Structures of transcription pre-initiation complex with TFIIH and Mediator.

For the initiation of transcription, RNA polymerase II (Pol II) assembles with general transcription factors on promoter DNA to form the pre-initiation complex (PIC). Here we report cryo-electron microscopy structures of the Saccharomyces cerevisiae PIC and PIC-core Mediator complex at nominal resolutions of 4.7 Å and 5.8 Å, respectively. The structures reveal transcription factor IIH (TFIIH), and suggest how the core and kinase TFIIH modules function in the opening of promoter DNA and the phosphorylation of Pol II, respectively. The TFIIH core subunit Ssl2 (a homologue of human XPB) is positioned on downstream DNA by the 'E-bridge' helix in TFIIE, consistent with TFIIE-stimulated DNA opening. The TFIIH kinase module subunit Tfb3 (MAT1 in human) anchors the kinase Kin28 (CDK7), which is mobile in the PIC but preferentially located between the Mediator hook and shoulder in the PIC-core Mediator complex. Open spaces between the Mediator head and middle modules may allow access of the kinase to its substrate, the C-terminal domain of Pol II.

Transcription initiation complex structures elucidate DNA opening.

Transcription of eukaryotic protein-coding genes begins with assembly of the RNA polymerase (Pol) II initiation complex and promoter DNA opening. Here we report cryo-electron microscopy (cryo-EM) structures of yeast initiation complexes containing closed and open DNA at resolutions of 8.8 Å and 3.6 Å, respectively. DNA is positioned and retained over the Pol II cleft by a network of interactions between the TATA-box-binding protein TBP and transcription factors TFIIA, TFIIB, TFIIE, and TFIIF. DNA opening occurs around the tip of the Pol II clamp and the TFIIE 'extended winged helix' domain, and can occur in the absence of TFIIH. Loading of the DNA template strand into the active centre may be facilitated by movements of obstructing protein elements triggered by allosteric binding of the TFIIE 'E-ribbon' domain. The results suggest a unified model for transcription initiation with a key event, the trapping of open promoter DNA by extended protein-protein and protein-DNA contacts.

Complete blood collection-based systemic inflammation biomarkers for patients with hidradenitis suppurativa.

BACKGROUND: Hidradenitis suppurativa (HS) is a relatively common chronic inflammatory condition of intertriginous skin. In recent years, the neutrophil/lymphocyte ratio (NLR), platelet/lymphocyte ratio (PLR), monocyte/lymphocyte ratio (MLR) and platelet/neutrophil ratio (PLR) have been shown to be indicators of systemic inflammation correlating with severity of inflammatory conditions. OBJECTIVES: We aimed to analyse for the first time the systemic inflammation biomarkers also including the pan-immune-inflammation value (PIV) and the systemic immune-inflammation index (SII) in HS patients and controls. METHODS: This study retrospectively investigated clinical and laboratory data of 142 patients with HS. Moreover, a sex-age-matched healthy control group was included. The severity of HS was routinely assessed by the Hurley staging, the mHSS and the SAHS score. All inflammation-based biomarkers were calculated from absolute values of complete blood counts. Receiver-operating characteristics analyses, including the Youden index, were performed in order to determine optimal cut-off values and test performance. RESULTS: Whereas PIV and SII were significantly higher in HS patients, PLR, MLR and PNR were significantly lower in HS patients when compared to controls. Almost all inflammation-based biomarkers significantly correlated with disease severity. However, PIV was the only test that was significantly associated with HS severity as indicated by a Youden index of 0.56 (associated criterion: 756.4; AUC: 0.79, P < 0.0001). CONCLUSIONS: Although all systemic inflammation-based biomarkers investigated are more or less associated with HS severity, the PIV appears to have the best performance in this regard. It may be employed in adjunction with the clinical scores for treatment decision making or clinical trial assessments.

Architecture of the RNA polymerase II-Mediator core initiation complex.

The conserved co-activator complex Mediator enables regulated transcription initiation by RNA polymerase (Pol) II. Here we reconstitute an active 15-subunit core Mediator (cMed) comprising all essential Mediator subunits from Saccharomyces cerevisiae. The cryo-electron microscopic structure of cMed bound to a core initiation complex was determined at 9.7 Å resolution. cMed binds Pol II around the Rpb4-Rpb7 stalk near the carboxy-terminal domain (CTD). The Mediator head module binds the Pol II dock and the TFIIB ribbon and stabilizes the initiation complex. The Mediator middle module extends to the Pol II foot with a 'plank' that may influence polymerase conformation. The Mediator subunit Med14 forms a 'beam' between the head and middle modules and connects to the tail module that is predicted to bind transcription activators located on upstream DNA. The Mediator 'arm' and 'hook' domains contribute to a 'cradle' that may position the CTD and TFIIH kinase to stimulate Pol II phosphorylation.

[Hidradenitis suppurativa /acne inversa-surgical options, reconstruction and combinations with drug therapies-an update]. / Hidradenitis suppurativa/Acne inversa ­ operative Optionen, Rekonstruktionen und Kombinationen mit medikamentösen Therapien ­ Ein Update.

Hidradenitis suppurativa/acne inversa (HS/AI) is a chronic inflammatory skin disease whose treatment includes both conservative and surgical treatment options. In Hurley stages II and III, surgical resection of irreversibly destroyed tissue should be the objective. For this purpose several resection techniques exist, which differ primarily with regard to their invasiveness and tendency to recur. To date, there is no generally accepted consensus on the use of different resection and reconstruction techniques or the inclusion of drug therapies in the overall therapeutic concept.

Sublinear scaling of the cellular proteome with ploidy.

Ploidy changes are frequent in nature and contribute to evolution, functional specialization and tumorigenesis. Analysis of model organisms of different ploidies revealed that increased ploidy leads to an increase in cell and nuclear volume, reduced proliferation, metabolic changes, lower fitness, and increased genomic instability, but the underlying mechanisms remain poorly understood. To investigate how gene expression changes with cellular ploidy, we analyzed isogenic series of budding yeasts from 1N to 4N. We show that mRNA and protein abundance scales allometrically with ploidy, with tetraploid cells showing only threefold increase in protein abundance compared to haploids. This ploidy-dependent sublinear scaling occurs via decreased rRNA and ribosomal protein abundance and reduced translation. We demonstrate that the activity of Tor1 is reduced with increasing ploidy, which leads to diminished rRNA gene repression via a Tor1-Sch9-Tup1 signaling pathway. mTORC1 and S6K activity are also reduced in human tetraploid cells and the concomitant increase of the Tup1 homolog Tle1 downregulates the rDNA transcription. Our results suggest that the mTORC1-Sch9/S6K-Tup1/TLE1 pathway ensures proteome remodeling in response to increased ploidy.

Richter transformation in chronic lymphocytic leukemia (CLL)-a pooled analysis of German CLL Study Group (GCLLSG) front line treatment trials.

Richter transformation (RT) is defined as development of aggressive lymphoma in patients (pts) with CLL. The incidence rates of RT among pts with CLL range from 2 to 10%. The aim of this analysis is to report the frequency, characteristics and outcomes of pts with RT enrolled in trials of the GCLLSG. A total of 2975 pts with advanced CLL were reviewed for incidence of RT. Clinical, laboratory, and genetic data were pooled. Time-to-event data, starting from time of CLL diagnosis, of first-line therapy or of RT diagnosis, were analyzed by Kaplan-Meier methodology. One hundred and three pts developed RT (3%): 95 pts diffuse large B-cell lymphoma (92%) and eight pts Hodgkin lymphoma (8%). Median observation time was 53 months (interquartile range 38.1-69.5). Median OS from initial CLL diagnosis for pts without RT was 167 months vs 71 months for pts with RT (HR 2.64, CI 2.09-3.33). Median OS after diagnosis of RT was 9 months. Forty-seven pts (46%) received CHOP-like regimens for RT treatment. Three pts subsequently underwent allogeneic and two pts autologous stem cell transplantation. Our findings show that within a large cohort of GCLLSG trial participants, 3% of the pts developed RT after receiving first-line chemo- or chemoimmunotherapy. This dataset confirms the ongoing poor prognosis and high mortality associated with RT.

Structural basis of transcription: RNA polymerase II at 2.8 angstrom resolution.

Structures of a 10-subunit yeast RNA polymerase II have been derived from two crystal forms at 2.8 and 3.1 angstrom resolution. Comparison of the structures reveals a division of the polymerase into four mobile modules, including a clamp, shown previously to swing over the active center. In the 2.8 angstrom structure, the clamp is in an open state, allowing entry of straight promoter DNA for the initiation of transcription. Three loops extending from the clamp may play roles in RNA unwinding and DNA rewinding during transcription. A 2.8 angstrom difference Fourier map reveals two metal ions at the active site, one persistently bound and the other possibly exchangeable during RNA synthesis. The results also provide evidence for RNA exit in the vicinity of the carboxyl-terminal repeat domain, coupling synthesis to RNA processing by enzymes bound to this domain.

Structural basis of transcription: an RNA polymerase II elongation complex at 3.3 A resolution.

The crystal structure of RNA polymerase II in the act of transcription was determined at 3.3 A resolution. Duplex DNA is seen entering the main cleft of the enzyme and unwinding before the active site. Nine base pairs of DNA-RNA hybrid extend from the active center at nearly right angles to the entering DNA, with the 3' end of the RNA in the nucleotide addition site. The 3' end is positioned above a pore, through which nucleotides may enter and through which RNA may be extruded during back-tracking. The 5'-most residue of the RNA is close to the point of entry to an exit groove. Changes in protein structure between the transcribing complex and free enzyme include closure of a clamp over the DNA and RNA and ordering of a series of "switches" at the base of the clamp to create a binding site complementary to the DNA-RNA hybrid. Protein-nucleic acid contacts help explain DNA and RNA strand separation, the specificity of RNA synthesis, "abortive cycling" during transcription initiation, and RNA and DNA translocation during transcription elongation.

Architecture of RNA polymerase II and implications for the transcription mechanism.

A backbone model of a 10-subunit yeast RNA polymerase II has been derived from x-ray diffraction data extending to 3 angstroms resolution. All 10 subunits exhibit a high degree of identity with the corresponding human proteins, and 9 of the 10 subunits are conserved among the three eukaryotic RNA polymerases I, II, and III. Notable features of the model include a pair of jaws, formed by subunits Rpb1, Rpb5, and Rpb9, that appear to grip DNA downstream of the active center. A clamp on the DNA nearer the active center, formed by Rpb1, Rpb2, and Rpb6, may be locked in the closed position by RNA, accounting for the great stability of transcribing complexes. A pore in the protein complex beneath the active center may allow entry of substrates for polymerization and exit of the transcript during proofreading and passage through pause sites in the DNA.

Updated results from the phase 3 HELIOS study of ibrutinib, bendamustine, and rituximab in relapsed chronic lymphocytic leukemia/small lymphocytic lymphoma.

We report follow-up results from the randomized, placebo-controlled, phase 3 HELIOS trial of ibrutinib+bendamustine and rituximab (BR) for previously treated chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL) without deletion 17p. Overall, 578 patients were randomized 1:1 to either ibrutinib (420 mg daily) or placebo, in combination with 6 cycles of BR, followed by ibrutinib or placebo alone. Median follow-up was 34.8 months (range: 0.1-45.8). Investigator-assessed median progression-free survival (PFS) was not reached for ibrutinib+BR, versus 14.3 months for placebo+BR (hazard ratio [HR] [95% CI], 0.206 [0.159-0.265]; P < 0.0001); 36-month PFS rates were 68.0% versus 13.9%, respectively. The results are consistent with the primary analysis findings (HR = 0.203, as assessed by independent review committee, with 17-month median follow-up). Median overall survival was not reached in either arm; HR (95% CI) for ibrutinib+BR versus placebo: 0.652 (0.454-0.935; P = 0.019). Minimal residual disease (MRD)-negative response rates were 26.3% for ibrutinib+BR and 6.2% for placebo+BR (P < 0.0001). Incidence of treatment-emergent adverse events (including grades 3-4) were generally consistent with the initial HELIOS report. These long-term data support improved survival outcomes and deepening responses with ibrutinib+BR compared with BR in relapsed CLL/SLL.

Architecture of a transcribing-translating expressome.

DNA transcription is functionally coupled to messenger RNA (mRNA) translation in bacteria, but how this is achieved remains unclear. Here we show that RNA polymerase (RNAP) and the ribosome of Escherichia coli can form a defined transcribing and translating "expressome" complex. The cryo-electron microscopic structure of the expressome reveals continuous protection of ~30 nucleotides of mRNA extending from the RNAP active center to the ribosome decoding center. The RNAP-ribosome interface includes the RNAP subunit α carboxyl-terminal domain, which is required for RNAP-ribosome interaction in vitro and for pronounced cell growth defects upon translation inhibition in vivo, consistent with its function in transcription-translation coupling. The expressome structure can only form during transcription elongation and explains how translation can prevent transcriptional pausing, backtracking, and termination.

A firm hand on NFkappaB: structures of the IkappaBalpha-NFkappaB complex.

Two crystal structures of an IkappaB-NFkappaB complex have recently been determined. The structures show in detail how IkappaB controls the subcellular localization and activity of the eukaryotic transcription factor NFkappaB.

Selenomethionine incorporation in Saccharomyces cerevisiae RNA polymerase II.

A protocol for the incorporation of SeMet into yeast proteins is described. Incorporation at a level of about 50% suffices for the location of Se sites in an anomalous difference Fourier map of the 0.5 MDa yeast RNA polymerase II. This shows the utility of the approach as an aid in the model-building of large protein complexes.

Structure of the specificity domain of the Dorsal homologue Gambif1 bound to DNA.

BACKGROUND: NF-kappa B/Rel transcription factors play important roles in immunity and development in mammals and insects. Their activity is regulated by their cellular localization, homo- and heterodimerization and association with other factors on their target gene promoters. Gambif1 from Anopheles gambiae is a member of the Rel family and a close homologue of the morphogen Dorsal, which establishes dorsoventral polarity in the Drosophila embryo. RESULTS: We present the crystal structure of the N-terminal specificity domain of Gambif1 bound to DNA. This first structure of an insect Rel protein-DNA complex shows that Gambif1 binds a GGG half-site element using a stack of three arginine sidechains. Differences in affinity to Dorsal binding sites in target gene promoters are predicted to arise from base changes in these GGG elements. An arginine that is conserved in class II Rel proteins (members of which contain a transcription activation domain) contacts the outermost guanines of the DNA site. This previously unseen specific contact contributes strongly to the DNA-binding affinity and might be responsible for differences in specificity between Rel proteins of class I and II. CONCLUSIONS: The Gambif1-DNA complex structure illustrates how differences in Dorsal affinity to binding sites in developmental gene promoters are achieved. Comparison with other Rel-DNA complex structures leads to a general model for DNA recognition by Rel proteins.

Correlation between seric antitumor activity and concomitant resistance in mice bearing nonimmunogenic tumors.

Serum from mice bearing five weakly immunogenic or nonimmunogenic tumors inducing concomitant resistance exhibited a growth-inhibitory activity on in vitro proliferation of the tumor cells. This activity was proportional to the intensity of concomitant resistance and correlated with the capacity to restrain metastatic development. It was not attributable to cytotoxic antibodies, was relatively nonspecific, and operated through a cytostatic and reversible mechanism. All attempts to transfer antitumor resistance in vivo by serum inoculation have failed, but this could be attained by parabiosis. Physical and chemical serum treatments suggest that heat-, acid-, and alkali-resistant peptide(s) with molecular weights ranging from 1000 to 3000 could account for this inhibitory effect.

Effect of first-line treatment on second primary malignancies and Richter's transformation in patients with CLL.

This study aimed to assess the frequency of and the contributing factors for second primary malignancies (SPMs) and Richter's transformations (RTs) following first-line treatment of chronic lymphocytic leukemia within four phase II/III trials of the GCLLSG evaluating fludarabine (F) vs F+cyclophosphamide (FC), chlorambucil vs F, FC without or with rituximab, and bendamustine+R (BR). Among 1458 patients, 239 (16.4%) experienced either an SPM (N=191) or a RT (N=75). Solid tumors (N=115; 43.2% of all second neoplasias) appeared most frequently, followed by RTs (N=75; 28.2%). Patients showed a 1.23-fold increased risk of solid tumors in comparison to the age-matched general population from the German cancer registry. Age>65 (hazard ratio (HR) 2.1; P<0.001), male sex (HR 1.7; P=0.01), co-morbidities (HR 1.6; P=0.01) and number of subsequent treatments⩾1 (HR 12.1; P<0.001) showed an independent adverse prognostic impact on SPM-free survival. Serum thymidine kinase>10 U/l at trial enrollment (HR 3.9; P=0.02), non-response to first-line treatment (HR 3.6; P<0.001) and number of subsequent treatments⩾1 (HR 30.2; P<0.001) were independently associated with increased risk for RT.

Knowledge about systemic inflammatory response syndrome and sepsis: a survey among Dutch emergency department nurses.

BACKGROUND: Sepsis has a high mortality. Early recognition and timely treatment are essential for patient survival. The aim of this study is to examine the factors that influence the knowledge and recognition of systemic inflammatory response syndrome (SIRS) criteria and sepsis by emergency department (ED) nurses. METHODS: A prospective, multi-center study including 216 ED nurses from 11 hospitals and academic medical centers in The Netherlands was conducted in 2013. A validated questionnaire was used to evaluate ED nurses' knowledge about SIRS and sepsis. Questions about demographic characteristics were also included, to investigate factors that may contribute to the knowledge about SIRS and sepsis. RESULTS: The mean total score was 15.9 points, with a maximum possible score of 29 points. ED nurses employed at hospitals with a level 3 intensive care unit (ICU) scored significantly higher than their colleagues employed at hospitals with a level 1 or 2 ICU. Recently completed education in sepsis was associated with a higher score. The employees in low ICU level hospitals who reported recent education did not score significantly lower than their ICU level 3 colleagues. ED nurses over the age of 50 scored significantly lower than their younger colleagues. CONCLUSIONS: The knowledge of ED nurses concerning SIRS and sepsis rises proportionally with the level of ICU in hospitals. Recent education in sepsis raises knowledge level as well. We recommend that when there is a low exposure rate to SIRS and sepsis, more emphasis should be placed on regular education.

Hodgkin's disease in childhood and adolescence: results of chemotherapy-radiotherapy in clinical stages IA-IIB.

From April 1972 to May 1980, 72 children and adolescents (aged 5 to 19 years old, median 16) with Hodgkin's disease, clinical stages IA-IIB (IA, 18; II2A, two areas involved on the same side of the diaphragm, 23; II3+A, three areas or more, 16; IIB, 15) were prospectively treated in two successive clinical trials (H 72 and H 77). Clinical stages IA and II2A received three courses of mechlorethamine, Oncovin, procarbazine, and prednisone (MOPP) and supradiaphragmatic radiotherapy (40 Gy), and no laparotomy was performed. Clinical stages II3+A and IIB received either six cycles of MOPP (H 72), three cycles of MOPP, or three cycles of CCNU, vinblastine, procarbazine, and prednisone (CVPP) (H 77) and subsequently had a laparotomy followed by supradiaphragmatic radiotherapy and a lumboaortic field if results of laparotomy were positive. Patients without evidence of mediastinal involvement did not have mediastinal radiotherapy. At the completion of therapy, the disease in 70 of 72 patients was in complete remission (one failure, one death during treatment). Eight patients relapsed (in situ, 1; marginal, 1; nonirradiated subdiaphragmatic area, 6) after three to 57 months of complete remission (median 20 months); one patient died after relapse. There were three deaths after complete remission of the disease (infection, two; acute nonlymphocytic leukemia [ANLL], one). As of June 1984 the median follow-up was 82 months (range, 49 to 145 months), the actuarial probabilities for survival and freedom from relapse for all patients being 91.6% and 87.6%, respectively. There was no statistical difference according to clinical stage, age (greater than 15 or less than 15 years), sex, or number of cycles of chemotherapy (six or three). Bone growth defects related to radiotherapy were reduced particularly in the 29 patients who did not receive mediastinal radiotherapy. None of these patients had a mediastinal relapse. Azoospermia was the rule for the male patients studied, but young girls and young women retained reproductive integrity.

Engineering of diffraction-quality crystals of the NF-kappaB P52 homodimer:DNA complex.

The eukaryotic transcription factors NF-kappaB P50 and NF-kappaB P52 are closely related members of the Rel family. Growth of diffraction-quality NF-kappaB P52:DNA co-crystals crucially depended on (a) extensive screens for the DNA fragment of optimal length and (b) engineering of the protein based on the two known NF-kappaB P50:DNA co-crystal structures [Müller et al. (1995) Nature 373, 311-317; Ghosh et al. (1995) Nature 373, 303-310]; namely, deletion of 12 C-terminal amino acid residues. These residues are part of the Rel homology region and comprise the nuclear localization signal. The approach might be of general use for the crystallization of other Rel protein: DNA complexes and in our case yielded co-crystals which diffract beyond 2.0 A resolution.