Pitchfork and Hopf bifurcation thresholds in stochastic equations with delayed feedback

The bifurcation diagram of a model stochastic differential equation with delayed feedback is presented. We are motivated by recent research on stochastic effects in models of transcriptional gene regulation. We start from the normal form for a pitchfork bifurcation, and add multiplicative or parametric noise and linear delayed feedback. The latter is sufficient to originate a Hopf bifurcation in that region of parameters in which there is a sufficiently strong negative feedback. We find a sharp bifurcation in parameter space, and define the threshold as the point in which the stationary distribution function p(x) changes from a delta function at the trivial state x=0 to p(x) ~ x^alpha at small x (with alpha = -1 exactly at threshold). We find that the bifurcation threshold is shifted by fluctuations relative to the deterministic limit by an amount that scales linearly with the noise intensity. Analytic calculations of the bifurcation threshold are also presented in the limit of small delay tau -> 0 that compare quite favorably with the numerical solutions even for tau = 1

Three dimensional characterization and archiving system

This system (3D-ICAS) is being developed as a remote system to perform rapid in situ analysis of hazardous organics and radionuclide contamination on structural materials. It is in the final phase of a 3-phase program to support Decontamination and Decommissioning (D&D) operations. Accurate physical characterization of surfaces and radioactive and organic contamination is a critical D&D task. Surface characterization includes identification of dangerous inorganic materials such as asbestos and transite. 3D-ICAS robotically conveys a multisensor probe near the surfaces to be inspected, using coherent laser radar tracking, which also provides 3D facility maps. High-speed automated organic analysis is provided by means of gas chromatograph-mass spectrometer sensor which can process a sample without contact in one minute. Volatile organics are extracted directly from contaminated surfaces without sample removal; multiple stage focusing is used for high time resolution. Additional discrimination is obtained through a final stage time-of-flight mass spectrometer. The radionuclide sensors combines {alpha}, {beta}, and {gamma} counting with energy discrimination of the {alpha} channel; this quantifies isotopes of U, Pu, Th, Tc, Np, and Am in one minute. The Molecular Vibrational Spectrometry sensor is used to characterize substrate material such as concrete, transite, wood, or asbestos; this can be used to provide estimates of the depth of contamination. The 3D-ICAS will be available for real-time monitoring immediately after each 1 to 2 minute sample period. After surface mapping, 3-D displays will be provided showing contours of detected contaminant concentrations. Permanent measurement and contaminant level archiving will be provided, assuring data integrity and allowing regulatory review before and after D&D operations

Solvated interaction energy: from small-molecule to antibody drug design

Scoring functions are ubiquitous in structure-based drug design as an aid to predicting binding modes and estimating binding affinities. Ideally, a scoring function should be broadly applicable, obviating the need to recalibrate and refit its parameters for every new target and class of ligands. Traditionally, drugs have been small molecules, but in recent years biologics, particularly antibodies, have become an increasingly important if not dominant class of therapeutics. This makes the goal of having a transferable scoring function, i.e., one that spans the range of small-molecule to protein ligands, even more challenging. One such broadly applicable scoring function is the Solvated Interaction Energy (SIE), which has been developed and applied in our lab for the last 15 years, leading to several important applications. This physics-based method arose from efforts to understand the physics governing binding events, with particular care given to the role played by solvation. SIE has been used by us and many independent labs worldwide for virtual screening and discovery of novel small-molecule binders or optimization of known drugs. Moreover, without any retraining, it is found to be transferrable to predictions of antibody-antigen relative binding affinities and as accurate as functions trained on protein-protein binding affinities. SIE has been incorporated in conjunction with other scoring functions into ADAPT (Assisted Design of Antibody and Protein Therapeutics), our platform for affinity modulation of antibodies. Application of ADAPT resulted in the optimization of several antibodies with 10-to-100-fold improvements in binding affinity. Further applications included broadening the specificity of a single-domain antibody to be cross-reactive with virus variants of both SARS-CoV-1 and SARS-CoV-2, and the design of safer antibodies by engineering of a pH switch to make them more selective towards acidic tumors while sparing normal tissues at physiological pH

Muscle activation during gait in children with Duchenne muscular dystrophy

The aim of this prospective study was to investigate changes in muscle activity during gait in children with Duchenne muscular Dystrophy (DMD). Dynamic surface electromyography recordings (EMGs) of 16 children with DMD and pathological gait were compared with those of 15 control children. The activity of the rectus femoris (RF), vastus lateralis (VL), medial hamstrings (HS), tibialis anterior (TA) and gastrocnemius soleus (GAS) muscles was recorded and analysed quantitatively and qualitatively. The overall muscle activity in the children with DMD was significantly different from that of the control group. Percentage activation amplitudes of RF, HS and TA were greater throughout the gait cycle in the children with DMD and the timing of GAS activity differed from the control children. Significantly greater muscle coactivation was found in the children with DMD. There were no significant differences between sides. Since the motor command is normal in DMD, the hyper-activity and co-contractions likely compensate for gait instability and muscle weakness, however may have negative consequences on the muscles and may increase the energy cost of gait. Simple rehabilitative strategies such as targeted physical therapies may improve stability and thus the pattern of muscle activity

Exploring rigid-backbone protein docking in biologics discovery: a test using the DARPin scaffold

Accurate protein-protein docking remains challenging, especially for artificial biologics not coevolved naturally against their protein targets, like antibodies and other engineered scaffolds. We previously developed ProPOSE, an exhaustive docker with full atomistic details, which delivers cutting-edge performance by allowing side-chain rearrangements upon docking. However, extensive protein backbone flexibility limits its practical applicability as indicated by unbound docking tests. To explore the usefulness of ProPOSE on systems with limited backbone flexibility, here we tested the engineered scaffold DARPin, which is characterized by its relatively rigid protein backbone. A prospective screening campaign was undertaken, in which sequence-diversified DARPins were docked and ranked against a directed epitope on the target protein BCL-W. In this proof-of-concept study, only a relatively small set of 2,213 diverse DARPin interfaces were selected for docking from the huge theoretical library from mutating 18 amino-acid positions. A computational selection protocol was then applied for enrichment of binders based on normalized computed binding scores and frequency of binding modes against the predefined epitope. The top-ranked 18 designed DARPin interfaces were selected for experimental validation. Three designs exhibited binding affinities to BCL-W in the nanomolar range comparable to control interfaces adopted from known DARPin binders. This result is encouraging for future screening and engineering campaigns of DARPins and possibly other similarly rigid scaffolds against targeted protein epitopes. Method limitations are discussed and directions for future refinements are proposed

N-(4-iodophenyl)-N′-(2-chloroethyl)urea as a microtubule disrupter: in vitro and in vivo profiling of antitumoral activity on CT-26 murine colon carcinoma cell line cultured and grafted to mice

The antitumoral profile of the microtubule disrupter N-(4-iodophenyl)-N′-(2-chloroethyl)urea (ICEU) was characterised in vitro and in vivo using the CT-26 colon carcinoma cell line, on the basis of the drug uptake by the cells, the modifications of cell cycle, and β-tubulin and lipid membrane profiles. N-(4-iodophenyl)-N′-(2-chloroethyl)urea exhibited a rapid and dose-dependent uptake by CT-26 cells suggesting its passive diffusion through the membranes. Intraperitoneally injected ICEU biodistributed into the grafted CT-26 tumour, resulting thus in a significant tumour growth inhibition (TGI). N-(4-iodophenyl)-N′-(2-chloroethyl)urea was also observed to accumulate within colon tissue. Tumour growth inhibition was associated with a slight increase in the number of G2 tetraploid tumour cells in vivo, whereas G2 blockage was more obvious in vitro. The phenotype of β-tubulin alkylation that was clearly demonstrated in vitro was undetectable in vivo. Nuclear magnetic resonance analysis showed that cells blocked in G2 phase underwent apoptosis, as confirmed by an increase in the methylene group resonance of mobile lipids, parallel to sub-G1 accumulation of the cells. In vivo, a decrease of the signals of both the phospholipid precursors and the products of membrane degradation occurred concomitantly with TGI. This multi-analysis established, at least partly, the ICEU activity profile, in vitro and in vivo, providing additional data in favour of ICEU as a tubulin-interacting drug accumulating within the intestinal tract. This may provide a starting point for researches for future efficacious tubulin-interacting drugs for the treatment of colorectal cancers

Involvement of TLR2 in Recognition of Acute Gammaherpesvirus-68 Infection

Toll-like receptors (TLRs) play a crucial role in the activation of innate immunity in response to many viruses. We previously reported the implication of TLR2 in the recognition of Epstein-Barr virus (EBV) by human monocytes. Because murine gammaherpesvirus-68 (MHV-68) is a useful model to study human gammaherpesvirus pathogenesis in vivo, we evaluated the importance of mouse TLR2 in the recognition of MHV-68.In studies using transfected HEK293 cells, MHV-68 lead to the activation of NF-κB reporter through TLR2. In addition, production of interleukin-6 (IL-6) and interferon-α (IFN-α) upon MHV-68 stimulation was reduced in murine embryonic fibroblasts (MEFs) derived from TLR2-/- and MyD88-/- mice as compared to their wild type (WT) counterpart. In transgenic mice expressing a luciferase reporter gene under the control of the mTLR2 promoter, MHV-68 challenge activated TLR2 transcription. Increased expression levels of TLR2 on blood granulocytes (CD115(-)Gr1(+)) and inflammatory monocytes (CD115(+)Gr1(+)), which mobilized to the lungs upon infection with MHV-68, was also confirmed by flow cytometry. Finally, TLR2 or MyD88 deficiency was associated with decreased IL-6 and type 1 IFN production as well as increased viral burden during short-term challenges with MHV-68.TLR2 contributes to the production of inflammatory cytokines and type 1 IFN as well as to the control of viral burden during infection with MHV-68. Taken together, our results suggest that the TLR2 pathway has a relevant role in the recognition of this virus and in the subsequent activation of the innate immune response

Determination of Molecular Structures of HIV Envelope Glycoproteins using Cryo-Electron Tomography and Automated Sub-tomogram Averaging

Since its discovery nearly 30 years ago, more than 60 million people have been infected with the human immunodeficiency virus (HIV) (www.usaid.gov). The virus infects and destroys CD4+ T-cells thereby crippling the immune system, and causing an acquired immunodeficiency syndrome (AIDS) 2. Infection begins when the HIV Envelope glycoprotein "spike" makes contact with the CD4 receptor on the surface of the CD4+ T-cell. This interaction induces a conformational change in the spike, which promotes interaction with a second cell surface co-receptor 5,9. The significance of these protein interactions in the HIV infection pathway makes them of profound importance in fundamental HIV research, and in the pursuit of an HIV vaccine

Evaluating the Distribution of African Swine Fever Virus Within a Feed Mill Environment Following Manufacture of Inoculated Feed

With the global spread of African swine fever virus (ASFV) and evidence that feed and/or ingredients may be potential vectors for pathogen transmission, it is critical to understand the role the feed manufacturing industry may have in regard to potential distribution of this highly virulent virus. A pilot-scale feed mill consisting of a mixer, bucket elevator, and relevant spouting was constructed in the Biosafety Level-3 Ag animal room at the Biosecurity Research Institute at Kansas State University. A total of 18 different sites on the equipment and in the room were swabbed to evaluate environmental contamination before and after introduction of ASFV-inoculated feedstuff. First, a batch of feed was manufactured through the system to confirm the feed mill was ASFV negative; then a feedstuff inoculated with ASFV was added into the mixer and manufactured with other, non-infected ingredients. Ingredients were mixed and discharged through the bucket elevator. Subsequently, four additional ASFV-free batches of feed were manufactured. Environmental swabs were collected after each batch of feed was discharged with locations categorized into four zones: A) feed contact surface, B) non-feed contact surface but \u3c 3.2 feet away from feed, C) non-feed contact surface \u3e 3.2 feet from feed, and D) transient surfaces such as worker shoes. Environmental swabs were analyzed using qPCR analysis for the P72 ASFV gene in a BSL-3 laboratory setting to detect ASFV-specific DNA. Environmental swabs collected prior to ASFV inoculation of feed were negative for ASFV DNA. Environmental swabs collected after the manufacture of ASFV-inoculated feed resulted in contamination of zones A-D. Contamination levels with ASFV-DNA are reported as Ct value or genomic copy number (CN) per mL. In this setup, there was no evidence of sampling zone × batch interaction and no difference in the proportion of ASFV positive reactions between sampling location or batch of feed throughout the experiment. This indicates that once ASFV contamination entered the facility, the contamination quickly becomes widespread and persists on the environmental surfaces, even during manufacturing of subsequent batches of ASFV non-inoculated feed. Samples from transient surfaces (Zone D) had more detectable ASFV (a lower Ct value) compared to all other surfaces (P \u3c 0.05), indicating high level of ASFV contamination (high CN values). Samples collected after manufacturing sequence 3 had less detectable ASFV (a greater Ct value) compared to samples collected immediately following manufacture of the ASFV-inoculated batch of feed (P \u3c 0.05), indicating lower levels of ASFV contamination (low CN values) in subsequent repeats of the feed production process. There was evidence of a sampling zone × batch interaction for the number of genomic copies/mL (P = 0.002). For samples collected after manufacture of the ASFV-inoculated batch of feed, a lower number of ASFV genomic copies/mL (higher Ct) was observed for swabs collected from non-feed contact surfaces \u3e 3.2 feet from feed (Zone C) compared to feed contact surfaces (zone A) (P \u3c 0.05), with other surfaces (zone B and D) having no evidence of a significant difference. Following manufacturing sequences 1, 2, and 3, samples collected from the transient surfaces (zone D) had a greater number of ASFV genomic copies/mL (low Ct) detected compared to other sampling locations (P \u3c 0.05). After manufacturing sequence 4, there was no evidence of a difference in the number of detected ASFV genomic copies/mL between sampling locations (P \u3e 0.05). In summary, once ASFV was experimentally introduced into a feed manufacturing environment, the virus became widely distributed throughout the facility with only minor changes in detection frequency as subsequent batches of feed were produced