Writing DNA with GenoCAD™

Chemical synthesis of custom DNA made to order calls for software streamlining the design of synthetic DNA sequences. GenoCAD™ (www.genocad.org) is a free web-based application to design protein expression vectors, artificial gene networks and other genetic constructs composed of multiple functional blocks called genetic parts. By capturing design strategies in grammatical models of DNA sequences, GenoCAD guides the user through the design process. By successively clicking on icons representing structural features or actual genetic parts, complex constructs composed of dozens of functional blocks can be designed in a matter of minutes. GenoCAD automatically derives the construct sequence from its comprehensive libraries of genetic parts. Upon completion of the design process, users can download the sequence for synthesis or further analysis. Users who elect to create a personal account on the system can customize their workspace by creating their own parts libraries, adding new parts to the libraries, or reusing designs to quickly generate sets of related constructs

DNA-binding and non-DNA-binding forms of the transformed glucocorticoid receptor

In this work we have probed the mechanism responsible for two non-DNA-binding states of the mouse glucocorticoid receptor. In the first case, transformed receptors were treated with hydrogen peroxide. It is known that oxidizing agents promote the formation of disulfide bonds in the glucocorticoid receptor, but it has not been determined what domains are involved in any disulfide bond formation that leads to inactivation of DNA-binding activity. We show here that hydrogen peroxide inhibits DNA-binding by the 15-kDa tryptic fragment containing the DNA-binding fingers with the same concentration dependency as it inhibits DNA-binding by the uncleaved receptor. This suggests that all of the effect of peroxide is on sulfhydryl groups within the zinc fingers. After dissociation (transformation) of cytosolic heteromeric glucocorticoid receptor complexes, only a portion (40-60%) of the dissociated receptors can bind to DNA-cellulose. We show that the 15-kDA tryptic fragment derived from the portion of transformed receptors that do not bind to DNA is itself competent at DNA-binding.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/30198/1/0000586.pd

The hsp56 immunophilin component of steroid receptor heterocomplexes: Could this be the elusive nuclear localization signal-binding protein?

In many cells, the glucocorticoid receptor undergoes rapid steroid-mediated translocation from the cytoplasm to the nucleus, and this receptor is an excellent model for studying the mechanism of targeted protein movement through the cytoplasm. For such unidirectional movement to occur, the receptor must attach to a retrograde movement system in a manner that involves the nuclear localization signal. It is improbable that such attachment occurs via a direct protein-protein interaction between the receptor and the movement system; rather, one or more linker proteins are likely to be involved. As with other steroid receptors, the glucocorticoid receptor is associated with several other proteins in a heterocomplex. Two of these receptor-associated proteins are the heat shock proteins hsp90 and hsp56, and a third heat shock protein, hsp70, is required for assembly of the receptor heterocomplex. The hormone binding domain of the steroid receptors determines the interaction with both hsp90 and hsp70. Hsp56 is known to bind to hsp90, but its potential site, or sites, of interaction with the receptor are undefined. Hsp56 has recently been cloned and demonstrated to be an immunophilin of the FK506/rapamycin binding class. The immunophilins have peptidyl-prolyl isomerase activity but their cellular functions are unknown. Herein, we review the literature on the hsp56 immunophilin component of the receptor heterocomplex and present a rationale for hsp56 being the protein that determines the direction of receptor movement via a direct protein-protein interaction with the nuclear localization signal.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/30603/1/0000240.pd

A Versatile Computational Pipeline for Bacterial Genome Annotation Improvement and Comparative Analysis, with \u3cem\u3eBrucella\u3c/em\u3e as a Use Case

We present a bacterial genome computational analysis pipeline, called GenVar. The pipeline, based on the program GeneWise, is designed to analyze an annotated genome and automatically identify missed gene calls and sequence variants such as genes with disrupted reading frames (split genes) and those with insertions and deletions (indels). For a given genome to be analyzed, GenVar relies on a database containing closely related genomes (such as other species or strains) as well as a few additional reference genomes. GenVar also helps identify gene disruptions probably caused by sequencing errors. We exemplify GenVar’s capabilities by presenting results from the analysis of four Brucella genomes. Brucella is an important human pathogen and zoonotic agent. The analysis revealed hundreds of missed gene calls, new split genes and indels, several of which are species specific and hence provide valuable clues to the understanding of the genome basis of Brucella pathogenicity and host specificity

Economic Losses From COVID-19 Cases in the Philippines: A Dynamic Model of Health and Economic Policy Trade-Offs

The COVID-19 pandemic forced governments globally to impose lockdown measures and mobility restrictions to curb the transmission of the virus. As economies slowly reopen, governments face a trade-off between implementing economic recovery and health policy measures to control the spread of the virus and to ensure it will not overwhelm the health system. We developed a mathematical model that measures the economic losses due to the spread of the disease and due to different lockdown policies. This is done by extending the subnational SEIR model to include two differential equations that capture economic losses due to COVID-19 infection and due to the lockdown measures imposed by the Philippine government. We then proceed to assess the trade-off policy space between health and economic measures faced by the Philippine government. The study simulates the cumulative economic losses for 3 months in 8 scenarios across 5 regions in the country, including the National Capital Region (NCR), to capture the trade-off mechanism. These scenarios present the various combinations of either retaining or easing lockdown policies in these regions. Per region, the trade-off policy space was assessed through minimising the 3-month cumulative economic losses subject to the constraint that the average health-care utilisation rate (HCUR) consistently falls below 70%, which is the threshold set by the government before declaring that the health system capacity is at high risk. The study finds that in NCR, a policy trade-off exists where the minimum cumulative economic losses comprise 10.66% of its Gross Regional Domestic Product. Meanwhile, for regions that are non-adjacent to NCR, a policy that hinges on trade-off analysis does not apply. Nevertheless, for all simulated regions, it is recommended to improve and expand the capacity of the health system to broaden the policy space for the government in easing lockdown measures

Current status and unanswered questions on the use of Denosumab in giant cell tumor of bone

Denosumab is a monoclonal antibody to RANK ligand approved for use in giant cell tumour (GCT) of bone. Due to its efficacy, Denosumab is recommended as the first option in inoperable or metastatic GCT. Denosumab has also been used pre-operatively to downstage tumours with large soft tissue extension to allow for less morbid surgery. The role of Denosumab for conventional limb GCT of bone is yet to be defined. Further studies are required to determine whether local recurrence rates will be decreased with the adjuvant use of Denosumab along with surgery. The long term use and toxicity of this agent is unknown as is the proportion of patients with primary or secondary resistance. It is advised that complicated cases of GCT requiring Denosumab treatment should be referred and followed up at expert centres. Collaborative studies involving further clinical trials and rigorous data collection are strongly recommended to identify the optimum use of this drug

Rickettsia Phylogenomics: Unwinding the Intricacies of Obligate Intracellular Life

BACKGROUND: Completed genome sequences are rapidly increasing for Rickettsia, obligate intracellular alpha-proteobacteria responsible for various human diseases, including epidemic typhus and Rocky Mountain spotted fever. In light of phylogeny, the establishment of orthologous groups (OGs) of open reading frames (ORFs) will distinguish the core rickettsial genes and other group specific genes (class 1 OGs or C1OGs) from those distributed indiscriminately throughout the rickettsial tree (class 2 OG or C2OGs). METHODOLOGY/PRINCIPAL FINDINGS: We present 1823 representative (no gene duplications) and 259 non-representative (at least one gene duplication) rickettsial OGs. While the highly reductive (approximately 1.2 MB) Rickettsia genomes range in predicted ORFs from 872 to 1512, a core of 752 OGs was identified, depicting the essential Rickettsia genes. Unsurprisingly, this core lacks many metabolic genes, reflecting the dependence on host resources for growth and survival. Additionally, we bolster our recent reclassification of Rickettsia by identifying OGs that define the AG (ancestral group), TG (typhus group), TRG (transitional group), and SFG (spotted fever group) rickettsiae. OGs for insect-associated species, tick-associated species and species that harbor plasmids were also predicted. Through superimposition of all OGs over robust phylogeny estimation, we discern between C1OGs and C2OGs, the latter depicting genes either decaying from the conserved C1OGs or acquired laterally. Finally, scrutiny of non-representative OGs revealed high levels of split genes versus gene duplications, with both phenomena confounding gene orthology assignment. Interestingly, non-representative OGs, as well as OGs comprised of several gene families typically involved in microbial pathogenicity and/or the acquisition of virulence factors, fall predominantly within C2OG distributions. CONCLUSION/SIGNIFICANCE: Collectively, we determined the relative conservation and distribution of 14354 predicted ORFs from 10 rickettsial genomes across robust phylogeny estimation. The data, available at PATRIC (PathoSystems Resource Integration Center), provide novel information for unwinding the intricacies associated with Rickettsia pathogenesis, expanding the range of potential diagnostic, vaccine and therapeutic targets

Cytoplasmic-nuclear transport of the glucocorticoid receptor: Implications forhsp56 and the cytoskeleton.

When steroid receptors are translated, they are assembled into heterocomplexes that contain hsp90, hsp56, p23, and often some hsp70, a complex which also exists independently of steroid receptors. The glucocorticoid receptor (GR) is a cytoplasmic steroid receptor that binds glucocorticoids and translocates to the nucleus to act as a ligand-dependent transcription factor. This thesis focuses on the role of hsp56 in the GR heterocomplex, and the process by which the GR moves to the nucleus. In Chapter 2, I determine the protein-protein interactions of the GR heterocomplex. Highly purified mouse hsp90, but not hsp70, binds to purified hsp56 in an equilibrium manner. hsp90 does not bind to purified hsp70 unless another factor is present. This is consistent with the model that hsp70 and hsp56 both bind to hsp90 at different sites and do not interact with each other. hsp56 is weakly associated with the GR complex isolated from WCL2 cytosol, and it is stabilized in the complex by the presence of molybdate. Localization of hsp56 might provide a clue to its function. Immunofluorescent localization of hsp56 reveals primarily a nonrandom nuclear localization identical to the nuclear GR of WCL2 cells indicating it may play a role in the nuclear activity of the GR. hsp56 also localizes to microtubules, a pathway along which the GR could potentially move into the nucleus following steroid-binding. A conserved region of hsp56 is electrostatically complementary to nuclear localization signals, and may recognize the GR's nuclear localization signal, which becomes active following steroid binding. Microinjection of antibodies against this region of hsp56 into L(M)tk\sp-cells impedes the steroid-dependent translocation of the GR that normally occurs with a half-time of approximately 5 minutes. The rapid translocation of the receptor might be directed along cytoskeletal fibrils, however disruption of the three cytoskeletal systems using colcemid in combination with cytochalasin does not change the rate of translocation. To determine if a cytoskeletal pathway exists following this treatment, rat pulmonary endothelial cells were prepared for immunofluorescence to visualize proteins of the three cytoskeletal systems and the receptor-associated proteins hsp90 and p23. While microtubules and microfilaments are completely depolymerized, intermediate filaments maintain a potentially viable pathway for receptor movement and hsp90 and p23 are localized to these filaments. In summary this thesis provides evidence that both hsp56 and the cytoskeleton play a role in the nuclear translocation of the glucocorticoid receptor.Ph.D.PharmacologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/104933/1/9624594.pdfDescription of 9624594.pdf : Restricted to UM users only

doi:10.1093/nar/gkp361 Writing DNA with GenoCAD TM

Chemical synthesis of custom DNA made to order calls for software streamlining the design of synthetic DNA sequences. GenoCAD TM (www. genocad.org) is a free web-based application to design protein expression vectors, artificial gene networks and other genetic constructs composed of multiple functional blocks called genetic parts. By capturing design strategies in grammatical models of DNA sequences, GenoCAD guides the user through the design process. By successively clicking on icons representing structural features or actual genetic parts, complex constructs composed of dozens of functional blocks can be designed in a matter of minutes. GenoCAD automatically derives the construct sequence from its comprehensive libraries of genetic parts. Upon completion of the design process, users can download the sequence for synthesis or further analysis. Users who elect to create a personal account on the system can customize their workspace by creating their own parts libraries, adding new parts to the libraries, or reusing designs to quickly generate sets of related constructs