Structure of vaccinia virus thymidine kinase in complex with dTTP: insights for drug design

BACKGROUND: Development of countermeasures to bioterrorist threats such as those posed by the smallpox virus (variola), include vaccination and drug development. Selective activation of nucleoside analogues by virus-encoded thymidine (dThd) kinases (TK) represents one of the most successful strategies for antiviral chemotherapy as demonstrated for anti-herpes drugs. Vaccinia virus TK is a close orthologue of variola TK but also shares a relatively high sequence identity to human type 2 TK (hTK), thus achieving drug selectivity relative to the host enzyme is challenging. RESULTS: In order to identify any differences compared to hTK that may be exploitable in drug design, we have determined the crystal structure of VVTK, in complex with thymidine 5'-triphosphate (dTTP). Although most of the active site residues are conserved between hTK and VVTK, we observe a difference in conformation of residues Asp-43 and Arg-45. The equivalent residues in hTK hydrogen bond to dTTP, whereas in subunit D of VVTK, Asp-43 and Arg-45 adopt a different conformation preventing interaction with this nucleotide. Asp-43 and Arg-45 are present in a flexible loop, which is disordered in subunits A, B and C. The observed difference in conformation and flexibility may also explain the ability of VVTK to phosphorylate (South)-methanocarbathymine whereas, in contrast, no substrate activity with hTK is reported for this compound. CONCLUSION: The difference in conformation for Asp-43 and Arg-45 could thus be used in drug design to generate VVTK/Variola TK-selective nucleoside analogue substrates and/or inhibitors that have lower affinity for hTK

Moving walls accelerate mixing

Mixing in viscous fluids is challenging, but chaotic advection in principle allows efficient mixing. In the best possible scenario,the decay rate of the concentration profile of a passive scalar should be exponential in time. In practice, several authors have found that the no-slip boundary condition at the walls of a vessel can slow down mixing considerably, turning an exponential decay into a power law. This slowdown affects the whole mixing region, and not just the vicinity of the wall. The reason is that when the chaotic mixing region extends to the wall, a separatrix connects to it. The approach to the wall along that separatrix is polynomial in time and dominates the long-time decay. However, if the walls are moved or rotated, closed orbits appear, separated from the central mixing region by a hyperbolic fixed point with a homoclinic orbit. The long-time approach to the fixed point is exponential, so an overall exponential decay is recovered, albeit with a thin unmixed region near the wall.Comment: 17 pages, 13 figures. PDFLaTeX with RevTeX 4-1 styl

Conformational flexibility of the oncogenic protein LMO2 primes the formation of the multi-protein transcription complex

LMO2 was discovered via chromosomal translocations in T-cell leukaemia and shown normally to be essential for haematopoiesis. LMO2 is made up of two LIM only domains (thus it is a LIM-only protein) and forms a bridge in a multi-protein complex. We have studied the mechanism of formation of this complex using a single domain antibody fragment that inhibits LMO2 by sequestering it in a non-functional form. The crystal structure of LMO2 with this antibody fragment has been solved revealing a conformational difference in the positioning and angle between the two LIM domains compared with its normal binding. This contortion occurs by bending at a central helical region of LMO2. This is a unique mechanism for inhibiting an intracellular protein function and the structural contusion implies a model in which newly synthesized, intrinsically disordered LMO2 binds to a partner protein nucleating further interactions and suggests approaches for therapeutic targeting of LMO2

Structure and assembly of the S-layer determine virulence in C. difficile

Many bacteria and archaea possess a cell surface layer – S-layer – made of a 2D protein array that covers the entire cell. As the outermost component of the cell envelope, S-layers play crucial roles in many aspects of cell physiology. Importantly, many clinically relevant bacterial pathogens possess a distinct S-layer that forms an initial interface with the host, making it a potential target for development of species-specific antimicrobials. Targeted therapeutics are particularly important for antibiotic resistant pathogens such as Clostridioides difficile, the most frequent cause of hospital acquired diarrhea, which relies on disruption of normal microbiota through antibiotic usage. Despite the ubiquity of S-layers, only partial structural information from a very limited number of species is available and their function and organization remains poorly understood. Here we report the first complete atomic level structure and in situ assembly model of an S-layer from a bacterial pathogen and reveal its role in disease severity. SlpA, the main C. difficile S-layer protein, assembles through tiling of triangular prisms abutting the cell wall, interlocked by distinct ridges facing the environment. This forms a tightly packed array, unlike the more porous S-layer models previously described. We report that removing one of the SlpA ridge features dramatically reduces disease severity, despite being dispensable for overall SlpA structure and S-layer assembly. Remarkably, the effect on disease severity is independent of toxin production and bacterial colonization within the mouse model of disease. Our work combines X-ray and electron crystallography to reveal a novel S-layer organization in atomic detail, highlighting the need for multiple technical approaches to obtain structural information on these paracrystalline arrays. These data also establish a direct link between specific structural elements of S-layer and virulence for the first time, in a crucial paradigm shift in our understanding of C. difficile disease, currently largely attributed to the action of potent toxins. This work highlights the crucial role of S-layers in pathogenicity and the importance of detailed structural information for providing new therapeutic avenues, targeting the S-layer. Understanding the interplay between S-layer and other virulence factors will further enhance our ability to tackle pathogens carrying an S-layer. We anticipate that this work provides a solid basis for development of new, C. difficile-specific therapeutics, targeting SlpA structure and S-layer assembly to reduce the healthcare burden of these infections.

High-resolution mapping of metal ions reveals principles of surface layer assembly in Caulobacter crescentus cells

Surface layers (S-layers) are proteinaceous crystalline coats that constitute the outermost component of most prokaryotic cell envelopes. In this study, we have investigated the role of metal ions in the formation of the Caulobacter crescentus S-layer using high-resolution structural and cell biology techniques, as well as molecular simulations. Utilizing optical microscopy of fluorescently tagged S-layers, we show that calcium ions facilitate S-layer lattice formation and cell-surface binding. We report all-atom molecular dynamics simulations of the S-layer lattice, revealing the importance of bound metal ions. Finally, using electron cryomicroscopy and long-wavelength X-ray diffraction experiments, we mapped the positions of metal ions in the S-layer at near-atomic resolution, supporting our insights from the cellular and simulations data. Our findings contribute to the understanding of how C. crescentus cells form a regularly arranged S-layer on their surface, with implications on fundamental S-layer biology and the synthetic biology of self-assembling biomaterials

Asuhan Kebidanan Berkelanjutan Pada Ny U.R Puskesmas Sagu Periode 22 April sampai 29 Juni 2019

Latar Belakang : Penyusunan Laporan Tugas Akhir adalah kegiatan belajar mengajar yang memberikan kesempatan kepada mahasiswa untuk mendapatkan pengalaman nyata dalam melaksanakan asuhan kebidanan yang komprehensif dalam lingkup kesehatan reproduksi.Data Puskesmas Sagu diperoleh tidak ada kematian bayi dan kematian ibu dalam 1 tahun terakhir. Sasaran ibu hamil di Puskesmas Sagu sebanyak 178 orang, jumlah persalinan sebanyak 194 orang, jumlah kunjungan nifas sebanyak 193 orang. Tujuan Penelitian : Diharapkan mahasiswa mampu menerapkan asuhan kebidanan berkelanjutan pada Ny.U.R Usia Kehamilan 37 Minggu 2 Hari Janin Hidup Tunggal Letak Kepala Intra Uterine Keadaan Jalan Lahir Normal Keadaan Ibu dan Janin Baik di Puskesmas Sagu Periode 22 April sampai 29 Juni 2019. Metode Penelitian : Jenis penelitian yang digunakan adalah studi penelaahan kasus. Studi kasus dilakukan pada Ny.U.R Usia Kehamilan 37 Minggu 2 Hari Janin Tunggal Hidup Intra Uterine Letak Kepala Keadaan Ibu dan Janin Baik di PuskesmaS Sagu Periode 22 April sampai 29 Juni 2019. Hasil : Setelah dilakukan asuhan kebidanan berkelanjutan pada Ny. U.R Usia Kehamilan 37 minggu 2 Hari Janin Tunggal Hidup Intra Uterine Letak Kepala Keadaan Ibu dan Janin Baik di Puskesmas Sagu Periode 22 April sampai 29 Juni 2019, ibu sudah melewati masa kehamilan, persalinan dan nifas dengan baik tanpa ada penyulit, bayi baru lahir dalam keadaan sehat, ibu belum menggunakan KB karena masih menunggu persetujuan suami. Kesimpulan : Asuhan kebidanan yang dilakukan pada Ny U.R mulai dari hamil, bersalin, BBL dan nifas, serta KB tidak ditemukan adanya kelainan dan penyulit yang menyertai

The ϕ6 Cystovirus Protein P7 Becomes Accessible to Antibodies in the Transcribing Nucleocapsid: A Probe for Viral Structural Elements

Protein P7 is a component of the cystovirus viral polymerase complex. In the unpackaged procapsid, the protein is situated in close proximity to the viral directed RNA polymerase, P2. Cryo-electron microscopy difference maps from the species ϕ6 procapsid have demonstrated that P7 and P2 likely interact prior to viral RNA packaging. The location of P7 in the post-packaged nucleocapsid (NC) remains unknown. P7 may translocate closer to the five-fold axis of a filled procapsid but this has not been directly visualized. We propose that monoclonal antibodies (Mabs) can be selected that serve as probe- reagents for viral assembly and structure. A set of Mabs have been isolated that recognize and bind to the ϕ6 P7. The antibody set contains five unique Mabs, four of which recognize a linear epitope and one which recognizes a conformational epitope. The four unique Mabs that recognize a linear epitope display restricted utilization of Vκ and VH genes. The restricted genetic range among 4 of the 5 antibodies implies that the antibody repertoire is limited. The limitation could be the consequence of a paucity of exposed antigenic sites on the ϕ6 P7 surface. It is further demonstrated that within ϕ6 nucleocapsids that are primed for early-phase transcription, P7 is partially accessible to the Mabs, indicating that the nucleocapsid shell (protein P8) has undergone partial disassembly exposing the protein’s antigenic sites

Contribution of DEAF1 Structural Domains to the Interaction with the Breast Cancer Oncogene LMO4

The proteins LMO4 and DEAF1 contribute to the proliferation of mammary epithelial cells. During breast cancer LMO4 is upregulated, affecting its interaction with other protein partners. This may set cells on a path to tumour formation. LMO4 and DEAF1 interact, but it is unknown how they cooperate to regulate cell proliferation. In this study, we identify a specific LMO4-binding domain in DEAF1. This domain contains an unstructured region that directly contacts LMO4, and a coiled coil that contains the DEAF1 nuclear export signal (NES). The coiled coil region can form tetramers and has the typical properties of a coiled coil domain. Using a simple cell-based assay, we show that LMO4 modulates the activity of the DEAF NES, causing nuclear accumulation of a construct containing the LMO4-interaction region of DEAF1

Comparative analysis of co-processed starches prepared by three different methods

Co-processing is currently of interest in the generation of high-functionality excipients for tablet formulation. In the present study, comparative analysis of the powder and tableting properties of three co-processed starches prepared by three different methods was carried out. The co-processed excipients consisting of maize starch (90%), acacia gum (7.5%) and colloidal silicon dioxide (2.5%) were prepared by co-dispersion (SAS-CD), co-fusion (SAS-CF) and co-granulation (SAS-CG). Powder properties of each co-processed excipient were characterized by measuring particle size, flow indices, particle density, dilution potential and lubricant sensitivity ratio. Heckel and Walker models were used to evaluate the compaction behaviour of the three co-processed starches. Tablets were produced with paracetamol as the model drug by direct compression on an eccentric Tablet Press fitted with 12 mm flat-faced punches and compressed at 216 MPa. The tablets were stored at room temperature for 24 h prior to evaluation. The results revealed that co-granulated co-processed excipient (SAS-CG) gave relatively better properties in terms of flow, compressibility, dilution potential, deformation, disintegration, crushing strength and friability. This study has shown that the method of co-processing influences the powder and tableting properties of the co-processed excipient