FirebotSLAM: Thermal SLAM to Increase Situational Awareness in Smoke-Filled Environments.

Operating in extreme environments is often challenging due to the lack of perceptual knowledge. During fire incidents in large buildings, the extreme levels of smoke can seriously impede a firefighter's vision, potentially leading to severe material damage and loss of life. To increase the safety of firefighters, research is conducted in collaboration with Dutch fire departments into the usability of Unmanned Ground Vehicles to increase situational awareness in hazardous environments. This paper proposes FirebotSLAM, the first algorithm capable of coherently computing a robot's odometry while creating a comprehensible 3D map solely using the information extracted from thermal images. The literature showed that the most challenging aspect of thermal Simultaneous Localization and Mapping (SLAM) is the extraction of robust features in thermal images. Therefore, a practical benchmark of feature extraction and description methods was performed on datasets recorded during a fire incident. The best-performing combination of extractor and descriptor is then implemented into a state-of-the-art visual SLAM algorithm. As a result, FirebotSLAM is the first thermal odometry algorithm able to perform global trajectory optimization by detecting loop closures. Finally, FirebotSLAM is the first thermal SLAM algorithm to be tested in a fiery environment to validate its applicability in an operational scenario.

Accurate Prediction of Transcriptional Activity of Single Missense Variants in HIV Tat with Deep Learning.

Tat is an essential gene for increasing the transcription of all HIV genes, and affects HIV replication, HIV exit from latency, and AIDS progression. The Tat gene frequently mutates in vivo and produces variants with diverse activities, contributing to HIV viral heterogeneity as well as drug-resistant clones. Thus, identifying the transcriptional activities of Tat variants will help to better understand AIDS pathology and treatment. We recently reported the missense mutation landscape of all single amino acid Tat variants. In these experiments, a fraction of double missense alleles exhibited intragenic epistasis. However, it is too time-consuming and costly to determine the effect of the variants for all double mutant alleles through experiments. Therefore, we propose a combined GigaAssay/deep learning approach. As a first step to determine activity landscapes for complex variants, we evaluated a deep learning framework using previously reported GigaAssay experiments to predict how transcription activity is affected by Tat variants with single missense substitutions. Our approach achieved a 0.94 Pearson correlation coefficient when comparing the predicted to experimental activities. This hybrid approach can be extensible to more complex Tat alleles for a better understanding of the genetic control of HIV genome transcription.

Most synonymous allelic variants in HIV tat are not silent.

The genetic code has degenerate codons that produce no change in the translated protein sequence and are generally thought to be silent. However, some synonymous variants are clearly not silent. Herein, we questioned the frequency of non-silent synonymous variants. We tested how random synonymous variants in the HIV Tat transcription factor effect transcription of an LTR-GFP reporter. Our model system has the advantage of directly measuring the function of the gene in human cells. Approximately, 67% of synonymous variants in Tat were non-silent, either having reduced activity or were full loss-of-function alleles. Eight mutant codons had higher codon usage than wild type, accompanied by reduced transcriptional activity. These were clustered on a loop in the Tat structure. We conclude that most synonymous Tat variants are not silent in human cells, and 25% are associated with changes in codon usage, likely effecting protein folding.

Data supporting a saturation mutagenesis assay for Tat-driven transcription with the GigaAssay.

The data in this article are associated with the research paper "GigaAssay - an adaptable high-throughput saturation mutagenesis assay" [1]. The raw data are sequence reads of HIV-1 Tat cDNA amplified from cellular genomic DNA in a new single-pot saturation mutagenesis assay designated the "GigaAssay". A bioinformatic pipeline and parameters used to analyze the data. Raw, processed, analyzed, and filtered data are reported. The data is processed to calculate the Tat-driven transcription activity for cells with each possible single amino acid substitution in Tat. This data can be reused to interpret Tat intermolecular interactions and HIV latency. This is one of the largest and most complete datasets regarding the impact of amino acid substitutions within a single protein on a molecular function.

GigaAssay - An adaptable high-throughput saturation mutagenesis assay platform.

High-throughput assay systems have had a large impact on understanding the mechanisms of basic cell functions. However, high-throughput assays that directly assess molecular functions are limited. Herein, we describe the "GigaAssay", a modular high-throughput one-pot assay system for measuring molecular functions of thousands of genetic variants at once. In this system, each cell was infected with one virus from a library encoding thousands of Tat mutant proteins, with each viral particle encoding a random unique molecular identifier (UMI). We demonstrate proof of concept by measuring transcription of a GFP reporter in an engineered reporter cell line driven by binding of the HIV Tat transcription factor to the HIV long terminal repeat. Infected cells were flow-sorted into 3 bins based on their GFP fluorescence readout. The transcriptional activity of each Tat mutant was calculated from the ratio of signals from each bin. The use of UMIs in the GigaAssay produced a high average accuracy (95%) and positive predictive value (98%) determined by comparison to literature benchmark data, known C-terminal truncations, and blinded independent mutant tests. Including the substitution tolerance with structure/function analysis shows restricted substitution types spatially concentrated in the Cys-rich region. Tat has abundant intragenic epistasis (10%) when single and double mutants are compared.

XRCC4 and MRE11 Roles and Transcriptional Response to Repair of TALEN-Induced Double-Strand DNA Breaks.

Double-strand breaks (DSB) are one of the most lethal forms of DNA damage that, if left unrepaired, can lead to genomic instability, cellular transformation, and cell death. In this work, we examined how repair of transcription activator-like effector nuclease (TALEN)-induced DNA damage was altered when knocking out, or inhibiting a function of, two DNA repair proteins, XRCC4 and MRE11, respectively. We developed a fluorescent reporter assay that uses TALENs to introduce DSB and detected repair by the presence of GFP fluorescence. We observed repair of TALEN-induced breaks in the XRCC4 knockout cells treated with mirin (a pharmacological inhibitor of MRE11 exonuclease activity), albeit with ~40% reduced efficiency compared to normal cells. Editing in the absence of XRCC4 or MRE11 exonuclease was robust, with little difference between the indel profiles amongst any of the groups. Reviewing the transcriptional profiles of the mirin-treated XRCC4 knockout cells showed 307 uniquely differentially expressed genes, a number far greater than for either of the other cell lines (the HeLa XRCC4 knockout sample had 83 genes, and the mirin-treated HeLa cells had 30 genes uniquely differentially expressed). Pathways unique to the XRCC4 knockout+mirin group included differential expression of p53 downstream pathways, and metabolic pathways indicating cell adaptation for energy regulation and stress response. In conclusion, our study showed that TALEN-induced DSBs are repaired, even when a key DSB repair protein or protein function is not operational, without a change in indel profiles. However, transcriptional profiles indicate the induction of unique cellular responses dependent upon the DNA repair protein(s) hampered.

Can Designer Indels Be Tailored by Gene Editing?: Can Indels Be Customized?

Genome editing with engineered nucleases (GEENs) introduce site-specific DNA double-strand breaks (DSBs) and repairs DSBs via nonhomologous end-joining (NHEJ) pathways that eventually create indels (insertions/deletions) in a genome. Whether the features of indels resulting from gene editing could be customized is asked. A review of the literature reveals how gene editing technologies via NHEJ pathways impact gene editing. The survey consolidates a body of literature that suggests that the type (insertion, deletion, and complex) and the approximate length of indel edits can be somewhat customized with different GEENs and by manipulating the expression of key NHEJ genes. Structural data suggest that binding of GEENs to DNA may interfere with binding of key components of DNA repair complexes, favoring either classical- or alternative-NHEJ. The hypotheses have some limitations, but if validated, will enable scientists to better control indel makeup, holding promise for basic science and clinical applications of gene editing. Also see the video abstract here https://youtu.be/vTkJtUsLi3w.

TALEN gene editing takes aim on HIV.

Transcription activator-like effector nucleases (TALENs) are one of several types of programmable, engineered nucleases that bind and cleave specific DNA sequences. Cellular machinery repairs the cleaved DNA by introducing indels. In this review, we emphasize the potential, explore progress, and identify challenges in using TALENs as a therapeutic tool to treat HIV infection. TALENs have less off-target editing and can be more effective at tolerating HIV escape mutations than CRISPR/Cas-9. Scientists have explored TALEN-mediated editing of host genes such as viral entry receptors (CCR5 and CXCR4) and a protein involved in proviral integration (LEDGF/p75). Viral targets include the proviral DNA, particularly focused on the long terminal repeats. Major challenges with translating gene therapy from bench to bedside are improving cleavage efficiency and delivery, while minimizing off-target editing, cytotoxicity, and immunogenicity. However, rapid improvements in TALEN technology are enhancing cleavage efficiency and specificity. Therapeutic testing in animal models of HIV infection will help determine whether TALENs are a viable HIV treatment therapy. TALENs or other engineered nucleases could shift the therapeutic paradigm from life-long antiretroviral therapy toward eradication of HIV infection.

Free energy cost of forming an interface between a crystal and its frozen version.

Using a thermodynamic integration scheme, we compute the free energy cost per unit area, γ, of forming an interface between a crystal and a frozen structured wall, formed by particles frozen into the same equilibrium structure as the crystal. Even though the structure and potential energy of the crystalline phase in the vicinity of the wall is the same as in the bulk, γ has a nonzero value and increases with increasing density of the crystal and the wall. Investigating the effect of several interaction potentials between the particles, we observe a positive γ at all crystalline densities if the potential is purely repulsive. For models with attractive interactions, such as the Lennard-Jones potential, a negative value for γ is obtained at low densities. A qualitative explanation for the change of sign of γ when going from repulsive to attractive interactions, at low crystal densities, is suggested.

Crystal growth kinetics in Lennard-Jones and Weeks-Chandler-Andersen systems along the solid-liquid coexistence line.

Kinetics of crystal-growth is investigated along the solid-liquid coexistence line for the (100), (110), and (111) orientations of the Lennard-Jones (LJ) and Weeks-Chandler-Andersen (WCA) fcc crystal-liquid interface, using non-equilibrium molecular dynamics simulations. A slowing down of the growth kinetics along the coexistence line is observed, which is due to the decrease of the melting enthalpy with increasing coexistence temperature and pressure. Other quantities such as the melting pressure and liquid self-diffusion coefficient have a comparatively lesser impact on the kinetic growth coefficient. Growth kinetics of the LJ and WCA potentials become similar at large values of the melting temperature and pressure, when both resemble a purely repulsive soft-sphere potential. Classical models of crystallization from the melt are in reasonable qualitative agreement with our simulation data. Finally, several one-phase empirical melting/freezing rules are studied with respect to their validity along the coexistence line.

Crystal-liquid interfacial free energy of hard spheres via a thermodynamic integration scheme.

The hard-sphere crystal-liquid interfacial free energy γcl is determined from molecular dynamics simulations using a thermodynamic integration (TI) scheme. The advantage of this TI scheme compared to previous methods is to successfully circumvent hysteresis effects due to the movement of the crystal-liquid interface. This is accomplished by the use of extremely-short-range and impenetrable Gaussian flat walls that prevent the drift of the interface while imposing a negligible free-energy penalty. We find that it is crucial to analyze finite-size effects in order to obtain reliable estimates of γcl in the thermodynamic limit.

Mycobacterial and HIV infections up-regulated human zinc finger protein 134, a novel positive regulator of HIV-1 LTR activity and viral propagation.

BACKGROUND: Concurrent occurrence of HIV and Tuberculosis (TB) infections influence the cellular environment of the host for synergistic existence. An elementary approach to understand such coalition at the molecular level is to understand the interactions of the host and the viral factors that subsequently effect viral replication. Long terminal repeats (LTR) of HIV genome serve as a template for binding trans-acting viral and cellular factors that regulate its transcriptional activity, thereby, deciding the fate of HIV pathogenesis, making it an ideal system to explore the interplay between HIV and the host. METHODOLOGY/PRINCIPAL FINDINGS: In this study, using biotinylated full length HIV-1 LTR sequence as bait followed by MALDI analyses, we identified and further characterized human-Zinc-finger-protein-134 (hZNF-134) as a novel positive regulator of HIV-1 that promoted LTR-driven transcription and viral production. Over-expression of hZNF-134 promoted LTR driven luciferase activity and viral transcripts, resulting in increased virus production while siRNA mediated knockdown reduced both the viral transcripts and the viral titers, establishing hZNF-134 as a positive effector of HIV-1. HIV, Mycobacteria and HIV-TB co-infections increased hZNF-134 expressions in PBMCs, the impact being highest by mycobacteria. Corroborating these observations, primary TB patients (n = 22) recorded extraordinarily high transcript levels of hZNF-134 as compared to healthy controls (n = 16). CONCLUSIONS/SIGNIFICANCE: With these observations, it was concluded that hZNF-134, which promoted HIV-1 LTR activity acted as a positive regulator of HIV propagation in human host. High titers of hZNF-134 transcripts in TB patients suggest that up-regulation of such positive effectors of HIV-1 upon mycobacterial infection can be yet another mechanism by which mycobacteria assists HIV-1 propagation during HIV-TB co-infections. hZNF-134, an uncharacterized host protein, thus assumes a novel regulatory role during HIV-host interactions. Our study provides new insights into the emerging role of zinc finger proteins in HIV-1 pathogenesis.

Crystal-liquid interfacial free energy via thermodynamic integration.

A novel thermodynamic integration (TI) scheme is presented to compute the crystal-liquid interfacial free energy (γcl) from molecular dynamics simulation. The scheme is applied to a Lennard-Jones system. By using extremely short-ranged and impenetrable Gaussian flat walls to confine the liquid and crystal phases, we overcome hysteresis problems of previous TI schemes that stem from the translational movement of the crystal-liquid interface. Our technique is applied to compute γcl for the (100), (110), and (111) orientation of the crystalline phase at three temperatures under coexistence conditions. For one case, namely, the (100) interface at the temperature T = 1.0 (in reduced units), we demonstrate that finite-size scaling in the framework of capillary wave theory can be used to estimate γcl in the thermodynamic limit. Thereby, we show that our TI scheme is not associated with the suppression of capillary wave fluctuations.

Human protein Staufen-2 promotes HIV-1 proliferation by positively regulating RNA export activity of viral protein Rev.

BACKGROUND: The export of intron containing viral RNAs from the nucleus to the cytoplasm is an essential step in the life cycle of Human Immunodeficiency Virus-1 (HIV-1). As the eukaryotic system does not permit the transport of intron containing RNA out of the nucleus, HIV-1 makes a regulatory protein, Rev, that mediates the transportation of unspliced and partially spliced viral mRNA from the nucleus to the cytoplasm, thereby playing a decisive role in the generation of new infectious virus particles. Therefore, the host factors modulating the RNA export activity of Rev can be major determinants of virus production in an infected cell. RESULTS: In this study, human Staufen-2 (hStau-2) was identified as a host factor interacting with HIV-1 Rev through affinity chromatography followed by MALDI analyses. Our experiments involving transient expressions, siRNA mediated knockdowns and infection assays conclusively established that hStau-2 is a positive regulator of HIV-1 pathogenesis. We demonstrated that Rev-hStau-2 interactions positively regulated the RNA export activity of Rev and promoted progeny virus synthesis. The Rev-hStau-2 interaction was independent of RNA despite both being RNA binding proteins. hStau-2 mutant, with mutations at Q314R-A318F-K319E, deficient of binding Rev, failed to promote hStau-2 dependent Rev activity and viral production, validating the essentiality of this protein-protein interaction. The expression of this positive regulator was elevated upon HIV-1 infection in both human T-lymphocyte and astrocyte cell lines. CONCLUSIONS: With this study, we establish that human Staufen-2, a host factor which is up-regulated upon HIV-1 infection, interacts with HIV-1 Rev, thereby promoting its RNA export activity and progeny virus formation. Altogether, our study provides new insights into the emerging role of the Staufen family of mRNA transporters in host-pathogen interaction and supports the notion that obliterating interactions between viral and host proteins that positively regulate HIV-1 proliferation can significantly contribute to anti-retroviral treatments.

Excess free energy of supercooled liquids at disordered walls.

Using a thermodynamic integration scheme, we compute the excess free energy, γ, of a glass-forming, binary Lennard-Jones liquid in contact with a frozen amorphous wall, formed by particles frozen into a similar structure as the liquid. We find that γ is nonzero, becoming negative at low temperature. This indicates that the thermodynamics of the system is perturbed by the effect of the amorphous wall.

Impact of viral factors on subcellular distribution and RNA export activity of HIV-1 rev in astrocytes 1321N1.

CNS associated cells are permissive to HIV-1 infection, but poor in virus production due to attenuated Rev activity. The temporal and the spatial distribution of Rev in human astrocyte 1321N1 and glioblastoma GO-G-CCM were monitored for explaining the reduced Rev activity and low viral production during HIV-1 infection. Rev remained localized to the nuclei of these cells upon infection, attenuating its export activity, as manifested by low copy number of RRE-containing viral-mRNA in the cytoplasm of these cells. In contrast to infection, when Rev alone was transiently expressed, it localized in the cytoplasm of 1321N1. The localization changed to the nucleus when Rev was expressed in the presence of other viral proteins through pro-viral DNA pNL4-3. This study, for the first time, revealed the impact of other HIV-1 proteins apart from host factors in regulating the subcellular localization of Rev in astrocytes and hence the fate of HIV-1 infection in these cells.

Lennard-Jones systems near solid walls: computing interfacial free energies from molecular simulation methods.

Different computational techniques in combination with molecular dynamics computer simulation are used to determine the wall-liquid and the wall-crystal interfacial free energies of a modified Lennard-Jones (LJ) system in contact with a solid wall. Two different kinds of solid walls are considered: a flat structureless wall and a structured wall consisting of an ideal crystal with the particles rigidly attached to fcc lattice sites. Interfacial free energies are determined by a thermodynamic integration scheme, the anisotropy of the pressure tensor, the non-equilibrium work method based on Bennett acceptance criteria, and a method using Cahn's adsorption equations based on the interfacial thermodynamics of Gibbs. For the flat wall, interfacial free energies as a function of different densities of the LJ liquid and as a function of temperature along the coexistence curve are calculated. In the case of a structured wall, the interaction strength between the wall and the LJ system and the lattice constant of the structured wall are varied. Using the values of the wall-liquid and wall-crystal interfacial energies along with the value for the crystal-liquid interfacial free energy determined previously for the same system by the "cleaving potential method," we obtain the contact angle as a function of various parameters; in particular, the conditions are found under which partial wetting occurs.

Discordance in CD4+T-cell levels and viral loads with co-occurrence of elevated peripheral TNF-α and IL-4 in newly diagnosed HIV-TB co-infected cases.

BACKGROUND: Cytokines are the hallmark of immune response to different pathogens and often dictate the disease outcome. HIV infection and tuberculosis (TB) are more destructive when confronted together than either alone. Clinical data related to the immune status of HIV-TB patients before the initiation of any drug therapy is not well documented. This study aimed to collect the baseline information pertaining to the immune status of HIV-TB co-infected patients and correlate the same with CD4+T cell levels and viral loads at the time of diagnosis prior to any drug therapy. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed the cytokines, CD4+T cell levels and viral loads to determine the immune environment in HIV-TB co-infection. The study involved four categories namely, Healthy controls (n=57), TB infected (n=57), HIV infected (n=59) and HIV-TB co-infected (n=57) patients. The multi-partite comparison and correlation between cytokines, CD4+T-cell levels and viral loads prior to drug therapy, showed an altered TH1 and TH2 response, as indicated by the cytokine profiles and skewed IFN-γ/IL-10 ratio. Inadequate CD4+T cell counts in HIV-TB patients did not correlate with high viral loads and vice-versa. When compared to HIV category, 34% of HIV-TB patients had concurrent high plasma levels of IL-4 and TNF-α at the time of diagnosis. TB relapse was observed in 5 of these HIV-TB co-infected patients who also displayed high IFN-γ/IL-10 ratio. CONCLUSION/SIGNIFICANCE: With these studies, we infer (i) CD4+T-cell levels as baseline criteria to report the disease progression in terms of viral load in HIV-TB co-infected patients can be misleading and (ii) co-occurrence of high TNF-α and IL-4 levels along with a high ratio of IFN-γ/IL-10, prior to drug therapy, may increase the susceptibility of HIV-TB co-infected patients to hyper-inflammation and TB relapse.

Wall-liquid and wall-crystal interfacial free energies via thermodynamic integration: a molecular dynamics simulation study.

A method is proposed to compute the interfacial free energy of a Lennard-Jones system in contact with a structured wall by molecular dynamics simulation. Both the bulk liquid and bulk face-centered-cubic crystal phase along the (111) orientation are considered. Our approach is based on a thermodynamic integration scheme where first the bulk Lennard-Jones system is reversibly transformed to a state where it interacts with a structureless flat wall. In a second step, the flat structureless wall is reversibly transformed into an atomistic wall with crystalline structure. The dependence of the interfacial free energy on various parameters such as the wall potential, the density and orientation of the wall is investigated. The conditions are indicated under which a Lennard-Jones crystal partially wets a flat wall.

Inertial effects in Büttiker-Landauer motor and refrigerator at the overdamped limit.

We investigate the energetics of a Brownian motor driven by position-dependent temperature, commonly known as the Büttiker-Landauer motor. Overdamped models (M=0) predict that the motor can attain Carnot efficiency. However, the overdamped limit (M-->0) contradicts the previous prediction due to the kinetic energy contribution to the heat transfer. Using molecular dynamics simulation and numerical solution of the inertial Langevin equation, we confirm that the motor can never achieve Carnot efficiency and verify that the heat flow via kinetic energy diverges as M{-1/2} in the overdamped limit. The reciprocal process of the motor, namely, the Büttiker-Landauer refrigerator, is also examined. In this case, the overdamped approach succeeds in predicting the heat transfer only when there is no temperature gradient. Its found that the Onsager symmetry between the motor and refrigerator does not suffer from the singular behavior of the kinetic energy contribution.