Challenges and shortcomings of antibacterial discovery projects.

OBJECTIVES: Antibacterial drug discovery activities are essential for filling clinical pipelines with promising clinical candidates. Little information is available about the challenges and shortcomings of small companies and academic institutions in performing these important discovery tasks. METHODS: We performed a content analysis of 463 reviewer comments on 91 funding applications of antibacterial drug discovery projects submitted to two major global funders between 2016 and 2020 that had not proceeded further in the selection process. This quality assessment was complemented with the inputs (via e-mail) from a panel involving six antibiotic research and development (R&D) experts with long-standing expertise and experience in antibiotic drug discovery. RESULTS: Common critical comments of reviewers are grouped into three main categories: scientific and technical shortcomings, unclear potential societal impact, and insufficient capability and expertise of the project team regarding the R&D process. Insufficient characterization of in vitro activity and/or testing of the hits/leads and insufficient antibacterial activity were the most common critical comments. Other areas of concern were insufficient or lack of differentiation from available drugs or projects with a long R&D history, and the research team's insufficient knowledge of a structured streamlined R&D process as reflected in severe gaps in the expertise of the R&D team. Little appreciation for the problem of the emergence of target-based resistance, especially in single-target approaches, and little awareness of toxicological issues, including approaches with historical liabilities were also commonly mentioned. The shortcomings identified through the analysis of funding applications are echoed by the results of the expert panel. DISCUSSION: Our analysis identified an urgent need of strengthening the support for antibacterial drug discovery teams to help more projects reach such a quality to be eligible for global funders and private investors.

Optimization of Vascular Supply in Free Flaps for Head and Neck Reconstruction: Analysis of a Young Team's Experience.

Background: For head and neck reconstructive procedures, free flap survival depends on microsurgical and anatomical choices besides multimodal clinical management. The aim of the present study is to identify relevant variables for flap survival in our initial consecutive series. Methods: A single-center, novel reconstructive team consecutive surgical series was revised. The outcome was analyzed in terms of flap survival observing variables considered more relevant: flap type, recipient artery, vein(s), and graft interposition were discussed for facial thirds to be reconstructed. Statistical analysis was performed with Chi-square, Mann-Whitney, and Odds ratio. Results: A total of 118 free flaps were performed in 115 microsurgical procedures (93.9% for malignancies) on 109 patients, with a flap survival rate of 91.5%. For reconstruction of the middle and lower third of the face, the facial artery was privileged, because it was already transected during lymph node dissection in order to save the superior thyroid artery for further microsurgical needs. Flap failure was 50% venous. Double vein anastomosis was not related to flap survival. Deep venous drainage (as the internal jugular vein system) required fewer revisions. Half of the re-explorations saved the flap. Grafts were a risk for flap survival. Bony flaps were more critical. Conclusion: At comparable reconstructive quality, flap choice should avoid a vascular graft. The facial artery is a preferable recipient vessel, since it saves other arteries both in the case of an arterial revision and in the case of recurrence, for further free flap reconstruction. For venous anastomosis, a deep venous recipient is safer, since it offers the possibility to choose the level of anastomosis optimizing the vascular pedicle geometry. A close postsurgical flap monitoring is advisable up to 7 days postoperatively to allow for timely flap salvage.

Activity-dependent spinal cord neuromodulation rapidly restores trunk and leg motor functions after complete paralysis.

Epidural electrical stimulation (EES) targeting the dorsal roots of lumbosacral segments restores walking in people with spinal cord injury (SCI). However, EES is delivered with multielectrode paddle leads that were originally designed to target the dorsal column of the spinal cord. Here, we hypothesized that an arrangement of electrodes targeting the ensemble of dorsal roots involved in leg and trunk movements would result in superior efficacy, restoring more diverse motor activities after the most severe SCI. To test this hypothesis, we established a computational framework that informed the optimal arrangement of electrodes on a new paddle lead and guided its neurosurgical positioning. We also developed software supporting the rapid configuration of activity-specific stimulation programs that reproduced the natural activation of motor neurons underlying each activity. We tested these neurotechnologies in three individuals with complete sensorimotor paralysis as part of an ongoing clinical trial ( www.clinicaltrials.gov identifier NCT02936453). Within a single day, activity-specific stimulation programs enabled these three individuals to stand, walk, cycle, swim and control trunk movements. Neurorehabilitation mediated sufficient improvement to restore these activities in community settings, opening a realistic path to support everyday mobility with EES in people with SCI.

Microsurgical Three-Dimensional Reconstruction of Complex Nasal and Midfacial Defect: Multistep Procedure Respecting Aesthetic Unit Criteria.

ABSTRACT: Extended tumor resection in the middle third of the face leads to complex defects: wide, 3-dimensional, and multitissutal. Appropriate reconstruction is challenging but mandatory to obtain a functional and aesthetic outcome for the preservation of an acceptable quality of life. Three-dimensional combined flaps and multistep procedures concur to reach this scope.This is exemplified on the treatment of an invasive recurrent skin malignancy involving the cheek and maxillary bone in association with a full-thickness nasal defect. Reconstruction was performed with 3-dimensional multifolded anterolateral tigh chimeric flap, followed by multistep procedure respecting the aesthetic nasal reconstruction guidelines. Reconstructive surgery had the following targets: targets: rebuilding the oral and nasal lining, filling the paranasal cavities, covering the facial skin defect respecting the aesthetic unit concept and providing a proper support to the facial structures.The aesthetic unit concept has to be respected throughout all steps, from tumor debulking, to reconstruction and even for the management of complications.

Simulating Market Entry Rewards for Antibiotics Development.

We design an agent based Monte Carlo model of antibiotics research and development (R&D) to explore the effects of the policy intervention known as Market Entry Reward (MER) on the likelihood that an antibiotic entering pre-clinical development reaches the market. By means of sensitivity analysis we explore the interaction between the MER and four key parameters: projected net revenues, R&D costs, venture capitalists discount rates, and large pharmaceutical organizations' financial thresholds. We show that improving revenues may be more efficient than reducing costs, and thus confirm that this pull-based policy intervention effectively stimulates antibiotics R&D.

Herpesvirus deconjugases inhibit the IFN response by promoting TRIM25 autoubiquitination and functional inactivation of the RIG-I signalosome.

The N-terminal domains of the herpesvirus large tegument proteins encode a conserved cysteine protease with ubiquitin- and NEDD8-specific deconjugase activity. The proteins are expressed during the productive virus cycle and are incorporated into infectious virus particles, being delivered to the target cells upon primary infection. Members of this viral enzyme family were shown to regulate different aspects of the virus life cycle and the innate anti-viral response. However, only few substrates have been identified and the mechanisms of these effects remain largely unknown. In order to gain insights on the substrates and signaling pathways targeted by the viral enzymes, we have used co-immunoprecipitation and mass spectrometry to identify cellular proteins that interact with the Epstein-Barr virus encoded homologue BPLF1. Several members of the 14-3-3-family of scaffold proteins were found amongst the top hits of the BPLF1 interactome, suggesting that, through this interaction, BPLF1 may regulate a variety of cellular signaling pathways. Analysis of the shared protein-interaction network revealed that BPLF1 promotes the assembly of a tri-molecular complex including, in addition to 14-3-3, the ubiquitin ligase TRIM25 that participates in the innate immune response via ubiquitination of cytosolic pattern recognition receptor, RIG-I. The involvement of BPLF1 in the regulation of this signaling pathway was confirmed by inhibition of the type-I IFN responses in cells transfected with a catalytically active BPLF1 N-terminal domain or expressing the endogenous protein upon reactivation of the productive virus cycle. We found that the active viral enzyme promotes the dimerization and autoubiquitination of TRIM25. Upon triggering of the IFN response, RIG-I is recruited to the complex but ubiquitination is severely impaired, which functionally inactivates the RIG-I signalosome. The capacity to bind to and functionally inactivate the RIG-I signalosome is shared by the homologues encoded by other human herpesviruses.

Individual-based modelling of population growth and diffusion in discrete time.

Individual-based models (IBMs) of human populations capture spatio-temporal dynamics using rules that govern the birth, behavior, and death of individuals. We explore a stochastic IBM of logistic growth-diffusion with constant time steps and independent, simultaneous actions of birth, death, and movement that approaches the Fisher-Kolmogorov model in the continuum limit. This model is well-suited to parallelization on high-performance computers. We explore its emergent properties with analytical approximations and numerical simulations in parameter ranges relevant to human population dynamics and ecology, and reproduce continuous-time results in the limit of small transition probabilities. Our model prediction indicates that the population density and dispersal speed are affected by fluctuations in the number of individuals. The discrete-time model displays novel properties owing to the binomial character of the fluctuations: in certain regimes of the growth model, a decrease in time step size drives the system away from the continuum limit. These effects are especially important at local population sizes of <50 individuals, which largely correspond to group sizes of hunter-gatherers. As an application scenario, we model the late Pleistocene dispersal of Homo sapiens into the Americas, and discuss the agreement of model-based estimates of first-arrival dates with archaeological dates in dependence of IBM model parameter settings.

The Epstein-Barr virus miR-BHRF1-1 targets RNF4 during productive infection to promote the accumulation of SUMO conjugates and the release of infectious virus.

Post-translational modification by the Small Ubiquitin-like Modifier (SUMO) regulates a variety of cellular functions, and is hijacked by viruses to remodel the host cell during latent and productive infection. Here we have monitored the activity of the SUMO conjugation machinery in cells productively infected with Epstein-Barr virus (EBV). We found that SUMO2/3 conjugates accumulate during the late phase of the productive virus cycle, and identified several viral proteins as bone fide SUMOylation substrates. Analysis of the mechanism involved in the accumulation of SUMOylated proteins revealed upregulation of several components of the SUMO-conjugation machinery and post-transcriptional downregulation of the SUMO-targeted ubiquitin ligase RNF4. The latter effect was mediated by selective inhibition of RNF4 protein expression by the viral miR-BHRF1-1. Reconstitution of RNF4 in cells expressing an inducible miR-BHRF1-1 sponge or a miR-BHRF1-1 resistant RNF4 was associated with reduced levels of early and late viral proteins and impaired virus release. These findings illustrate a novel strategy for viral interference with the SUMO pathway, and identify the EBV miR-BHRF1-1 and the cellular RNF4 as regulators of the productive virus cycle.

Electric field induced structural colour tuning of a silver/titanium dioxide nanoparticle one-dimensional photonic crystal.

An electric field is employed for the active tuning of the structural colour in photonic crystals, which acts as an effective external stimulus with an impact on light transmission manipulation. In this work, we demonstrate structural colour in a photonic crystal device comprised of alternating layers of silver nanoparticles and titanium dioxide nanoparticles, exhibiting spectral shifts of around 10 nm for an applied voltage of only 10 V. The accumulation of charge at the metal/dielectric interface with an applied electric field leads to an effective increase of the charges contributing to the plasma frequency in silver. This initiates a blue shift of the silver plasmon band with a simultaneous blue shift of the photonic band gap as a result of the change in the silver dielectric function (i.e. decrease of the effective refractive index). These results are the first demonstration of active colour tuning in silver/titanium dioxide nanoparticle-based photonic crystals and open the route to metal/dielectric-based photonic crystals as electro-optic switches.

Epstein-Barr virus encoded microRNAs target SUMO-regulated cellular functions.

Post-translational modification by the small ubiquitin-like modifier (SUMO) regulates the cellular response to different types of stress and plays a pivotal role in the control of oncogenic viral infections. Here we investigated the capacity of microRNAs (miRNAs) encoded by Epstein-Barr virus to interfere with the SUMO signaling network. Using a computational strategy that scores different properties of miRNA-mRNA target pairs, we identified a minimal set of 575 members of the SUMO interactome that may be targeted by one or more Epstein-Barr virus miRNAs. A significant proportion of the candidates cluster in a functional network that controls chromatin organization, stress, DNA damage and immune responses, apoptosis and transforming growth factor beta signaling. Multiple components of the transforming growth factor beta signaling pathway were inhibited upon upregulation of the BamHI-H rightward open reading frame 1 (BHRF1) encoded miRNAs in cells transduced with recombinant lentiviruses or entering the productive virus cycle. These findings point to the capacity of viral miRNAs to interfere with SUMO-regulated cellular functions that control key aspects of viral replication and pathogenesis.

Caspase-1 promotes Epstein-Barr virus replication by targeting the large tegument protein deneddylase to the nucleus of productively infected cells.

The large tegument proteins of herpesviruses contain N-terminal cysteine proteases with potent ubiquitin and NEDD8-specific deconjugase activities, but the function of the enzymes during virus replication remains largely unknown. Using as model BPLF1, the homologue encoded by Epstein-Barr virus (EBV), we found that induction of the productive virus cycle does not affect the total level of ubiquitin-conjugation but is accompanied by a BPLF1-dependent decrease of NEDD8-adducts and accumulation of free NEDD8. Expression of BPLF1 promotes cullin degradation and the stabilization of cullin-RING ligases (CRLs) substrates in the nucleus, while cytoplasmic CRLs and their substrates are not affected. The inactivation of nuclear CRLs is reversed by the N-terminus of CAND1, which inhibits the binding of BPLF1 to cullins and prevents efficient viral DNA replication. Targeting of the deneddylase activity to the nucleus is dependent on processing of the catalytic N-terminus by caspase-1. Inhibition of caspase-1 severely impairs viral DNA synthesis and the release of infectious virus, pointing a previously unrecognized role of the cellular response to danger signals triggered by EBV reactivation in promoting virus replication.

Interaction of gamma-herpesvirus genome maintenance proteins with cellular chromatin.

The capacity of gamma-herpesviruses to establish lifelong infections is dependent on the expression of genome maintenance proteins (GMPs) that tether the viral episomes to cellular chromatin and allow their persistence in latently infected proliferating cells. Here we have characterized the chromatin interaction of GMPs encoded by viruses belonging to the genera Lymphocryptovirus (LCV) and Rhadinovirus (RHV). We found that, in addition to a similar diffuse nuclear localization and comparable detergent resistant interaction with chromatin in transfected cells, all GMPs shared the capacity to promote the decondensation of heterochromatin in the A03-1 reporter cell line. They differed, however, in their mobility measured by fluorescence recovery after photobleaching (FRAP), and in the capacity to recruit accessory molecules required for the chromatin remodeling function. While the AT-hook containing GMPs of LCVs were highly mobile, a great variability was observed among GMPs encoded by RHV, ranging from virtually immobile to significantly reduced mobility compared to LCV GMPs. Only the RHV GMPs recruited the bromo- and extra terminal domain (BET) proteins BRD2 and BRD4 to the site of chromatin remodeling. These findings suggest that differences in the mode of interaction with cellular chromatin may underlie different strategies adopted by these viruses for reprogramming of the host cells during latency.

An N-terminal SIAH-interacting motif regulates the stability of the ubiquitin specific protease (USP)-19.

The Ubiquitin Specific Protease-19 (USP19) regulates cell cycle progression and is involved in the cellular response to different types of stress, including the unfolded protein response (UPR), hypoxia and muscle atrophy. Using the unique N-terminal domain as bait in a yeast-two hybrid screen we have identified the ubiquitin ligases Seven In Absentia Homolog (SIAH)-1 and SIAH2 as binding partners of USP19. The interaction is mediated by a SIAH-consensus binding motif and promotes USP19 ubiquitylation and proteasome-dependent degradation. These findings identify USP19 as a common substrate of the SIAH ubiquitin ligases.

The Epstein-Barr virus nuclear antigen-1 reprograms transcription by mimicry of high mobility group A proteins.

Viral proteins reprogram their host cells by hijacking regulatory components of protein networks. Here we describe a novel property of the Epstein-Barr virus (EBV) nuclear antigen-1 (EBNA1) that may underlie the capacity of the virus to promote a global remodeling of chromatin architecture and cellular transcription. We found that the expression of EBNA1 in transfected human and mouse cells is associated with decreased prevalence of heterochromatin foci, enhanced accessibility of cellular DNA to micrococcal nuclease digestion and decreased average length of nucleosome repeats, suggesting de-protection of the nucleosome linker regions. This is a direct effect of EBNA1 because targeting the viral protein to heterochromatin promotes large-scale chromatin decondensation with slow kinetics and independent of the recruitment of adenosine triphosphate-dependent chromatin remodelers. The remodeling function is mediated by a bipartite Gly-Arg rich domain of EBNA1 that resembles the AT-hook of High Mobility Group A (HMGA) architectural transcription factors. Similar to HMGAs, EBNA1 is highly mobile in interphase nuclei and promotes the mobility of linker histone H1, which counteracts chromatin condensation and alters the transcription of numerous cellular genes. Thus, by regulating chromatin compaction, EBNA1 may reset cellular transcription during infection and prime the infected cells for malignant transformation.

Herpes virus deneddylases interrupt the cullin-RING ligase neddylation cycle by inhibiting the binding of CAND1.

The conserved N-terminal domains of the major tegument proteins of herpes viridae encode cysteine proteases with potent ubiquitin and NEDD8-specific deconjugase activity. Here we show that the Epstein-Barr virus-encoded member of this enzyme family, BPLF1, is targeted to cullin-RING ubiquitin ligases (CRLs) via the interaction of the conserved helix-2 with helix-23 of the C-terminal domain (CTD) of cullins, at a site involved in electrostatic interaction with helix-8 of the CRL regulator CAND1. Mutation of the solvent-exposed Asp86 and Asp90 of helix-2 to Arg does not affect the enzymatic activity of BPLF1 but abolishes cullin binding and prevents CRL inactivation. The binding of the catalytically active BPLF1 to cullins inhibits the recruitment of CAND1 to the deneddylated CRLs and promotes the selective degradation of cullins by the proteasome. Cullin proteolysis is rescued by the overexpression of CAND1 or its CTD-binding N-terminal domain. These findings illustrate a new strategy for viral modulation of CRL activity where the combined effects of cullin deneddylation and their targeting for proteasomal degradation drive stable inactivation of the ligases.

Bacterial genotoxin triggers FEN1-dependent RhoA activation, cytoskeleton remodeling and cell survival.

The DNA damage response triggered by bacterial cytolethal distending toxins (CDTs) is associated with activation of the actin-regulating protein RhoA and phosphorylation of the downstream-regulated mitogen-activated protein kinase (MAPK) p38, which promotes the survival of intoxicated (i.e. cells exposed to a bacterial toxin) cells. To identify the effectors of this CDT-induced survival response, we screened a library of 4492 Saccharomyces cerevisiae mutants that carry deletions in nonessential genes for reduced growth following inducible expression of CdtB. We identified 78 genes whose deletion confers hypersensitivity to toxin. Bioinformatics analysis revealed that DNA repair and endocytosis were the two most overrepresented signaling pathways. Among the human orthologs present in our data set, FEN1 and TSG101 regulate DNA repair and endocytosis, respectively, and also share common interacting partners with RhoA. We further demonstrate that FEN1, but not TSG101, regulates cell survival, MAPK p38 phosphorylation, RhoA activation and actin cytoskeleton reorganization in response to DNA damage. Our data reveal a previously unrecognized crosstalk between DNA damage and cytoskeleton dynamics in the regulation of cell survival, and might provide new insights on the role of chronic bacteria infection in carcinogenesis.

Ultrasound-guided percutaneous injection of triamcinolone acetonide for treating capsular contracture in patients with augmented and reconstructed breast.

OBJECTIVES: To evaluate ultrasound (US)-guided treatment of capsular contracture (CC) in patients with reconstructed/augmented breast. METHODS: Twenty-five patients with grade IV CC were treated with peri-implant US-guided injection of triamcinolone acetonide. Before/after treatment, maximum capsular thickness (MCT) was measured by ultrasound and pain assessed with visual analogue score (pain-VAS). Patients with pain relief at 1 month were considered early responders (ERs). Another injection was performed in patients without pain relief at 1 month (late responders, LRs). RESULTS: One patient (treated with chemo-radiotherapy) experienced severe pain and local reaction after the second injection, requiring surgery. Twenty-four patients had baseline MCT of 1.8 ± 0.3 mm and pain-VAS of 4.9 ± 0.5, the baseline MCT of 19 ERs (1.7 ± 0.2 mm) being significantly lower than that of 5 LRs (2.1 ± 0.2 mm) (p = 0.030). ERs had significantly reduced MCT and pain-VAS at one (1.1 ± 0.3 mm; 1.5 ± 0.5) and 6 months (1.1 ± 0.2 mm; 0.9 ± 0.7, respectively) (p < 0.001). At 1 month, LRs had a significantly reduced MCT (1.6 ± 0.1 mm, p = 0.042) but non-significantly changed pain-VAS (4.7 ± 0.2); 5 months later, MCT reached 1.0 ± 0.1 mm, pain-VAS reached 0.8 ± 0.5 (p < 0.044). Significant correlation between the relative variation of MCT and pain-VAS (1 month/baseline) was found. CONCLUSIONS: US-guided injection of triamcinolone acetonide is effective in treating grade IV CC.

Transcription profiling of Epstein-Barr virus nuclear antigen (EBNA)-1 expressing cells suggests targeting of chromatin remodeling complexes.

The Epstein-Barr virus (EBV) encoded nuclear antigen (EBNA)-1 regulates virus replication and transcription, and participates in the remodeling of the cellular environment that accompanies EBV induced B-cell immortalization and malignant transformation. The putative cellular targets of these effects of EBNA-1 are largely unknown. To address this issue we have profiled the transcriptional changes induced by short- and long-term expression of EBNA-1 in the EBV negative B-cell lymphoma BJAB. Three hundred and nineteen cellular genes were regulated in a conditional transfectant shortly after EBNA-1 induction while a ten fold higher number of genes was regulated upon continuous EBNA-1 expression. Promoter analysis of the differentially regulated genes demonstrated a significant enrichment of putative EBNA-1 binding sites suggesting that EBNA-1 may directly influence the transcription of a subset of genes. Gene ontology analysis of forty seven genes that were consistently regulated independently on the time of EBNA-1 expression revealed an unexpected enrichment of genes involved in the maintenance of chromatin architecture. The interaction network of the affected gene products suggests that EBNA-1 may promote a broad rearrangement of the cellular transcription landscape by altering the expression of key components of chromatin remodeling complexes.

MPRAP: an accessibility predictor for a-helical transmembrane proteins that performs well inside and outside the membrane.

BACKGROUND: In water-soluble proteins it is energetically favorable to bury hydrophobic residues and to expose polar and charged residues. In contrast to water soluble proteins, transmembrane proteins face three distinct environments; a hydrophobic lipid environment inside the membrane, a hydrophilic water environment outside the membrane and an interface region rich in phospholipid head-groups. Therefore, it is energetically favorable for transmembrane proteins to expose different types of residues in the different regions. RESULTS: Investigations of a set of structurally determined transmembrane proteins showed that the composition of solvent exposed residues differs significantly inside and outside the membrane. In contrast, residues buried within the interior of a protein show a much smaller difference. However, in all regions exposed residues are less conserved than buried residues. Further, we found that current state-of-the-art predictors for surface area are optimized for one of the regions and perform badly in the other regions. To circumvent this limitation we developed a new predictor, MPRAP, that performs well in all regions. In addition, MPRAP performs better on complete membrane proteins than a combination of specialized predictors and acceptably on water-soluble proteins. A web-server of MPRAP is available at http://mprap.cbr.su.se/ CONCLUSION: By including complete a-helical transmembrane proteins in the training MPRAP is able to predict surface accessibility accurately both inside and outside the membrane. This predictor can aid in the prediction of 3D-structure, and in the identification of erroneous protein structures.

A deneddylase encoded by Epstein-Barr virus promotes viral DNA replication by regulating the activity of cullin-RING ligases.

The large tegument proteins of herpesviruses encode conserved cysteine proteases of unknown function. Here we show that BPLF1, the Epstein-Barr-virus-encoded member of this protease family, is a deneddylase that regulates virus production by modulating the activity of cullin-RING ligases (CRLs). BPLF1 hydrolyses NEDD8 conjugates in vitro, acts as a deneddylase in vivo, binds to cullins and stabilizes CRL substrates. Expression of BPLF1 alone or in the context of the productive virus cycle induces accumulation of the licensing factor CDT1 and deregulates S-phase DNA synthesis. Inhibition of BPLF1 during the productive virus cycle prevents cellular DNA re-replication and inhibits virus replication. Viral DNA synthesis is restored by overexpression of CDT1. Homologues encoded by other herpesviruses share the deneddylase activity. Thus, these enzymes are likely to have a key function in the virus life cycle by inducing a replication-permissive S-phase-like cellular environment.