DNA Structure-Specific Cleavage of DNA-Protein Crosslinks by the SPRTN Protease.

Repair of covalent DNA-protein crosslinks (DPCs) by DNA-dependent proteases has emerged as an essential genome maintenance mechanism required for cellular viability and tumor suppression. However, how proteolysis is restricted to the crosslinked protein while leaving surrounding chromatin proteins unharmed has remained unknown. Using defined DPC model substrates, we show that the DPC protease SPRTN displays strict DNA structure-specific activity. Strikingly, SPRTN cleaves DPCs at or in direct proximity to disruptions within double-stranded DNA. In contrast, proteins crosslinked to intact double- or single-stranded DNA are not cleaved by SPRTN. NMR spectroscopy data suggest that specificity is not merely affinity-driven but achieved through a flexible bipartite strategy based on two DNA binding interfaces recognizing distinct structural features. This couples DNA context to activation of the enzyme, tightly confining SPRTN's action to biologically relevant scenarios.

The Aspartic Protease Ddi1 Contributes to DNA-Protein Crosslink Repair in Yeast.

Naturally occurring or drug-induced DNA-protein crosslinks (DPCs) interfere with key DNA transactions if not repaired in a timely manner. The unique family of DPC-specific proteases Wss1/SPRTN targets DPC protein moieties for degradation, including stabilized topoisomerase-1 cleavage complexes (Top1ccs). Here, we describe that the efficient DPC disassembly requires Ddi1, another conserved predicted protease in Saccharomyces cerevisiae. We found Ddi1 in a genetic screen of the tdp1 wss1 mutant defective in Top1cc processing. Ddi1 is recruited to a persistent Top1cc-like DPC lesion in an S phase-dependent manner to assist in the eviction of crosslinked protein from DNA. Loss of Ddi1 or its putative protease activity hypersensitizes cells to DPC trapping agents independently from Wss1 and 26S proteasome, implying its broader role in DPC repair. Among the potential Ddi1 targets, we found the core component of Pol II and show that its genotoxin-induced degradation is impaired in ddi1. We propose that the Ddi1 protease contributes to DPC proteolysis.

RAD51-WSS1-dependent genetic pathways are essential for DNA-protein crosslink repair and pathogenesis in Candida albicans.

Genetic analyses in Saccharomyces cerevisiae suggest that nucleotide excision repair (NER), homologous recombination (HR), and protease-dependent repair pathways coordinately function to remove DNA-protein crosslinks (DPCs) from the genome. DPCs are genomic cytotoxic lesions generated because of the covalent linkage of proteins with DNA. Although NER and HR processes have been studied in pathogenic Candida albicans, their roles in DPC repair (DPCR) are yet to be explored. Proteases like Wss1 and Tdp1 (tyrosyl-DNA phosphodiesterase-1) are known to be involved in DPCR; however, Tdp1 that selectively removes topoisomerase-DNA complexes is intrinsically absent in C. albicans. Therefore, the mechanism of DPCR might have evolved differently in C. albicans. Herein, we investigated the interplay of three genetic pathways and found that RAD51-WSS1-dependent HR and protease-dependent repair pathways are essential for DPC removal, and their absence caused an increased rate of loss of heterozygosity in C. albicans. RAD1 but not RAD2 of NER is critical for DPCR. In addition, we observed truncation of chromosome #6 in the cells defective in both RAD51 and WSS1 genes. While the protease and DNA-binding activities are essential, a direct interaction of Wss1 with the eukaryotic DNA clamp proliferating cell nuclear antigen is not a requisite for the function of Wss1. DPCR-defective C. albicans cells exhibited filamentous morphology, reduced immune cell evasion, and attenuation in virulence. Thus, we concluded that RAD51-WSS1-dependent DPCR pathways are essential for genome stability and candidiasis development. Since no vaccine against candidiasis is available for human use yet, we propose to explore DPCR-defective attenuated strains (rad51ΔΔwss1ΔΔ and rad2ΔΔrad51ΔΔwss1ΔΔ) for whole-cell vaccine development.

Hemodynamic Characteristics of a Tortuous Microvessel Using High-Fidelity Red Blood Cell Resolved Simulations.

OBJECTIVE: Tortuous microvessels are characteristic of microvascular remodeling associated with numerous physiological and pathological scenarios. Three-dimensional (3D) hemodynamics in tortuous microvessels influenced by red blood cells (RBCs), however, are largely unknown, and important questions remain. Is blood viscosity influenced by vessel tortuosity? How do RBC dynamics affect wall shear stress (WSS) patterns and the near-wall cell-free layer (CFL) over a range of conditions? The objective of this work was to parameterize hemodynamic characteristics unique to a tortuous microvessel. METHODS: RBC-resolved simulations were performed using an immersed boundary method-based 3D fluid dynamics solver. A representative tortuous microvessel was selected from a stimulated angiogenic network obtained from imaging of the rat mesentery and digitally reconstructed for the simulations. The representative microvessel was a venule with a diameter of approximately 20 µm. The model assumes a constant diameter along the vessel length and does not consider variations due to endothelial cell shapes or the endothelial surface layer. RESULTS: Microvessel tortuosity was observed to increase blood apparent viscosity compared to a straight tube by up to 26%. WSS spatial variations in high curvature regions reached 23.6 dyne/cm2 over the vessel cross-section. The magnitudes of WSS and CFL thickness variations due to tortuosity were strongly influenced by shear rate and negligibly influenced by tube hematocrit levels. CONCLUSIONS: New findings from this work reveal unique tortuosity-dependent hemodynamic characteristics over a range of conditions. The results provide new thought-provoking information to better understand the contribution of tortuous vessels in physiological and pathological processes and help improve reduced-order models.

Mechanism and Regulation of DNA-Protein Crosslink Repair by the DNA-Dependent Metalloprotease SPRTN.

Covalent DNA-protein crosslinks (DPCs) are toxic DNA lesions that interfere with essential chromatin transactions, such as replication and transcription. Little was known about DPC-specific repair mechanisms until the recent identification of a DPC-processing protease in yeast. The existence of a DPC protease in higher eukaryotes is inferred from data in Xenopus laevis egg extracts, but its identity remains elusive. Here we identify the metalloprotease SPRTN as the DPC protease acting in metazoans. Loss of SPRTN results in failure to repair DPCs and hypersensitivity to DPC-inducing agents. SPRTN accomplishes DPC processing through a unique DNA-induced protease activity, which is controlled by several sophisticated regulatory mechanisms. Cellular, biochemical, and structural studies define a DNA switch triggering its protease activity, a ubiquitin switch controlling SPRTN chromatin accessibility, and regulatory autocatalytic cleavage. Our data also provide a molecular explanation on how SPRTN deficiency causes the premature aging and cancer predisposition disorder Ruijs-Aalfs syndrome.

Ubiquitin-directed AAA+ ATPase p97/VCP unfolds stable proteins crosslinked to DNA for proteolysis by SPRTN.

The protease SPRTN degrades DNA-protein crosslinks (DPCs) that threaten genome stability. SPRTN has been connected to the ubiquitin-directed protein unfoldase p97 (also called VCP or Cdc48), but a functional cooperation has not been demonstrated directly. Here, we biochemically reconstituted p97-assisted proteolysis with purified proteins and showed that p97 targets ubiquitin-modified DPCs and unfolds them to prepare them for proteolysis by SPRTN. We demonstrate that purified SPRTN alone was unable to degrade a tightly-folded Eos fluorescent reporter protein even when Eos was crosslinked to DNA (Eos-DPC). However, when present, p97 unfolded poly-ubiquitinated Eos-DPC in a manner requiring its ubiquitin adapter, Ufd1-Npl4. Notably, we show that, in cooperation with p97 and Ufd1-Npl4, SPRTN proteolyzed unfolded Eos-DPC, which relied on recognition of the DNA-crosslink by SPRTN. In a simplified unfolding assay, we further demonstrate that p97, while unfolding a protein substrate, can surmount the obstacle of a DNA crosslink site in the substrate. Thus, our data demonstrate that p97, in conjunction with Ufd1-Npl4, assists SPRTN-mediated proteolysis of tightly-folded proteins crosslinked to DNA, even threading bulky protein-DNA adducts. These findings will be relevant for understanding how cells handle DPCs to ensure genome stability and for designing strategies that target p97 in combination cancer therapy.

Wheat Long Noncoding RNAs from Organelle and Nuclear Genomes Carry Conserved microRNA Precursors Which May Together Comprise Intricate Networks in Insect Responses.

Long noncoding RNAs (lncRNAs) are a diverse class of noncoding RNAs that are typically longer than 200 nucleotides but lack coding potentials. Advances in deep sequencing technologies enabled a better exploration of this type of noncoding transcripts. The poor sequence conservation, however, complicates the identification and annotation of lncRNAs at a large scale. Wheat is among the leading food staples worldwide whose production is threatened by both biotic and abiotic stressors. Here, we identified putative lncRNAs from durum wheat varieties that differ in stem solidness, a major source of defense against wheat stem sawfly, a devastating insect pest. We also analyzed and annotated lncRNAs from two bread wheat varieties, resistant and susceptible to another destructive pest, orange wheat blossom midge, with and without infestation. Several putative lncRNAs contained potential precursor sequences and/or target regions for microRNAs, another type of regulatory noncoding RNAs, which may indicate functional networks. Interestingly, in contrast to lncRNAs themselves, microRNAs with potential precursors within the lncRNA sequences appeared to be highly conserved at the sequence and family levels. We also observed a few putative lncRNAs that have perfect to near-perfect matches to organellar genomes, supporting the recent observations that organellar genomes may contribute to the noncoding transcript pool of the cell.

Improvement and validation of a computational model of flow in the swirling well cell culture model.

Effects of fluid dynamics on cells are often studied by growing the cells on the base of cylindrical wells or dishes that are swirled on the horizontal platform of an orbital shaker. The swirling culture medium applies a shear stress to the cells that varies in magnitude and directionality from the center to the edge of the vessel. Computational fluid dynamics methods are used to simulate the flow and hence calculate shear stresses at the base of the well. The shear characteristics at each radial location are then compared with cell behavior at the same position. Previous simulations have generally ignored effects of surface tension and wetting, and results have only occasionally been experimentally validated. We investigated whether such idealized simulations are sufficiently accurate, examining a commonly-used swirling well configuration. The breaking wave predicted by earlier simulations was not seen, and the edge-to-center difference in shear magnitude (but not directionality) almost disappeared, when surface tension and wetting were included. Optical measurements of fluid height and velocity agreed well only with the computational model that incorporated surface tension and wetting. These results demonstrate the importance of including accurate fluid properties in computational models of the swirling well method.

Novel splicing-site mutation in DCAF17 gene causing Woodhouse-Sakati syndrome in a large consanguineous family.

BACKGROUND: Woodhouse-Sakati syndrome is a rare autosomal recessive disease with endocrine and neuroectodermal aberrations with heterogeneous phenotypes and disease course. The most common phenotypes of the disease are progressive sensorineural hearing loss and alopecia, mild-to-moderate mental retardation and hypogonadism. The disease results from mutations in the DCAF17 gene. METHOD: Here, we reported a large consanguineous pedigree with multiple affected individuals with Woodhouse-Sakati syndrome phenotypes. Laboratory tests confirmed the endocrine perturbance in affected individuals. To find out the underlying genetic change, whole-exome sequencing was carried out. RESULT: Analysis of the exome data identified a splicing-site deletion NM_025000.3:c.1423-1_1425delGACA in DCAF17 gene. Sanger sequencing confirmed the co-segregation of the variant with the disease phenotypes in the family. CONCLUSION: The variant is predicted to cause aberrant splicing, i.e., exon skipping, resulting in the translation of a truncated functionless protein which results in appearance of typical phenotypic features and clinical laboratory findings of Woodhouse-Sakati syndrome in affected members of the family.

A Mobility Model for a 3D Non-Stationary Geometry Cluster-Based Channel Model for High Speed Trains in MIMO Wireless Channels.

During channel modeling for high-mobility channels, such as high-speed train (HST) channels, the velocity of the mobile radio station is assumed to be constant. However, this might not be realistic due to the dynamic movement of the train along the track. Therefore, in this paper, an enhanced Gauss-Markov mobility model with a 3D non-stationary geometry based stochastic model (GBSM) for HST in MIMO Wireless Channels is proposed. The non-isotropic scatterers within a cluster are assumed to be around the sphere in which the mobile relay station (MRS) is located. The multi-path components (MPCs) are modeled with varying velocities, whereas the mobility model is a function of time. The MPCs are represented in a death-birth cluster using the Markov process. Furthermore, the channel statistics, i.e., the space-time correlation function, the root-mean-square Doppler shift, and the quasi-stationary interval, are derived from the non-stationary model. The model shows how the quasi-stationary time increases from 0.21 to 0.451 s with a decreasing acceleration of 0.6 to 0.2 m/s2 of the HST. In addition, the impact of the distribution of the angles on the channel statistics is presented. Finally, the simulated results are compared with the measured results. Therefore, there is a close relationship between the proposed model and the measured results, and the model can be used to characterize the channel's properties.

First Discovery of Beta-Sitosterol as a Novel Antiviral Agent against White Spot Syndrome Virus.

The outbreak of white spot syndrome (WSS) is a looming challenge, due to dramatic losses to the crustacean aquaculture industry. However, at present, there are no prophylactic or therapeutic means to control this infectious viral disease. Here, we screened fifteen medicinal plants for their inhibitory activity on the white spot syndrome virus (WSSV), using red swamp crayfish (Procambarus clarkii) as a model species. The results showed that the crude extracts of Pinellia ternata (Thunb.) Breit. had the highest inhibitory effect (91.59%, 100 mg/kg) on WSSV proliferation, and its main component, beta-sitosterol, showed a much higher activity (95.79%, 50 mg/kg). Further, beta-sitosterol potently reduced (p < 0.01) viral loads and viral gene transcription levels in a concentration-dependent fashion, and significantly promoted the survival rate of WSSV-challenged crayfish (57.14%, 50 mg/kg). The co-incubation assay indicated that beta-sitosterol did not influence the infectivity of WSSV particles. Both pre- and post-treatment of beta-sitosterol exerted a significant inhibitory effect (p < 0.01) on the viral load in vivo. Mechanistically, beta-sitosterol not only interfered with the expression of viral genes (immediate early gene 1, ie1; DNA polymerase, DNApol) that are important in initiating WSSV transcription, but it also attenuated the hijacking of innate immune signaling pathways (Toll, IMD, and JAK/STAT pathways) by viral genes to block WSSV replication. Moreover, the expression of several antiviral immune, antioxidant, pro-inflammatory, and apoptosis-related genes changed significantly in beta-sitosterol-treated crayfish. Beta-sitosterol is a potent WSSV inhibitor and has the potential to be developed as an effective anti-WSSV agent against a WSS outbreak in crustacean aquaculture.

Low-Complexity Alternatives to the Optimal Linear Coding Scheme for Transmitting ARMA Sources.

In the era of the Internet of Things, there are many applications where numerous devices are deployed to acquire information and send it to analyse the data and make informed decisions. In these applications, the power consumption and price of the devices are often an issue. In this work, analog coding schemes are considered, so that an ADC is not needed, allowing the size and power consumption of the devices to be reduced. In addition, linear and DFT-based transmission schemes are proposed, so that the complexity of the operations involved is lowered, thus reducing the requirements in terms of processing capacity and the price of the hardware. The proposed schemes are proved to be asymptotically optimal among the linear ones for WSS, MA, AR and ARMA sources.

DNA-Protein Crosslink Proteolysis Repair.

Proteins that are covalently bound to DNA constitute a specific type of DNA lesion known as DNA-protein crosslinks (DPCs). DPCs represent physical obstacles to the progression of DNA replication. If not repaired, DPCs cause stalling of DNA replication forks that consequently leads to DNA double-strand breaks, the most cytotoxic DNA lesion. Although DPCs are common DNA lesions, the mechanism of DPC repair was unclear until now. Recent work unveiled that DPC repair is orchestrated by proteolysis performed by two distinct metalloproteases, SPARTAN in metazoans and Wss1 in yeast. This review summarizes recent discoveries on two proteases in DNA replication-coupled DPC repair and establishes DPC proteolysis repair as a separate DNA repair pathway for genome stability and protection from accelerated aging and cancer.

Wss1 homolog from Candida albicans and its role in DNA-protein crosslink tolerance.

Candida albicans is an opportunistic yeast that can cause life-threatening systemic infection in immunocompromised individuals. During infections, C. albicans has to cope with genotoxic stresses generated by the host immune system. DNA-protein crosslink (DPC), the covalent linkage of proteins with DNA, is one type of DNA damages that can be caused by the host immune response. DPCs are bulky lesions that interfere with the progression of replication and transcription machineries, and hence threaten genomic integrity. Accordingly, either a DPC tolerance mechanism or a DPC repair pathway is essential for C. albicans to maintain genomic stability and survive in the host. Here, we identified Wss1 (weak suppressor of Smt3) in C. albicans (CaWss1) using bioinformatics, genetic complementation, and biochemical studies. We showed that CaWss1 promotes cell survival under genotoxic stress conditions that generate DPCs and that the catalytic metalloprotease domain of CaWss1 is essential for its cellular function. Interactions of CaWss1 with Cdc48 and small ubiquitin-like modifier, although not strictly required, contribute to the function of CaWss1 in the suppression of the growth defects under DPC-inducing conditions. This report is the first investigation of the role of CaWss1 in DPC tolerance in C. albicans.

Emerging roles of Wss1 in the survival of Candida albicans under genotoxic stresses.

This perspective aims to discuss the potential physiological roles and regulation mechanisms of the recently identified Candida albicans Wss1 protease important in DNA-protein crosslink (DPC) tolerance and repair. DPC is a bulky DNA lesion that blocks essential DNA transactions; thus, it poses a significant threat to genome integrity if left unrepaired. Discoveries of Wss1 in Saccharomyces cerevisiae and SPRTN in human as DPC proteases have demonstrated the importance of protease function in DPC repair. Our recent study revealed that Wss1 in C. albicans, an opportunistic pathogen that can cause life-threatening infection in immunocompromised individuals, also promotes DPC tolerance similarly to both S. cerevisiae Wss1 and human SPRTN. However, its molecular mechanism and regulation are still poorly understood. Here, we briefly discuss the recent insights into C. albicans Wss1 based on the information from S. cerevisiae, as well as outline the aspect of this protein that could make it a potential target for antifungal drug development.

Analysis and outcomes of wrong site thyroid surgery.

BACKGROUND: In thyroid surgery, wrong-site surgery (WSS) is considered a rare event and seldom reported in the literature. CASE PRESENTATION: This report presents 5 WSS cases following thyroid surgery in a 20-year period. We stratified the subtypes of WSS in wrong target, wrong side, wrong procedure and wrong patient. Only planned and elective thyroid surgeries present WSS cases. The interventions were performed in low-volume hospitals, and subsequently, the patients were referred to our centres. Four cases of wrong-target procedures (thymectomies [n = 3] and lymph node excision [n = 1] performed instead of thyroidectomies) and one case of wrong-side procedure were observed in this study. Two wrong target cases resulting additionally in wrong procedure were noted. Wrong patient cases were not detected in the review. Patients experienced benign, malignant, or suspicious pathology and underwent traditional surgery (no endoscopic or robotic surgery). 40% of WSS led to legal action against the surgeon or a monetary settlement. CONCLUSION: WSS is also observed in thyroid surgery. Considering that reports regarding the serious complications of WSS are not yet available, these complications should be discussed with the surgical community. Etiologic causes, outcomes, preventive strategies of WSS and expert opinion are presented.

Comparative Analysis of Coding and Non-Coding Features within Insect Tolerance Loci in Wheat with Their Homologs in Cereal Genomes.

Food insecurity and malnutrition have reached critical levels with increased human population, climate fluctuations, water shortage; therefore, higher-yielding crops are in the spotlight of numerous studies. Abiotic factors affect the yield of staple food crops; among all, wheat stem sawfly (Cephus cinctus Norton) and orange wheat blossom midge (Sitodiplosis mosellana) are two of the most economically and agronomically harmful insect pests which cause yield loss in cereals, especially in wheat in North America. There is no effective strategy for suppressing this pest damage yet, and only the plants with intrinsic tolerance mechanisms such as solid stem phenotypes for WSS and antixenosis and/or antibiosis mechanisms for OWBM can limit damage. A major QTL and a causal gene for WSS resistance were previously identified in wheat, and 3 major QTLs and a causal gene for OWBM resistance. Here, we present a comparative analysis of coding and non-coding features of these loci of wheat across important cereal crops, barley, rye, oat, and rice. This research paves the way for our cloning and editing of additional WSS and OWBM tolerance gene(s), proteins, and metabolites.

Numerical investigations of temperature and hemodynamics in carotid arteries with and without atherosclerotic plaque during open surgery.

Intraoperative monitoring is essential for providing safe and effective care during open surgery. In this paper, numerical simulation is performed to track the flow and heat transfer of carotid arteries with and without atherosclerotic plaque in a real physiological system during surgery, in which the heat transport is first considered to couple to the blood flow due to the temperature dependence of the blood viscosity. The impacts of the operating room temperature and hematocrit (H) on the viscosity, velocity, temperature, wall shear stress (WSS), pressure drop and oscillation are investigated. The results demonstrate that the presence of plaque in the carotid artery induces a greater blood flow velocity, pressure drop, WSS, and oscillation, as well as a smaller viscosity and temperature variations. A decreasing ambient temperature leads to a decrease in the temperature and an increase in the low-WSS area, which implies a greater risk of hypothermia and atherosclerosis. As H increases, the high-WSS areas substantially expand; when H varies from 65% to 80%, WSSave increases by 70.02% and 68.57% for the arteries with and without plaque, respectively, which indicates a higher risk of vascular injury. The results obtained can serve as a guideline regarding the selection of an operating room temperature for carotid disease patients with distinct hematocrits.

On the Asymptotic Optimality of a Low-Complexity Coding Strategy for WSS, MA, and AR Vector Sources.

In this paper, we study the asymptotic optimality of a low-complexity coding strategy for Gaussian vector sources. Specifically, we study the convergence speed of the rate of such a coding strategy when it is used to encode the most relevant vector sources, namely wide sense stationary (WSS), moving average (MA), and autoregressive (AR) vector sources. We also study how the coding strategy considered performs when it is used to encode perturbed versions of those relevant sources. More precisely, we give a sufficient condition for such perturbed versions so that the convergence speed of the rate remains unaltered.

DNA-protein crosslink repair: proteases as DNA repair enzymes.

DNA-protein crosslinks (DPCs) are highly toxic DNA lesions because they interfere with DNA transactions. The recent discovery of a yeast protease that processes DPCs proteolytically raises the question whether DPC proteases also exist in higher eukaryotes. We argue here that the yeast enzyme, Wss1 (weak suppressor of smt3), is a member of a protease family whose mammalian representative is Spartan (SprT-like domain-containing protein)/DVC1 (DNA damage protein targeting VCP). DPC proteases may thus be common to all eukaryotes where they function as novel guardians of the genome.