Structure of a Complete Mediator-RNA Polymerase II Pre-Initiation Complex.

A complete, 52-protein, 2.5 million dalton, Mediator-RNA polymerase II pre-initiation complex (Med-PIC) was assembled and analyzed by cryo-electron microscopy and by chemical cross-linking and mass spectrometry. The resulting complete Med-PIC structure reveals two components of functional significance, absent from previous structures, a protein kinase complex and the Mediator-activator interaction region. It thereby shows how the kinase and its target, the C-terminal domain of the polymerase, control Med-PIC interaction and transcription.

Structure of mitotic chromosomes.

Chromatin fibers must fold or coil in the process of chromosome condensation. Patterns of coiling have been demonstrated for reconstituted chromatin, but the actual trajectories of fibers in condensed states of chromosomes could not be visualized because of the high density of the material. We have exploited partial decondensation of mitotic chromosomes to reveal their internal structure at sub-nucleosomal resolution by cryo-electron tomography, without the use of stains, fixatives, milling, or sectioning. DNA gyres around nucleosomes were visible, allowing the nucleosomes to be identified and their orientations to be determined. Linker DNA regions were traced, revealing the trajectories of the chromatin fibers. The trajectories were irregular, with almost no evidence of coiling and no short- or long-range order of the chromosomal material. The 146-bp core particle, long known as a product of nuclease digestion, is identified as the native state of the nucleosome, with no regular spacing along the chromatin fibers.

Uncoupling Promoter Opening from Start-Site Scanning.

Whereas RNA polymerase II (Pol II) transcription start sites (TSSs) occur about 30-35 bp downstream of the TATA box in metazoans, TSSs are located 40-120 bp downstream in S. cerevisiae. Promoter melting begins about 12 bp downstream in all eukaryotes, so Pol II is presumed to "scan" further downstream before starting transcription in yeast. Here we report that removal of the kinase complex TFIIK from TFIIH shifts the TSS in a yeast system upstream to the location observed in metazoans. Conversely, moving the normal TSS to an upstream location enables a high level of TFIIK-independent transcription in the yeast system. We distinguish two stages of the transcription initiation process: bubble formation by TFIIH, which fills the Pol II active center with single-stranded DNA, and subsequent scanning downstream, also driven by TFIIH, which requires displacement of the initial bubble. Omission of TFIIK uncouples the two stages of the process.

Adjuvant brachytherapy for oral squamous cell carcinomas: a single-center experience comparing low-dose and pulsed-dose-rate techniques.

OBJECTIVE: This study aims to assess the outcomes of adjuvant interstitial brachytherapy (BT) to the tumor bed for oral cavity squamous cell carcinoma (SCC), and to compare the oncological outcomes and toxicity profile of low-dose-rate (LDR) and pulsed-dose-rate (PDR) BT. DESIGN: This retrospective single-center study included all patients who underwent postoperative LDR- or PDR-BT to the tumor bed as the sole adjuvant treatment for an oral tongue or floor of the mouth SCC between January 2000 and December 2020. RESULTS: A total of 79 patients were eligible for this study. The cohort was divided into an LDR group (n = 38) and a PDR group (n = 41). The median time interval between surgery and brachytherapy was 55 days. Median delivered total dose was 55 Gy and median hospital stay was 5 days. Five patients (8.3%) experienced grade 3-4 early toxicity, 2 in the LDR group and 3 in the PDR group. Late toxicities were present in 28 patients (44.4%) and were dominated by grade 1-2 residual pain and dysesthesia, without a statistical difference between the groups. After a median follow-up of 65.1 months, 5­year local control (LC), disease-free survival (DFS), and overall survival (OS) for the whole cohort were 76.3% (95% CI = 63.4-85.1), 61.6% (95% CI = 49.0-72.0), and 71.4% (95% CI = 58.6-80.8), respectively. CONCLUSION: Adjuvant BT after excision of oral cavity SCC provides satisfactory oncological outcomes along with good tolerance. In our study, PDR-BT showed similar oncological and functional results to LDR-BT in this indication.

Therapeutic strategies, oncologic and swallowing outcomes and their predictive factors in patients with locally advanced hypopharyngeal cancer.

INTRODUCTION: Hypopharyngeal cancer (HC) is an aggressive and life-threatening malignancy that requires a complex multimodal treatment. The aims of the present study were to analyze, in locally advanced HC patients, the oncologic and swallowing outcomes and their predictive factors according to the therapeutic strategy. METHODS: All patients with locally advanced HC (T3/T4, N0-3, M0) treated at our institution between 2000 and 2020 were included in this retrospective study. Patients were classified in 3 groups according to the therapeutic strategy: primary radical surgery (RS), induction chemotherapy (ICT) or definitive (chemo)-radiation therapy ((C)RT). Predictive factors of oncologic outcomes (overall, cause-specific and recurrence-free survival: OS, CSS and RFS) and swallowing outcome (dysphagia outcome and severity scale: DOSS) were investigated in univariate and multivariate analysis. RESULTS: A total of 217 patients were included in this study (RS: 40; ICT: 106; (C)RT: 71). 5-year OS, CSS and RFS rates were 36, 38 and 32%, respectively. ICT was associated with improved oncologic and swallowing outcomes in univariate analysis. After multivariate analysis, patient age ≥ 70 years (p = 0.0002) was the only factor significantly associated with a worse OS, whereas patient age ≥ 70 years (p = 0.002) and N stage ≥ 2 (p = 0.01) were significantly associated with a worse CSS. Comorbidity level (KFI ≥ 2; p = 0.01) and N stage (≥ 2; p = 0.02) were significantly associated with worse swallowing outcomes. CONCLUSION: In selected locally advanced HC patients, an ICT-based therapeutic strategy offers acceptable oncologic and functional outcomes. Patient age, N stage and comorbidity level are the main determinants of oncologic and functional outcomes.

Characterization of acoustic sources in a corrugated pipe flow with linear stochastic estimation.

The intense whistling of corrugated pipe under flow is related to a coherence between the dynamics of structures developing in the shear layer over cavities and acoustic eigenmodes of the pipe. In order to highlight the coupling between aerodynamics and acoustics, three measurement systems with complementary characteristics in terms of space and time resolutions are synchronized. The simultaneity of the measurements of velocity and acoustic pressure provided by five local probes, as two hot-wires and three microphones, with the velocity fields measured by PIV in the same flow region is used to estimate the velocity fields at frequencies compatible with the space-time characterization of the acoustic sources. The linear stochastic estimation (LSE) is performed to reconstruct these high frequency velocity fields. Two rectangular corrugated pipes with different corrugation geometry are investigated. Thanks to the LSE velocity field reconstruction, the contribution of the flow structures, both jet flapping and vortex shedding, to acoustic level is highlighted.

Chromatin potentiates transcription.

Chromatin isolated from the chromosomal locus of the PHO5 gene of yeast in a transcriptionally repressed state was transcribed with 12 pure proteins (80 polypeptides): RNA polymerase II, six general transcription factors, TFIIS, the Pho4 gene activator protein, and the SAGA, SWI/SNF, and Mediator complexes. Contrary to expectation, a nucleosome occluding the TATA box and transcription start sites did not impede transcription but rather, enhanced it: the level of chromatin transcription was at least sevenfold greater than that of naked DNA, and chromatin gave patterns of transcription start sites closely similar to those occurring in vivo, whereas naked DNA gave many aberrant transcripts. Both histone acetylation and trimethylation of H3K4 (H3K4me3) were important for chromatin transcription. The nucleosome, long known to serve as a general gene repressor, thus also performs an important positive role in transcription.

Docetaxel-Cisplatin-Fluorouracil Induction Chemotherapy for Larynx Preservation in Patients with Locally Advanced Hypopharyngeal Cancer: Predictive Factors of Oncologic and Functional Outcomes.

BACKGROUND: The aims of this study were to evaluate the clinical outcomes and their predictive factors in locally advanced hypopharyngeal cancer (HC) patients included in a docetaxel-cisplatin-fluorouracil induction chemotherapy (ICT)-based larynx preservation (LP) program. METHODS: Between 2005 and 2021, 82 patients with a locally advanced resectable HC who received ICT in an LP program were included in this retrospective study. The predictors of oncologic and swallowing outcomes were determined in univariate and multivariate analyses. RESULTS: The three- and five-year overall survival (OS) rates were 67 and 54%, respectively. The T4 tumor stage was the only predictive factor of poor response to ICT (p = 0.03). In multivariate analysis, a T stage = 4 (p = 0.02), an ICT cycle number < 3 (p = 0.003) and the absence of a response to ICT (p = 0.03) were significantly associated with worse OS. A low body mass index before therapy (p = 0.003) and enteral nutrition during therapy (p = 0.005) were significantly associated with severity of dysphagia 6 months after treatment. CONCLUSIONS: The T stage, number of ICT cycles performed and response to ICT are the main predictors of oncologic outcomes. Patients with T4 HC are poor candidates for LP and should be referred to immediate radical surgery.

Should an elective contralateral neck dissection be performed in midline-reaching squamous cell carcinomas of the oral cavity and oropharynx?

OBJECTIVE: to compare the rate of occult contralateral neck metastases (OCNM) in oral and oropharyngeal squamous cell carcinomas (SCC) reaching or crossing the midline and to identify risk factors for OCNM. MATERIALS AND METHODS: we conducted a single-center retrospective study of oral and oropharyngeal SCC with contralateral cN0 neck. The cohort was divided into a midline-reaching (MR; approaching the midline from up to 10 mm) group and a midline-crossing (MC; exceeding the midline by up to 10 mm) group. Clinical N-status was assessed by a radiologist specializing in head and neck imaging. All patients underwent contralateral elective neck dissection (END). RESULTS: A total of 98 patients were included in this study, 59 in the MR group and 39 in the MC group. OCNM were present in 17.3% of patients, 20.3% in the MR group and 12.8% in the MC group (p = 0.336). In multivariable analysis, MR/MC status as well as distance from the midline (in mm) were not identified as risk factors for OCNM. Conversely, oropharyngeal primary and clinical N-status above N1 were significantly associated with a higher risk of OCNM, with odds ratios (OR) of 3.98 (95% CI = 1.08-14.60; p = 0.037) and 3.41 (95% CI = 1.07-10.85; p = 0.038) respectively. CONCLUSION: in patients with oral and oropharyngeal SCC extending close to or beyond the midline, tumor origin and clinical N-status should carry the most weight when dictating the indications for contralateral END, rather than the midline involvement in itself.

Characterization of the elongasome core PBP2 : MreC complex of Helicobacter pylori.

The definition of bacterial cell shape is a complex process requiring the participation of multiple components of an intricate macromolecular machinery. We aimed at characterizing the determinants involved in cell shape of the helical bacterium Helicobacter pylori. Using a yeast two-hybrid screen with the key cell elongation protein PBP2 as bait, we identified an interaction between PBP2 and MreC. The minimal region of MreC required for this interaction ranges from amino acids 116 to 226. Using recombinant proteins, we showed by affinity and size exclusion chromatographies and surface plasmon resonance that PBP2 and MreC form a stable complex. In vivo, the two proteins display a similar spatial localization and their complex has an apparent 1:1 stoichiometry; these results were confirmed in vitro by analytical ultracentrifugation and chemical cross-linking. Small angle X-ray scattering analyses of the PBP2 : MreC complex suggest that MreC interacts directly with the C-terminal region of PBP2. Depletion of either PBP2 or MreC leads to transition into spherical cells that lose viability. Finally, the specific expression in trans of the minimal interacting domain of MreC with PBP2 in the periplasmic space leads to cell rounding, suggesting that the PBP2/MreC complex formation in vivo is essential for cell morphology.

Unobserved classes and extra variables in high-dimensional discriminant analysis.

In supervised classification problems, the test set may contain data points belonging to classes not observed in the learning phase. Moreover, the same units in the test data may be measured on a set of additional variables recorded at a subsequent stage with respect to when the learning sample was collected. In this situation, the classifier built in the learning phase needs to adapt to handle potential unknown classes and the extra dimensions. We introduce a model-based discriminant approach, Dimension-Adaptive Mixture Discriminant Analysis (D-AMDA), which can detect unobserved classes and adapt to the increasing dimensionality. Model estimation is carried out via a full inductive approach based on an EM algorithm. The method is then embedded in a more general framework for adaptive variable selection and classification suitable for data of large dimensions. A simulation study and an artificial experiment related to classification of adulterated honey samples are used to validate the ability of the proposed framework to deal with complex situations.

Impact of pulmonary hypertension on lung cancer management.

INTRODUCTION: The diagnosis and management of lung cancer is challenging among patients followed-up for pulmonary hypertension (PH). Many interventional procedures are not suitable for severely ill patients, thus limiting the diagnosis and treatment of cancer. We report on patients diagnosed with both conditions in our Institution. METHODS: We conducted a retrospective observational cohort study at Toulouse University Hospital. We analysed both management and outcome for patients followed-up for precapillary PH following diagnosis of primary lung cancer. RESULTS: Out of 764 patients followed-up for PH, 25 went on to develop bronchopulmonary neoplasia. The median age was 69 years with a predominance of males (56%) and smokers (92%). Fifty-two percent had group 1 PH and 36% severe group 3 PH. The comorbidity burden was high and 76% were oxygen-dependent. Twenty-eight percent of patients were considered ineligible for tissue biopsy, the diagnosis being made by a multidisciplinary team (MDT) based on radio-clinical presentation. Fifty-four percent of patients did not benefit from any treatment. Sixteen percent of pulmonary diagnostic procedures were associated with complications (severe hypoxaemia, intra-alveolar hemorrhage, haemothorax). Patients were undertreated compared to disease stage guidelines (2 surgical procedures for 9 localised stages). Median survival after cancer diagnosis was 6 months. CONCLUSION: The management of lung cancer is complex in PH patients. The high rate of complications during the diagnosis and therapy steps coupled with very poor patient outcome for both conditions should prompt physicians to thoroughly discuss the benefit/risk benefit in each case.

Paratracheal lymph node dissection during total (pharyngo-)laryngectomy: A systematic review and meta-analysis.

OBJECTIVES: The objective of this review was to determine the rate and risk factors of paratracheal lymph node (PTLN) involvement during total laryngectomy (TL) or total pharyngolaryngectomy (TPL). In addition, we aimed to assess its prognostic significance in terms of survival and peristomal recurrence. METHODS: A comprehensive electronic search was performed on PubMed, EMBASE, and CENTRAL databases. We searched for studies reporting outcomes of PTLN dissection during radical laryngeal surgery for squamous cell carcinoma of the larynx, hypopharynx or cervical oesophagus. RESULTS: We included a total of ten studies (838 patients). The overall rate of PTLN dissection positivity was 18.6% (20.7% for primary TL, 8.7% for salvage TL). Random-effects meta-analysis identified T4 stage, N+ stage of the lateral neck, subglottis involvement and primary tumour arising from the hypopharynx or cervical oesophagus as significant risk factors for PTLN involvement. CONCLUSIONS: This meta-analysis allowed to better define the risk of PTLN involvement during TL or TPL, in a bid to guide indication for PTLN dissection. There is a need for further large studies reporting rigorously the outcomes of PTLN dissection in order to establish stronger evidence-based recommendations.

Structural basis of chaperone recognition of type III secretion system minor translocator proteins.

The type III secretion system (T3SS) is a complex nanomachine employed by many Gram-negative pathogens, including the nosocomial agent Pseudomonas aeruginosa, to inject toxins directly into the cytoplasm of eukaryotic cells. A key component of all T3SS is the translocon, a proteinaceous channel that is inserted into the target membrane, which allows passage of toxins into target cells. In most bacterial species, two distinct membrane proteins (the "translocators") are involved in translocon formation, whereas in the bacterial cytoplasm, however, they remain associated to a common chaperone. To date, the strategy employed by a single chaperone to recognize two distinct translocators is unknown. Here, we report the crystal structure of a complex between the Pseudomonas translocator chaperone PcrH and a short region from the minor translocator PopD. PcrH displays a 7-helical tetratricopeptide repeat fold that harbors the PopD peptide within its concave region, originally believed to be involved in recognition of the major translocator, PopB. Point mutations introduced into the PcrH-interacting region of PopD impede translocator-chaperone recognition in vitro and lead to impairment of bacterial cytotoxicity toward macrophages in vivo. These results indicate that T3SS translocator chaperones form binary complexes with their partner molecules, and the stability of their interaction regions must be strictly maintained to guarantee bacterial infectivity. The PcrH-PopD complex displays homologs among a number of pathogenic strains and could represent a novel, potential target for antibiotic development.

Experimental study of the influence of low frequency flow modulation on the whistling behavior of a corrugated pipe.

It is well known that airflow in a corrugated pipe can excite whistling at the frequencies of the pipe's longitudinal acoustic modes. This short contribution reports on the results of experiments where a low frequency, oscillating flow with velocity magnitudes of the same order as the airflow has been added. Depending on the oscillation strength, it has been found that this flow may silence the pipe or move the whistling to higher harmonics. It is also shown that the low frequency oscillation itself may excite higher frequency whistling sounds in the pipe.

3D genomics across the tree of life reveals condensin II as a determinant of architecture type.

We investigated genome folding across the eukaryotic tree of life. We find two types of three-dimensional (3D) genome architectures at the chromosome scale. Each type appears and disappears repeatedly during eukaryotic evolution. The type of genome architecture that an organism exhibits correlates with the absence of condensin II subunits. Moreover, condensin II depletion converts the architecture of the human genome to a state resembling that seen in organisms such as fungi or mosquitoes. In this state, centromeres cluster together at nucleoli, and heterochromatin domains merge. We propose a physical model in which lengthwise compaction of chromosomes by condensin II during mitosis determines chromosome-scale genome architecture, with effects that are retained during the subsequent interphase. This mechanism likely has been conserved since the last common ancestor of all eukaryotes.

Molecular architecture of the PBP2-MreC core bacterial cell wall synthesis complex.

Bacterial cell wall biosynthesis is an essential process that requires the coordinated activity of peptidoglycan biosynthesis enzymes within multi-protein complexes involved in cell division (the "divisome") and lateral wall growth (the "elongasome"). MreC is a structural protein that serves as a platform during wall elongation, scaffolding other essential peptidoglycan biosynthesis macromolecules, such as penicillin-binding proteins. Despite the importance of these multi-partite complexes, details of their architecture have remained elusive due to the transitory nature of their interactions. Here, we present the crystal structures of the soluble PBP2:MreC core elongasome complex from Helicobacter pylori, and of uncomplexed PBP2. PBP2 recognizes the two-winged MreC molecule upon opening of its N-terminal region, revealing a hydrophobic zipper that serves as binding platform. The PBP2:MreC interface is essential both for protein recognition in vitro and maintenance of bacterial shape and growth. This work allows visualization as to how peptidoglycan machinery proteins are scaffolded, revealing interaction regions that could be targeted by tailored inhibitors.Bacterial wall biosynthesis is a complex process that requires the coordination of multiple enzymes. Here, the authors structurally characterize the PBP2:MreC complex involved in peptidoglycan elongation and cross-linking, and demonstrate that its disruption leads to loss of H. pylori shape and inability to sustain growth.

Membrane targeting and pore formation by the type III secretion system translocon.

The type III secretion system (T3SS) is a complex macromolecular machinery employed by a number of Gram-negative species to initiate infection. Toxins secreted through the system are synthesized in the bacterial cytoplasm and utilize the T3SS to pass through both bacterial membranes and the periplasm, thus being introduced directly into the eukaryotic cytoplasm. A key element of the T3SS of all bacterial pathogens is the translocon, which comprises a pore that is inserted into the membrane of the target cell, allowing toxin injection. Three macromolecular partners associate to form the translocon: two are hydrophobic and one is hydrophilic, and the latter also associates with the T3SS needle. In this review, we discuss recent advances on the biochemical and structural characterization of the proteins involved in translocon formation, as well as their participation in the modification of intracellular signalling pathways upon infection. Models of translocon assembly and regulation are also discussed.

A bacterial protein targets the BAHD1 chromatin complex to stimulate type III interferon response.

Intracellular pathogens such as Listeria monocytogenes subvert cellular functions through the interaction of bacterial effectors with host components. Here we found that a secreted listerial virulence factor, LntA, could target the chromatin repressor BAHD1 in the host cell nucleus to activate interferon (IFN)-stimulated genes (ISGs). IFN-λ expression was induced in response to infection of epithelial cells with bacteria lacking LntA; however, the BAHD1-chromatin associated complex repressed downstream ISGs. In contrast, in cells infected with lntA-expressing bacteria, LntA prevented BAHD1 recruitment to ISGs and stimulated their expression. Murine listeriosis decreased in BAHD1(+/-) mice or when lntA was constitutively expressed. Thus, the LntA-BAHD1 interplay may modulate IFN-λ-mediated immune response to control bacterial colonization of the host.

Bridging cell wall biosynthesis and bacterial morphogenesis.

The bacterial cell wall is a complex three-dimensional structure that protects the cell from environmental stress and ensures its shape. The biosynthesis of its main component, the peptidoglycan, involves the coordination of activities of proteins present in the cytoplasm, the membrane, and the periplasm, some of which also interact with the bacterial cytoskeleton. The sheer complexity of the cell wall elongation process, which is the main focus of this review, has created a significant challenge for the study of the macromolecular interactions that regulate peptidoglycan biosynthesis. The availability of new structural and biochemical data on a number of components of peptidoglycan assembly machineries, including a complex between MreB and RodZ as well as structures of penicillin-binding proteins (PBPs) from a number of pathogenic species, now provide novel insight into the underpinnings of an intricate molecular machinery.