryo-EM) reconstructions reveal an elongated inchworm-like architecture. Sen1 is composed of an amino terminal helical repeat Sen1 N-terminal (Sen1N) regulatory domain that is flexibly linked to its C-terminal SF1B helicase motor core (Sen1Hel) via an intrinsically disordered tether. In an autoinhibited state, the Sen1Sen1N domain regulates substrate engagement by promoting occlusion of the RNA substrate-binding cleft. The X-ray structure of an activated Sen1Hel engaging single-stranded RNA and ADP-SO4 shows that the enzyme encircles RNA and implicates a single-nucleotide power stroke in the Sen1 RNA translocation mechanism. Together, our data unveil dynamic protein-protein and protein-RNA interfaces underpinning helicase regulation and inactivation of human SETX activity by RNA-binding-deficient mutants in ataxia with oculomotor apraxia 2 neurodegenerative disease.

Endogenous DNA 3' Blocks Are Vulnerabilities for BRCA1 and BRCA2 Deficiency and Are Reversed by the APE2 Nuclease.

The APEX2 gene encodes APE2, a nuclease related to APE1, the apurinic/apyrimidinic endonuclease acting in base excision repair. Loss of APE2 is lethal in cells with mutated BRCA1 or BRCA2, making APE2 a prime target for homologous recombination-defective cancers. However, because the function of APE2 in DNA repair is poorly understood, it is unclear why BRCA-deficient cells require APE2 for viability. Here we present the genetic interaction profiles of APE2, APE1, and TDP1 deficiency coupled to biochemical and structural dissection of APE2. We conclude that the main role of APE2 is to reverse blocked 3' DNA ends, problematic lesions that preclude DNA synthesis. Our work also suggests that TOP1 processing of genomic ribonucleotides is the main source of 3'-blocking lesions relevant to APEX2-BRCA1/2 synthetic lethality. The exquisite sensitivity of BRCA-deficient cells to 3' blocks indicates that they represent a tractable vulnerability in homologous recombination-deficient tumor cells.

ForMAX - a beamline for multiscale and multimodal structural characterization of hierarchical materials.

The ForMAX beamline at the MAX IV Laboratory provides multiscale and multimodal structural characterization of hierarchical materials in the nanometre to millimetre range by combining small- and wide-angle X-ray scattering with full-field microtomography. The modular design of the beamline is optimized for easy switching between different experimental modalities. The beamline has a special focus on the development of novel fibrous materials from forest resources, but it is also well suited for studies within, for example, food science and biomedical research.

Ubiquitin stimulated reversal of topoisomerase 2 DNA-protein crosslinks by TDP2.

Tyrosyl-DNA phosphodiesterase 2 (TDP2) reverses Topoisomerase 2 DNA-protein crosslinks (TOP2-DPCs) in a direct-reversal pathway licensed by ZATTZNF451 SUMO2 E3 ligase and SUMOylation of TOP2. TDP2 also binds ubiquitin (Ub), but how Ub regulates TDP2 functions is unknown. Here, we show that TDP2 co-purifies with K63 and K27 poly-Ubiquitinated cellular proteins independently of, and separately from SUMOylated TOP2 complexes. Poly-ubiquitin chains of ≥ Ub3 stimulate TDP2 catalytic activity in nuclear extracts and enhance TDP2 binding of DNA-protein crosslinks in vitro. X-ray crystal structures and small-angle X-ray scattering analysis of TDP2-Ub complexes reveal that the TDP2 UBA domain binds K63-Ub3 in a 1:1 stoichiometric complex that relieves a UBA-regulated autoinhibitory state of TDP2. Our data indicates that that poly-Ub regulates TDP2-catalyzed TOP2-DPC removal, and TDP2 single nucleotide polymorphisms can disrupt the TDP2-Ubiquitin interface.

APE2 Zf-GRF facilitates 3'-5' resection of DNA damage following oxidative stress.

The Xenopus laevis APE2 (apurinic/apyrimidinic endonuclease 2) nuclease participates in 3'-5' nucleolytic resection of oxidative DNA damage and activation of the ATR-Chk1 DNA damage response (DDR) pathway via ill-defined mechanisms. Here we report that APE2 resection activity is regulated by DNA interactions in its Zf-GRF domain, a region sharing high homology with DDR proteins Topoisomerase 3α (TOP3α) and NEIL3 (Nei-like DNA glycosylase 3), as well as transcription and RNA regulatory proteins, such as TTF2 (transcription termination factor 2), TFIIS, and RPB9. Biochemical and NMR results establish the nucleic acid-binding activity of the Zf-GRF domain. Moreover, an APE2 Zf-GRF X-ray structure and small-angle X-ray scattering analyses show that the Zf-GRF fold is typified by a crescent-shaped ssDNA binding claw that is flexibly appended to an APE2 endonuclease/exonuclease/phosphatase (EEP) catalytic core. Structure-guided Zf-GRF mutations impact APE2 DNA binding and 3'-5' exonuclease processing, and also prevent efficient APE2-dependent RPA recruitment to damaged chromatin and activation of the ATR-Chk1 DDR pathway in response to oxidative stress in Xenopus egg extracts. Collectively, our data unveil the APE2 Zf-GRF domain as a nucleic acid interaction module in the regulation of a key single-strand break resection function of APE2, and also reveal topologic similarity of the Zf-GRF to the zinc ribbon domains of TFIIS and RPB9.

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

Mammalian Tyrosyl-DNA phosphodiesterase 2 (Tdp2) reverses Topoisomerase 2 (Top2) DNA-protein crosslinks triggered by Top2 engagement of DNA damage or poisoning by anticancer drugs. Tdp2 deficiencies are linked to neurological disease and cellular sensitivity to Top2 poisons. Herein, we report X-ray crystal structures of ligand-free Tdp2 and Tdp2-DNA complexes with alkylated and abasic DNA that unveil a dynamic Tdp2 active site lid and deep substrate binding trench well-suited for engaging the diverse DNA damage triggers of abortive Top2 reactions. Modeling of a proposed Tdp2 reaction coordinate, combined with mutagenesis and biochemical studies support a single Mg(2+)-ion mechanism assisted by a phosphotyrosyl-arginine cation-π interface. We further identify a Tdp2 active site SNP that ablates Tdp2 Mg(2+) binding and catalytic activity, impairs Tdp2 mediated NHEJ of tyrosine blocked termini, and renders cells sensitive to the anticancer agent etoposide. Collectively, our results provide a structural mechanism for Tdp2 engagement of heterogeneous DNA damage that causes Top2 poisoning, and indicate that evaluation of Tdp2 status may be an important personalized medicine biomarker informing on individual sensitivities to chemotherapeutic Top2 poisons.

Deficiency of terminal ADP-ribose protein glycohydrolase TARG1/C6orf130 in neurodegenerative disease.

Adenosine diphosphate (ADP)-ribosylation is a post-translational protein modification implicated in the regulation of a range of cellular processes. A family of proteins that catalyse ADP-ribosylation reactions are the poly(ADP-ribose) (PAR) polymerases (PARPs). PARPs covalently attach an ADP-ribose nucleotide to target proteins and some PARP family members can subsequently add additional ADP-ribose units to generate a PAR chain. The hydrolysis of PAR chains is catalysed by PAR glycohydrolase (PARG). PARG is unable to cleave the mono(ADP-ribose) unit directly linked to the protein and although the enzymatic activity that catalyses this reaction has been detected in mammalian cell extracts, the protein(s) responsible remain unknown. Here, we report the homozygous mutation of the c6orf130 gene in patients with severe neurodegeneration, and identify C6orf130 as a PARP-interacting protein that removes mono(ADP-ribosyl)ation on glutamate amino acid residues in PARP-modified proteins. X-ray structures and biochemical analysis of C6orf130 suggest a mechanism of catalytic reversal involving a transient C6orf130 lysyl-(ADP-ribose) intermediate. Furthermore, depletion of C6orf130 protein in cells leads to proliferation and DNA repair defects. Collectively, our data suggest that C6orf130 enzymatic activity has a role in the turnover and recycling of protein ADP-ribosylation, and we have implicated the importance of this protein in supporting normal cellular function in humans.

[Strain experience and religiosity. A comparison of patients with chronic pain and breast cancer]. / Subjektive Belastung und Religiosität bei chronischen Schmerzen und Brustkrebs. Ein Stichprobenvergleich.

BACKGROUND: Studies show that especially ill people turn to their religious faith to find help in dealing with their diseases. However, religiousness is assumed to vary in its extent and effect depending on different kinds of strain. MATERIAL AND METHODS: In order to differentiate patterns of strain and coping, a sample of 178 patients with chronic pain was compared with 167 breast cancer patients. RESULTS: Pain patients show higher strain and impairment on almost all variables. Regression analyses indicate that patients with chronic pain are less religious in comparison to the breast cancer patients. CONCLUSIONS: Different values of the religious variables can be explained by different characteristics of the strain: Due to the threat to life experienced by the patients, the breast cancer group is more likely to turn to religiousness for help. Specific characteristics of chronic pain (e.g. longer illness duration, a stronger impairment in everyday activities) lead to higher resignation, also concerning religious efforts.

Virus transfer from surf to wind.

Bubbles in the sea surf adsorb and carry viruses to the surface where they are propelled into the air on tiny jets of seawater when the bubble bursts. The ejected jets become tiny drops of aerosol. The buble adsorption and virus concentration in the surf is analagous to industrial bubble levitation processes that concentrate metallic ores, enzymes, and finely divided organic crystals. Bubble levitation of viruses delibrately injected into the surf produced 200 times more virus per milliliter in the aerosol than were present in samples from the surf. Some aerosol drops created by the surf and carried by the wind fall out on the beach. The frequency of virus-bearing drops, that is, the number of plaques on seeded plates exposed on the beach, decreased exponentially with the distance downwind from the surf.

Structure of the sirtuin-linked macrodomain SAV0325 from Staphylococcus aureus.

Cells use the post-translational modification ADP-ribosylation to control a host of biological activities. In some pathogenic bacteria, an operon-encoded mono-ADP-ribosylation cycle mediates response to host-induced oxidative stress. In this system, reversible mono ADP-ribosylation of a lipoylated target protein represses oxidative stress response. An NAD(+) -dependent sirtuin catalyzes the single ADP-ribose (ADPr) addition, while a linked macrodomain-containing protein removes the ADPr. Here we report the crystal structure of the sitruin-linked macrodomain protein from Staphylococcus aureus, SauMacro (also known as SAV0325) to 1.75-Å resolution. The monomeric SauMacro bears a previously unidentified Zn(2+) -binding site that putatively aids in substrate recognition and catalysis. An amino-terminal three-helix bundle motif unique to this class of macrodomain proteins provides a structural scaffold for the Zn(2+) site. Structural features of the enzyme further indicate a cleft proximal to the Zn(2+) binding site appears well suited for ADPr binding, while a deep hydrophobic channel in the protein core is suitable for binding the lipoate of the lipoylated protein target.

Phase II evaluation of oral estramustine and oral etoposide in hormone-refractory adenocarcinoma of the prostate.

PURPOSE: Estramustine and etoposide (VP-16) have been demonstrated to inhibit the growth of prostate cancer cells in experimental models. This led us to evaluate the effectiveness of this combination in the treatment of patients with metastatic prostate carcinoma refractory to hormone therapy. PATIENTS AND METHODS: Estramustine 15 mg/kg/d and VP-16 50 mg/m2/d, were administered orally in divided doses for 21 days. Patients were then taken off therapy for 7 days and the cycle then repeated. Therapy continued until evidence of disease progression. RESULTS: Forty-two patients have been enrolled onto this trial with a minimum of 40 weeks follow-up. Of 18 patients with measurable soft tissue disease, three demonstrated a complete response (CR) and six a partial response (PR) for longer than 2 months. Of these 18 patients, pretreatment prostate-specific antigen (PSA) levels decreased by at least 75% in five men (28%) and by at least 50% in nine (50%). The median survival duration has not been reached in those patients who demonstrated a response either by soft tissue or PSA criteria. Of 24 patients with disease limited to bone, six (25%) demonstrated improvement and nine (38%) demonstrated stability in their bone scans. Five men (21%) demonstrated a decrease of at least 75% in pretreatment PSA levels and 14 (58%) demonstrated at least a 50% decrease; the median survival duration has not been reached in these patients. Pretreatment performance status is an important predictor of survival. CONCLUSION: We conclude that the combination of estramustine and VP-16 is an active oral regimen in hormone-refractory prostate cancer.

Outpatient biochemotherapy with interleukin-2 and interferon alfa-2b in patients with metastatic malignant melanoma: results of two phase II cytokine working group trials.

PURPOSE: The Cytokine Working Group performed a randomized phase II trial of two outpatient biochemotherapy regimens to identify an outpatient regimen with high antitumor activity and less toxicity than inpatient regimens which might be compared with chemotherapy or inpatient biochemotherapy regimens in future phase III trials. PATIENTS AND METHODS: Eighty-one patients with metastatic malignant melanoma received dacarbazine 250 mg/m(2)/d intravenously (IV) and cisplatin 25 mg/m(2)/d IV on days 1, 2, and 3, plus interferon (IFN) alfa-2b 5 mU/m(2) subcutaneously (SC) on days 6, 8, 10, 13, and 15, given every 28 days. Interleukin-2 (IL-2) was given daily on days 6 to 10 and 13 to 15. In group 1, IV IL-2 was given at 18.0 MU/m(2), and in group 2, SC IL-2 was given at 5.0 mU/m(2). RESULTS: In group 1 (IV IL-2), there were five complete responses (CRs) and 11 partial responses (PRs) among 44 patients (objective response rate [ORR], 36%; 95% confidence interval [CI], 22% to 51%). In group 2 (SC IL-2), there was one CR and five PRs among the 36 patients (ORR, 17%; 95% CI, 4% to 29%). The median survival was 10.7 months in group 1 and 7.3 months in group 2. Eleven patients in group 1 and four patients in group 2 remain alive as of the last follow-up. Toxicities in both groups were similar. No patient required hospitalization for neutropenic fever. CONCLUSION: Biochemotherapy has activity in these outpatient regimens with acceptable toxicity. The antitumor activity observed with the IV IL-2 regimen seems similar to that of inpatient biochemotherapy regimens. If inpatient biochemotherapy regimens develop an established role in the management of melanoma, future phase III trial comparisons with this outpatient IV IL-2 regimen would be appropriate.

Randall Selitto pressure algometry for assessment of bone-related pain in rats.

BACKGROUND: Deep pain is neglected compared with cutaneous sources. Pressure algometry has been validated in the clinic for assessment of bone-related pain in humans. In animal models of bone-related pain, we have validated the Randall Selitto behavioural test for assessment of acute and pathological bone pain and compared the outcome with more traditional pain-related behaviour measures. METHODS: Randall Selitto pressure algometry was performed over the anteromedial part of the tibia in naïve rats, sham-operated rats, and rats inoculated with MRMT-1 carcinoma cells in the left tibia, and the effect of morphine was investigated. Randall Selitto measures of cancer-induced bone pain were supplemented by von Frey testing, weight-bearing and limb use test. Contribution of cutaneous nociception to Randall Selitto measures were examined by local anaesthesia. RESULTS: Randall Selitto pressure algometry over the tibia resulted in reproducible withdrawal thresholds, which were dose-dependently increased by morphine. Cutaneous nociception did not contribute to Randall Selitto measures. In cancer-bearing animals, compared with sham, significant differences in pain-related behaviours were demonstrated by the Randall Selitto test on day 17 and 21 post-surgery. A difference was also demonstrated by von Frey testing, weight-bearing and limb use tests. CONCLUSION: Our results indicate that pressure applied by the Randall Selitto algometer on a region, where the bone is close to the skin, may offer a way to measure bone-related pain in animal models and could provide a supplement to the traditional behavioural tests and a means to study deep pain.

Tetrameric Ctp1 coordinates DNA binding and DNA bridging in DNA double-strand-break repair.

Ctp1 (also known as CtIP or Sae2) collaborates with Mre11-Rad50-Nbs1 to initiate repair of DNA double-strand breaks (DSBs), but its functions remain enigmatic. We report that tetrameric Schizosaccharomyces pombe Ctp1 contains multivalent DNA-binding and DNA-bridging activities. Through structural and biophysical analyses of the Ctp1 tetramer, we define the salient features of Ctp1 architecture: an N-terminal interlocking tetrameric helical dimer-of-dimers (THDD) domain and a central intrinsically disordered region (IDR) linked to C-terminal 'RHR' DNA-interaction motifs. The THDD, IDR and RHR are required for Ctp1 DNA-bridging activity in vitro, and both the THDD and RHR are required for efficient DSB repair in S. pombe. Our results establish non-nucleolytic roles of Ctp1 in binding and coordination of DSB-repair intermediates and suggest that ablation of human CtIP DNA binding by truncating mutations underlie the CtIP-linked Seckel and Jawad syndromes.

Increased risk of antidepressant use in childhood cancer survivors: a Danish population-based cohort study.

AIM: Childhood cancer survivors are at risk of both somatic and mental late effects, but large population-based studies of depression are lacking. METHODS: Risk of antidepressant use was evaluated in a population-based cohort of 5452 Danish children treated for cancer in 1975-2009 by linkage to the National Prescription Drug Database, which worldwide is the oldest nationwide registry of prescription medication. Hazard ratios (HRs) for antidepressant use were estimated in a Cox proportional hazards model stratified on sex, with population comparisons as referents. RESULTS: Overall, childhood cancer survivors were at increased risk of having antidepressants prescribed (HR, 1.4; 95% confidence interval (CI), 1.3-1.5). The excess absolute risk of antidepressant use was 2.5 per 1000 person-years (95% CI, 1.7-3.3), equivalent to an excess of 2.5 survivors for every 100 survivors followed for 10years. Increased HRs of 30-50% were seen for survivors of cancers of all main groups (haematological malignancies, central nervous system (CNS) and solid tumors); the highest risk was among children treated with haematopoietic stem cell transplantation (HR, 1.9; 95% CI, 1.2-3.1). Our data suggested that the risk was most pronounced for children treated in the most recent calendar periods (test for interaction between cancer and calendar periods: P<0.001), especially for survivors of haematological cancers (P=0.007). Interaction analysis of the effect of parental socioeconomic position and psychiatric disease on the association between childhood cancer and antidepressant use indicated no modifying effect. CONCLUSION: Childhood cancer survivors should be followed-up for depression. Our results indicate an increasing need for follow-up especially in survivors treated by more recent, intensive anticancer treatment.

Caffeine-induced hypokalemic paralysis in pregnancy.

BACKGROUND: Excessive ingestion of caffeine can induce hypokalemia, which affects the neuromuscular system and can lead to paralysis. CASE: A 24-year-old woman, gravida 3, para 2-0-0-2 at 33 weeks' gestation presented with muscular paralysis and hypokalemia secondary to drinking 6 to 7 L of cola per day with little other oral intake. After potassium replacement and stopping caffeine ingestion, the symptoms resolved quickly. CONCLUSION: The physiologic changes of pregnancy might potentiate the effect of caffeine on serum potassium concentration.

Long-term outpatient treatment in alcoholics with previous suicidal behavior.

The importance of previous suicidal behavior for the treatment of alcoholics was analyzed in a long-term outpatient treatment study. In a series of 72 patients, 21 patients, (29%), 17 men and 4 women, had previously made at least one suicide attempt or had seriously threatened to kill themselves (3 cases). In comparison with the other alcoholics, those with previous suicidal behavior had a similar attrition rate, they were not more troublesome in treatment, and they had the same rate of favorable outcome during the third year after start of treatment. They differed from the other alcoholics in having a more severe abuse and a less favorable outcome during the initial part of the treatment. In conclusion, our results support the possibility of a long-term outpatient treatment strategy in alcoholics with previous suicide attempts.

[Neurohumoral influences of steroid hormones on sexual responsiveness in women (author's transl)]. / Uber neuro-humorale Einflüsse von Steroidhormonen auf die Sexualsphäre der Frau

An analysis has been carried out on the basis of endocrinological and psychosomatic studies of the influence of steroid hormones, acting via probable transmitter substances, on the sexual response in women. From a review in the literature it can be concluded that among other factors the endocrine state of the women determines her sexual response and behaviour. However, the present lack of specific psychological tests is pointed out, as well as the absence of relevant hormonal data to the sexual sphere, both normal and pathological.

PIP: Based on current literature on the subject, an analysis is made of the endocrinological and psychosomatic aspects of the influence of steroid hormones, which probably act through the action of transmitter substances, upon the sexual responsiveness of women. Endocrinological studies center on the role of androgens, estrogens, and progesterones, and the possible transmitter substances. The physiological basis of abnormal changes in the sexual responsiveness, such as frigidity, is the prime concern of such studies. Among other factors, it is concluded that the state of the endocrinal system does determine a woman's sexual responsiveness and behavior. The present lack of psychological tests is noted as relevant, however, as well as the absence of hormonal data relevant to the sexual sphere, both for normal and pathological states.

Psychologic consequences of breast cancer on partner and family.

Breast cancer can have psychologic consequences not only for patients but also for the entire family system. Research indicates a major impact on the husband, the marital relationship, the children, and family roles and responsibilities. Greater attention needs to be given to the family members to ensure that they get the support they need, and to enable them to maintain their supportive roles with the patient.

Mechanism of repair of 5'-topoisomerase II-DNA adducts by mammalian tyrosyl-DNA phosphodiesterase 2.

The topoisomerase II (topo II) DNA incision-and-ligation cycle can be poisoned (for example following treatment with cancer chemotherapeutics) to generate cytotoxic DNA double-strand breaks (DSBs) with topo II covalently conjugated to DNA. Tyrosyl-DNA phosphodiesterase 2 (Tdp2) protects genomic integrity by reversing 5'-phosphotyrosyl-linked topo II-DNA adducts. Here, X-ray structures of mouse Tdp2-DNA complexes reveal that Tdp2 ß-2-helix-ß DNA damage-binding 'grasp', helical 'cap' and DNA lesion-binding elements fuse to form an elongated protein-DNA conjugate substrate-interaction groove. The Tdp2 DNA-binding surface is highly tailored for engagement of 5'-adducted single-stranded DNA ends and restricts nonspecific endonucleolytic or exonucleolytic processing. Structural, mutational and functional analyses support a single-metal ion catalytic mechanism for the exonuclease-endonuclease-phosphatase (EEP) nuclease superfamily and establish a molecular framework for targeted small-molecule blockade of Tdp2-mediated resistance to anticancer topoisomerase drugs.