X-Ray Structure Determination Of A Dimeric Hemoglobin From Caudina arenicola

The X-ray structure of a dimeric, cyanomet-liganded hemoglobin D-chain (Hb-D) from Caudina arenicola has been determined by the molecular-replacement method. The search model was a concatenated model of three hemoglobin structures superimposed on the backbone of monomeric, hemichrome hemoglobin C-chain (Hb-C) from the same organism. Hb-D crystallizes in space group P4(1)2(1)2 with cell constants a = b = 77.0 and c = 61.5 Angstrom with one subunit in the asymmetric unit. The dimer twofold axis corresponds to a crystallographic twofold along one of the body diagonals of the unit cell. Rotation and translation searches as well as model refinement were carried out in X-PLOR with the final model having an R value of 0.19 using the data from 5.0 to 2.9 Angstrom resolution (R = 0.26 for 10.0 to 2.9 Angstrom resolution). The homodimeric structure of Caudina Hb-D features close heme-heme contacts with an Fe-Fe distance of 19.0 Angstrom. The subunit-subunit interface involves both the E and F helices from each subunit with the E helices oriented antiparallel at 50 degrees with respect to one another, similar to the quaternary structure observed for the homodimeric hemoglobin from Scapharca inaequivalvis.NIH GM 30105Robert A. Welch FoundationChemistryBiochemistr

Pol5 is required for recycling of small subunit biogenesis factors and for formation of the peptide exit tunnel of the large ribosomal subunit

More than 200 assembly factors (AFs) are required for the production of ribosomes in yeast. The step-wise association and dissociation of these AFs with the pre-ribosomal subunits occurs in a hierarchical manner to ensure correct maturation of the prerRNAs and assembly of the ribosomal proteins. Although decades of research have provided a wealth of insights into the functions of many AFs, others remain poorly characterized. Pol5 was initially classified with B-type DNA polymerases, however, several lines of evidence indicate the involvement of this protein in ribosome assembly. Here, we show that depletion of Pol5 affects the processing of pre-rRNAs destined for the both the large and small subunits. Furthermore, we identify binding sites for Pol5 in the 5' external transcribed spacer and within domain III of the 25S rRNA sequence. Consistent with this, we reveal that Pol5 is required for recruitment of ribosomal proteins that form the polypeptide exit tunnel in the LSU and that depletion of Pol5 impairs the release of 5' ETS fragments from early pre-40S particles. The dual functions of Pol5 in 60S assembly and recycling of pre-40S AFs suggest that this factor could contribute to ensuring the stoichiometric production of ribosomal subunits

Ribosome-bound Get4/5 facilitates the capture of tail-anchored proteins by Sgt2 in yeast

The guided entry of tail-anchored proteins (GET) pathway assists in the posttranslational delivery of tail-anchored proteins, containing a single C-terminal transmembrane domain, to the ER. Here we uncover how the yeast GET pathway component Get4/5 facilitates capture of tail-anchored proteins by Sgt2, which interacts with tail-anchors and hands them over to the targeting component Get3. Get4/5 binds directly and with high affinity to ribosomes, positions Sgt2 close to the ribosomal tunnel exit, and facilitates the capture of tail-anchored proteins by Sgt2. The contact sites of Get4/5 on the ribosome overlap with those of SRP, the factor mediating cotranslational ER-targeting. Exposure of internal transmembrane domains at the tunnel exit induces high-affinity ribosome binding of SRP, which in turn prevents ribosome binding of Get4/5. In this way, the position of a transmembrane domain within nascent ER-targeted proteins mediates partitioning into either the GET or SRP pathway directly at the ribosomal tunnel exit

Global Survey on Pancreatic Surgery During the COVID-19 Pandemic.

OBJECTIVE:The aim of this study was to clarify the role of pancreatic surgery during the COVID-19 pandemic to optimize patients\u27 and clinicians\u27 safety and safeguard health care capacity. SUMMARY BACKGROUND DATA:The COVID-19 pandemic heavily impacts health care systems worldwide. Cancer patients appear to have an increased risk for adverse events when infected by COVID-19, but the inability to receive oncological care seems may be an even larger threat, particularly in case of pancreatic cancer. METHODS:An online survey was submitted to all members of seven international pancreatic associations and study groups, investigating the impact of the COVID-19 pandemic on pancreatic surgery using 21 statements (April, 2020). Consensus was defined as \u3e80% agreement among respondents and moderate agreement as 60% to 80% agreement. RESULTS:A total of 337 respondents from 267 centers and 37 countries spanning 5 continents completed the survey. Most respondents were surgeons (n = 302, 89.6%) and working in an academic center (n = 286, 84.9%). The majority of centers (n = 166, 62.2%) performed less pancreatic surgery because of the COVID-19 pandemic, reducing the weekly pancreatic resection rate from 3 [interquartile range (IQR) 2-5] to 1 (IQR 0-2) (P \u3c 0.001). Most centers screened for COVID-19 before pancreatic surgery (n = 233, 87.3%). Consensus was reached on 13 statements and 5 statements achieved moderate agreement. CONCLUSIONS:This global survey elucidates the role of pancreatic surgery during the COVID-19 pandemic, regarding patient selection for the surgical and oncological treatment of pancreatic diseases to support clinical decision-making and creating a starting point for further discussion

Early epigenetic downregulation of microRNA-192 expression promotes pancreatic cancer progression

Pancreatic ductal adenocarcinoma (PDAC) is characterized by very early metastasis, suggesting the hypothesis that metastasis-associated changes may occur prior to actual tumor formation. In this study, we identified miR-192 as an epigenetically regulated suppressor gene with predictive value in this disease. miR-192 was downregulated by promoter methylation in both PDAC and chronic pancreatitis, the latter of which is a major risk factor for the development of PDAC. Functional studies in vitro and in vivo in mouse models of PDAC showed that overexpression of miR-192 was sufficient to reduce cell proliferation and invasion. Mechanistic analyses correlated changes in miR-192 promoter methylation and expression with epithelial–mesenchymal transition. Cell proliferation and invasion were linked to altered expression of the miR-192 target gene SERPINE1 that is encoding the protein plasminogen activator inhibitor-1 (PAI-1), an established regulator of these properties in PDAC cells. Notably, our data suggested that invasive capacity was altered even before neoplastic transformation occurred, as triggered by miR-192 downregulation. Overall, our results highlighted a role for miR-192 in explaining the early metastatic behavior of PDAC and suggested its relevance as a target to develop for early diagnostics and therapy. Cancer Res; 76(14); 4149–59. ©2016 AACR

Heidelberg standard examination and "Heidelberg standard procedures" - Development of faculty-wide standards for physical examination techniques and clinical procedures in undergraduate medical education

The competent physical examination of patients and the safe and professional implementation of clinical procedures constitute essential components of medical practice in nearly all areas of medicine. The central objective of the projects "Heidelberg standard examination" and "Heidelberg standard procedures", which were initiated by students, was to establish uniform interdisciplinary standards for physical examination and clinical procedures, and to distribute them in coordination with all clinical disciplines at the Heidelberg University Hospital. The presented project report illuminates the background of the initiative and its methodological implementation. Moreover, it describes the multimedia documentation in the form of pocketbooks and a multimedia internet-based platform, as well as the integration into the curriculum. The project presentation aims to provide orientation and action guidelines to facilitate similar processes in other faculties

Electrolyte design for suspended particulates in electrolyte jet processing

The addition of particles (<1 μm) to electrolyte feedstocks results in marked changes to the morphology of deposits when electrolyte jet processing (EJP) compared to electrolyte only feedstocks. Through the use of a ‘carrier’ electrolyte in additive mode these particulates may become embedded within a deposited matrix. These also serve as nucleation points for crystallisation. This allows opportunities for creating complex surface coatings and incorporating materials independent of crystallisation phenomena. Control of the microscale morphology of these is demonstrated here through the addition of the brightening agent – thiourea and the buffer – sodium sulfate. Here acidity and hence electric surface potential are modified and the response upon deposition evaluated

Platelet Function in Acute Experimental Pancreatitis

Acute pancreatitis (AP) is characterized by disturbances of pancreatic microcirculation. It remains unclear whether platelets contribute to these perfusion disturbances. The aim of our study was to investigate platelet activation and function in experimental AP. Acute pancreatitis was induced in rats: (1) control (n = 18; Ringer’s solution), (2) mild AP (n = 18; cerulein), and (3) severe AP (n = 18; glycodeoxycholic acid (GDOC) + cerulein). After 12 h, intravital microscopy was performed. Rhodamine-stained platelets were used to investigate velocity and endothelial adhesion in capillaries and venules. In addition, erythrocyte velocity and leukocyte adhesion were evaluated. Serum amylase, thromboxane A2, and histology were evaluated after 24 h in additional animals of each group. Results showed that 24 h after cerulein application, histology exhibited a mild AP, whereas GDOC induced severe necrotizing AP. Intravital microscopy showed significantly more platelet–endothelium interaction, reduced erythrocyte velocity, and increased leukocyte adherence in animals with AP compared to control animals. Thromboxane levels were significantly elevated in all AP animals and correlated with the extent of platelet activation and severity of AP. In conclusion, platelet activation plays an important role in acute, especially necrotizing, pancreatitis. Mainly temporary platelet–endothelium interaction is observed during mild AP, whereas severe AP is characterized by firm adhesion with consecutive coagulatory activation and perfusion failure

Structural basis for potency differences between GDF8 and GDF11.

BACKGROUND: Growth/differentiation factor 8 (GDF8) and GDF11 are two highly similar members of the transforming growth factor β (TGFβ) family. While GDF8 has been recognized as a negative regulator of muscle growth and differentiation, there are conflicting studies on the function of GDF11 and whether GDF11 has beneficial effects on age-related dysfunction. To address whether GDF8 and GDF11 are functionally identical, we compared their signaling and structural properties. RESULTS: Here we show that, despite their high similarity, GDF11 is a more potent activator of SMAD2/3 and signals more effectively through the type I activin-like receptor kinase receptors ALK4/5/7 than GDF8. Resolution of the GDF11:FS288 complex, apo-GDF8, and apo-GDF11 crystal structures reveals unique properties of both ligands, specifically in the type I receptor binding site. Lastly, substitution of GDF11 residues into GDF8 confers enhanced activity to GDF8. CONCLUSIONS: These studies identify distinctive structural features of GDF11 that enhance its potency, relative to GDF8; however, the biological consequences of these differences remain to be determined

A pre-ribosomal RNA interaction network involving snoRNAs and the Rok1 helicase

Ribosome biogenesis in yeast requires 75 small nucleolar RNAs (snoRNAs) and a myriad of cofactors for processing, modification, and folding of the ribosomal RNAs (rRNAs). For the 19 RNA helicases implicated in ribosome synthesis, their sites of action and molecular functions have largely remained unknown. Here, we have used UV cross-linking and analysis of cDNA (CRAC) to reveal the pre-rRNA binding sites of the RNA helicase Rok1, which is involved in early small subunit biogenesis. Several contact sites were identified in the 18S rRNA sequence, which interestingly all cluster in the “foot” region of the small ribosomal subunit. These include a major binding site in the eukaryotic expansion segment ES6, where Rok1 is required for release of the snR30 snoRNA. Rok1 directly contacts snR30 and other snoRNAs required for pre-rRNA processing. Using cross-linking, ligation and sequencing of hybrids (CLASH) we identified several novel pre-rRNA base-pairing sites for the snoRNAs snR30, snR10, U3, and U14, which cluster in the expansion segments of the 18S rRNA. Our data suggest that these snoRNAs bridge interactions between the expansion segments, thereby forming an extensive interaction network that likely promotes pre-rRNA maturation and folding in early pre-ribosomal complexes and establishes long-range rRNA interactions during ribosome synthesis