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As one of the newest fields of engineering, synthetic biology relies upon a trial-and-error Design–Build–Test–Learn (DBTL) approach to simultaneously learn how a function is encoded in biology and attempt to engineer it. Many software and hardware platforms have been developed to automate, optimize and algorithmically perform each step of the DBTL cycle. However, there are many fewer options for automating the build step. Build typically involves deoxyribonucleic acid (DNA) assembly, which remains manual, low throughput and unreliable in most cases and limits our ability to advance the science and engineering of biology. Here, we present AssemblyTron, an open-source Python package to integrate j5 DNA assembly design software outputs with build implementation in Opentrons liquid handling robotics with minimal human intervention. We demonstrate the versatility of AssemblyTron through several scarless, multipart DNA assemblies, beginning from fragment amplification. We show that AssemblyTron can perform polymerase chain reactions across a range of fragment lengths and annealing temperatures by using an optimal annealing temperature gradient calculation algorithm. We then demonstrate that AssemblyTron can perform Golden Gate and homology-dependent in vivo assemblies (IVAs) with comparable fidelity to manual assemblies by simultaneously building four four-fragment assemblies of chromoprotein reporter expression plasmids. Finally, we used AssemblyTron to perform site-directed mutagenesis reactions via homology-dependent IVA also achieving comparable fidelity to manual assemblies as assessed by sequencing. AssemblyTron can reduce the time, training, costs and wastes associated with synthetic biology, which, along with open-source and affordable automation, will further foster the accessibility of synthetic biology and accelerate biological research and engineering. [ABSTRACT FROM AUTHOR]

Abstract Background Similar to other local therapeutic methods, local interstitial radiotherapy (IRT) also suffers from insufficient systematic immune activation, resulting in tumor metastasis. Results Mn-based IRT radiosensitizers consisting of 131I, MnO2 and bovine serum albumin (BSA) (131I-MnO2-BSA) were engineered. Such Mn-based IRT radiosensitizers successfully unlocked radiogenetics to magnify systematic immune responses of local IRT via remodeling hypoxic and immunosuppressive microenvironments and resist tumor metastasis. The MnO2 in 131I-MnO2-BSA caused decomposition of H2O2 enriched in tumors to generate O2 for alleviating hypoxic microenvironment and removing tumor resistances to IRT. Concurrently, hypoxia mitigation by such radiosensitizers-unlocked radiogenetics can effectively remodel immunosuppressive microenvironment associated with regulatory T (Treg) cells and tumor-associated macrophages (TAMs) infiltration inhibition to induce immunogenic cell death (ICD), which, along with hypoxia mitigation, activates systematic immune responses. More intriguingly, 131I-MnO2-BSA-enabled radiogenetics can upregulate PD-L1 expression, which allows anti-PD-L1-combined therapy to exert a robust antitumor effect on primary tumors and elicit memory effects to suppress metastatic tumors in both tumor models (4T1 and CT26). Conclusions IRT radiosensitizer-unlocked radiogenetics and the corresponding design principle provide a general pathway to address the insufficient systematic immune responses of local IRT. Graphical Abstract

Background: Similar to other local therapeutic methods, local interstitial radiotherapy (IRT) also suffers from insufficient systematic immune activation, resulting in tumor metastasis. Results: Mn-based IRT radiosensitizers consisting of 131I, MnO2 and bovine serum albumin (BSA) (131I-MnO2-BSA) were engineered. Such Mn-based IRT radiosensitizers successfully unlocked radiogenetics to magnify systematic immune responses of local IRT via remodeling hypoxic and immunosuppressive microenvironments and resist tumor metastasis. The MnO2 in 131I-MnO2-BSA caused decomposition of H2O2 enriched in tumors to generate O2 for alleviating hypoxic microenvironment and removing tumor resistances to IRT. Concurrently, hypoxia mitigation by such radiosensitizers-unlocked radiogenetics can effectively remodel immunosuppressive microenvironment associated with regulatory T (Treg) cells and tumor-associated macrophages (TAMs) infiltration inhibition to induce immunogenic cell death (ICD), which, along with hypoxia mitigation, activates systematic immune responses. More intriguingly, 131I-MnO2-BSA-enabled radiogenetics can upregulate PD-L1 expression, which allows anti-PD-L1-combined therapy to exert a robust antitumor effect on primary tumors and elicit memory effects to suppress metastatic tumors in both tumor models (4T1 and CT26). Conclusions: IRT radiosensitizer-unlocked radiogenetics and the corresponding design principle provide a general pathway to address the insufficient systematic immune responses of local IRT. [ABSTRACT FROM AUTHOR]

Objectives: We aimed to investigate the feasibility of predicting high-risk cytogenetic abnormalities (HRCAs) in patients with multiple myeloma (MM) using a spinal MRI-based radiomics method. Materials and methods: In this retrospective study, we analyzed the radiomic features of 248 lesions (HRCA [n = 111] and non-HRCA [n = 137]) using T1WI, T2WI, and fat suppression T2WI. To construct the radiomics model, the top nine most frequent radiomic features were selected using logistic regression (LR) machine-learning processes. A combined LR model incorporating radiomic features and basic clinical characteristics (age and sex) was also built. Fivefold external cross-validation was performed, and a comparative analysis of 10 random fivefold cross-validation sets was used to verify result stability. Model performance was compared by plotting receiver operating characteristic curves and the area under the curve (AUC). Results: Comparable AUC values were observed between the radiomics model and the combined model in validation cohorts (AUC: 0.863 vs. 0.870, respectively, p = 0.206). The radiomics model had an AUC of 0.863, with a sensitivity of 0.789, a specificity of 0.787, a positive predictive value of 0.753, a negative predictive value of 0.824, and an accuracy of 0.788 in the validation cohort, which were comparable with the performance in the training cohorts. Conclusions: Radiomic features of routine spinal MRI reflect differences between HRCAs and non-HRCAs in patients with MM. This MRI-based radiomics model might be a useful and independent tool to predict HRCAs in patients MM. [ABSTRACT FROM AUTHOR]

A critical limitation of Salmonella typhimurium (S. typhimurium) as an anti-cancer agent is the loss of their invasive or replicative activities, which results in no or less delivery of anti-cancer agents inside cancer cells in cancer therapy. Here we developed an oxytolerant attenuated Salmonella strain (KST0650) from the parental KST0649 (ΔptsIΔcrr) strain using radiation mutation technology (RMT). The oxytolerant KST0650 strain possessed 20-times higher replication activity in CT26 cancer cells and was less virulent than KST0649. Furthermore, KST0650 migrated effectively into tumor tissues in mice. KST0650 was further equipped with a plasmid harboring a spliced form of the intracellular pro-apoptotic protein sATF6, and the expression of sATF6 was controlled by the radiation-inducible recN promoter. The new strain was named as KST0652, in which sATF6 protein expression was induced in response to radiation in a dose-dependent manner. This strain was effectively delivered inside cancer cells and tumor tissues via the Salmonella type III secretion system (T3SS). In addition, combination treatment with KST0652 and radiation showed a synergistic anti-tumor effect in murine tumor model with complete inhibition of tumor growth and protection against death. In conclusion, we showed that RMT can be used to effectively develop an anti-tumor Salmonella strain for delivering anti-cancer agents inside tumors. [ABSTRACT FROM AUTHOR]

Chemical mutagenesis is a useful tool for inducing mutations in plants. Seeds are often used as the material for chemical mutagenesis. The biological effect of a chemical mutagen on seeds is determined by absorption dose (the product of mutagen concentration and acting time, which starts after the mutagen is absorbed by the seeds). In practice, however, the concept of exposure dose (the product of mutagen concentration and treating time) is usually used instead because the time for absorbing mutagen is unknown. In this study, we conducted an experiment using ethyl methane sulphonate (EMS) to treat cauliflower seeds, in which five EMS concentrations (0%, 0.5%, 1.0%, 1.5% and 2.0%), three treating time lengths (4 h, 6 h and 8 h) and two pretreatments (non-presoaking and presoaking of seeds for 2 h) were set. We obtained a well-fitted nonlinear regression model for the relationship between seedling survival rate and the EMS treatment, and its marginal models for the two pretreatments. Based on the models, we determined the EMS absorption doses under the two different pretreatments and identified their 50% lethality dose (LD50). We found that presoaking could delay EMS absorption and therefore reduce the injury caused by EMS within a given treating time, but could hardly change the biological effect of EMS after it is absorbed. The conclusions about absorption dose and presoaking effect obtained in this study might be generally applicable to plant chemical mutagenesis in principle. [ABSTRACT FROM AUTHOR]

The Precambrian‐Cambrian (PЄ‐Є) transition is a unique period in Earth's history known for the "Cambrian Explosion." Here we present a continuous radiogenic and stable isotope (87Sr/86Sr, δ13CVPDB, carb, and δ18OVPDB, carb) and elemental (including rare earth elements) record across the PЄ‐Є transition, preserved in phosphatic and carbonate shallow‐water deposits from two sections (Koksu and Kyrshabakty) in Kazakhstan. Microstructure, trace, and rare earth elements concentrations reflect near‐primary preservation. δ13CVPDB, carb data (−12.1‰ to 3.3‰) record formation conditions at a time of severe geodynamic changes. Negative δ13CVPDB, carb at the PЄ‐Є boundary resemble the Basal Cambrian Carbon Anomaly. Average δ18OVPDB, carb values of −11.7 ± 3.8‰ are suggested to represent near‐primary signals and can be explained by isotopically lighter past sea water and elevated temperature during precipitation compared to present‐day conditions. Initial 87Sr/86Sr of the carbonate and phosphate fraction are in the range of 0.7086–0.7112 at the Koksu section and 0.7082–0.7136 at the Kyrshabakty section. This is in general agreement with the seawater 87Sr/86Sr evolution during the Early Cambrian. In this study, we present a refined 87Sr/86Sr seawater evolution, highlighting an extreme positive Sr isotope excursion at the PЄ‐Є transition, which is suggested to be indicative for enhanced continental weathering and an increase in nutrient runoff into shallow ocean waters. This extreme geodynamic event may have served as a driving force for enhanced biologic activity in the lead up to the Cambrian Explosion, where phosphatic shallow‐water deposits from Kazakhstan constitute a valuable paleo‐environmental archive. Key Points: We present the first continuous 87Sr/86Sr record from Kazakhstan across the Pc‐C transitionA refined 87Sr/86Sr seawater evolution is presented with distinct positive excursion at the Pc‐C transition corresponding to a negative δ13C anomaly (BACE)Enhanced continental weathering was a driving force for the Cambrian Explosion [ABSTRACT FROM AUTHOR]

Quorum sensing is a cell-cell communication process that bacteria use to orchestrate group behaviors. Quorum sensing is mediated by signal molecules called autoinducers. Autoinducers are often structurally similar, raising questions concerning how bacteria distinguish among them. Here, we use the Pseudomonas aeruginosa LasR quorum-sensing receptor to explore signal discrimination. The cognate autoinducer, 3OC12 homoserine lactone (3OC12HSL), is a more potent activator of LasR than other homoserine lactones. However, other homoserine lactones can elicit LasR-dependent quorum-sensing responses, showing that LasR displays ligand promiscuity. We identify mutants that alter which homoserine lactones LasR detects. Substitution at residue S129 decreases the LasR response to 3OC12HSL, while enhancing discrimination against noncognate autoinducers. Conversely, the LasR L130F mutation increases the potency of 3OC12HSL and other homoserine lactones. We solve crystal structures of LasR ligand-binding domains complexed with noncognate autoinducers. Comparison with existing structures reveals that ligand selectivity/sensitivity is mediated by a flexible loop near the ligand-binding site. We show that LasR variants with modified ligand preferences exhibit altered quorum-sensing responses to autoinducers in vivo. We suggest that possessing some ligand promiscuity endows LasR with the ability to optimally regulate quorum-sensing traits. [ABSTRACT FROM AUTHOR]

The article discusses the genetic effects of radiation. A gene is remarkably stable. Ordinarily it is transmitted unchanged from parent to offspring for generation after generation. But rarely, perhaps once in a hundred thousand generations, a gene changes, or mutates. Mutations and mutation rates have been studied in a wide variety of experimental animals and plants, and in man. Studies have confirmed that almost all mutations are harmful. In 1927, H. J. Muller, made the discovery that X-rays produced a large increase in the mutation rate in the fruit fly, Drosophila. The results were soon confirmed by studies on various animals and plants and with various ionizing radiations.

The article discusses the significance of genetic damage produced by radiation, based from an address given before the National Academy of Sciences in Washington, D.C. on April 25, 1955. According to sources, it is no longer a matter of doubt among scientists working in this field that radiation, of the types derived from radioactive substances or X-ray machines, does produce permanent changes, including mutations. Thus, the most important of these changes, occurring in the individual hereditary particles or genes called gene mutations, arise with a frequency depending proportionately on the total dose of radiation. Therefore, it is necessary to admit the damage, and weigh estimates of it against the benefits to be derived from tests, or against the damage which follows from the policy.

The article reports on the exposure of the genetic material in relation to the advent of radiology in medicine in the U.S. According to experts, there is a corresponding increase of radiation exposure relative to the widespread utilization of X-rays for diagnostic and therapeutic purposes. An amount many times greater than those produced by all atomic and thermonuclear weapons tests combined. The estimates, however, on which this conclusion is based, are of necessity uncertain. Strangely enough, it was the alarm caused among geneticists by the thermonuclear bomb tests that has drawn attention to the exposures which have long occurred in medicine.

The central nervous system relies on a continual supply of glucose, and must be able to detect glucose levels and regulate peripheral organ functions to ensure that its energy requirements are met. Specialized glucose-sensing neurons, first described half a century ago, use glucose as a signal and modulate their firing rates as glucose levels change. Glucose-excited neurons are activated by increasing glucose concentrations, while glucose-inhibited neurons increase their firing rate as glucose concentrations fall and decrease their firing rate as glucose concentrations rise. Glucose-sensing neurons are present in multiple brain regions and are highly expressed in hypothalamic regions, where they are involved in functions related to glucose homeostasis. However, the roles of glucose-sensing neurons in healthy and disease states remain poorly understood. Technologies that can rapidly and reversibly activate or inhibit defined neural populations provide invaluable tools to investigate how specific neural populations regulate metabolism and other physiological roles. Optogenetics has high temporal and spatial resolutions, requires implants for neural stimulation, and is suitable for modulating local neural populations. Chemogenetics, which requires injection of a synthetic ligand, can target both local and widespread populations. Radio- and magnetogenetics offer rapid neural activation in localized or widespread neural populations without the need for implants or injections. These tools will allow us to better understand glucosesensing neurons and their metabolism-regulating circuits. [ABSTRACT FROM AUTHOR]

Purpose: Broomrape produces serious damage to many legume crops and, particularly, becomes a limiting factor for faba bean (Vicia faba L.) production in the Mediterranean basin. Currently, several traditional methods of control have been developed, but none has proved to be effective for this parasite. However, breeding for resistance to this pest remains as one of the most feasible and environment-friendly methods for managing broomrape, but the mechanisms governing the interaction between the parasite and the host are not yet well understood. Therefore, we studied the behavior and molecular and enzymatic changes associated with the resistance to Orobanche foetida in faba bean mutants, which were obtained through radiation mutagenesis. Materials and methods: Three faba bean genotypes were used in this study, the variety ‘Badï’, characterized by high productivity in Orobanche-free soils and susceptibility to O. foetida, and two mutant lines P2M3 and P7M3 (derived from radio mutagenesis program), selected for their higher resistance to O. foetida in a field evaluation. The infection progress and the relative changes in the co-culture response, the enzymatic activities changes and the efficiency of the root extract stimulants from the host plant were followed and evaluated in all the genotypes. Results: Experiments showed that low induction of seed germination is a major component of resistance in these lines against O. foetida. This is confirmed by the in vitro experiments with root exudates. The parallel reduction in infection was accompanied by the continuous enhancement of the peroxidase activity, the polyphenol oxidase activity and the phenylalanine ammonia lyase activity in faba bean roots. Conclusion: These data suggest the contribution of these enzymes in faba bean resistance to O. foetida broomrape induced by the use of gamma rays. Management of Orobanche by way of crop selection, based on these enzyme systems is a possible option. [ABSTRACT FROM AUTHOR]

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Abstract: The central nervous system (CNS) plays a vital role in regulating energy balance and metabolism. Over the last 50 years, studies in animal models have allowed us to identify critical CNS regions involved in these processes and even crucial cell populations. Now, techniques for genetically and anatomically targeted manipulation of specific neural populations using light (optogenetic), ligands (chemogenetic), or magnetic fields (radiogenetic/magnetogenetic) allow detailed investigation of circuits involved in metabolic regulation. In this review, we provide a brief overview of recent studies using light‐ and magnetic field–regulated neural activity to investigate the neural circuits contributing to metabolic control. [ABSTRACT FROM AUTHOR]

The aim of the present study is to analyze the global research trend of radiation-responsive genes and identify the highly reproducible radiation-responsive genes. Bibliometric methods were applied to analyze the global research trend of radiation-responsive genes. We found 79 publications on radiation-responsive genes from 2000 to 2017. A total of 35 highly reproducible radiation-responsive genes were identified. Most genes are involved in response to DNA damage, cell proliferation, cell cycle regulation, and DNA repair. The p53 signal pathway was the top enriched pathway. The expression levels of 18 genes in human B lymphoblastoid cell line (AHH-1) cells were significantly up-regulated in a dose-dependent manner at 24 h after exposure to 0-5 Gy 60 Co γ-ray irradiation. Our results indicate that developing a gene expression panel with the 35 high reproducibility radiation-responsive genes may be necessary for qualitative and quantitative assessment after exposure. [ABSTRACT FROM AUTHOR]

This study examines strontium, oxygen, and carbon isotope ratios (Sr/Sr, δO, δC) in dental enamel and bone apatite from 82 individuals interred at Late Neolithic, Chalcolithic, and Bronze Age burial sites near Madrid, Spain, to discern variations in dietary patterns and identify possible migrants. Questions about mobility patterns and subsistence practices have played a central role in the scholarship of Late Prehistoric central Iberia in the last 20 years, but the archaeological record has still not been able to provide clear answers. This study adds valuable data to this line of research. The results of this study suggest that migration from regions with different geologic landscapes was uncommon in these communities. For the identified migrants, based upon the Sr/Sr values, several of the identified non-local individuals originate from regions with substantially older lithological features and possible places of origin are being investigated. As it is not possible to discern individuals who may have moved from regions with similar geologic landscapes using this methodology, these data provide the minimum number of migrants, and it is conceivable that the number of non-locals in this sample may be higher. Combining multiple lines of material and biological evidence and the completion of Sr isotope mapping in the Iberian Peninsula will help to clarify these findings. Stable carbon isotope data provide new and direct evidence of regional changes in consumption patterns. In particular, this study provides some possible evidence for the consumption of C plants in third-millennium bc central Spain. [ABSTRACT FROM AUTHOR]

Purpose: This study investigated relationships between neuroblastomas (NBs) imaging phenotypes, tumor genomic profile and patient outcome. Patients and methods: This IRB-approved retrospective observational study included 133 NB patients (73 M, 60 F; median age 15 months, range 0–151) treated in a single institution between 1998 and 2012. A consensus review of imaging (CT-scan, MRI) categorized tumors according to both the primarily involved compartment (i.e., neck, chest, abdomen or pelvis) and the sympathetic anatomical structure the tumors rose from (i.e., cervical, paravertebral or periarterial chains, or adrenal gland). Tumor shape, volume and image-defined surgical risk factors (IDRFs) at diagnosis were recorded. Genomic profiles were assessed using array-based comparative genomic hybridization and divided into three groups: “numerical-only chromosome alterations” (NCA), “segmental chromosome alterations” (SCA) and “MYCN amplification” (MNA). Statistical analyses included Kruskal–Wallis, Chi2 and Fisher’s exact tests and the Kaplan-Meier method with log-rank tests and Cox model for univariate and multivariate survival analyses. Results: A significant association between the sympathetic structure origin of tumors and genomic profiles was demonstrated. NBs arising from cervical sympathetic chains were all NCA. Paravertebral NBs were NCA or SCA in 75% and 25%, respectively and none were MNA. Periarterial NBs were NCA, SCA or MNA in 33%, 56% and 11%, respectively. Adrenal NBs were NCA, SCA or MNA in 16%, 36% and 48%, respectively. Among MNA NBs, 92% originated from the adrenal gland. The sympathetic anatomical classification was significantly better correlated to overall survival than the compartmental classification (P < .0003). The tumor volume of MNA NBs was significantly higher than NCA or SCA NBs (P < .0001). Patients with initial volume less than 160 mL had significantly better overall survival (P < .009). A “single mass” pattern was significantly more frequent in NCA NBs (P = .0003). The number of IDRFs was significantly higher in MNA NBs (P < .0001). Conclusion: Imaging phenotypes of neuroblastomas, including tumor origin along the sympathetic system, correlate with tumor genomic profile and patient outcome. [ABSTRACT FROM AUTHOR]

The need for generating precisely designed mutations is common in genetics, biochemistry, and molecular biology. Here, I describe a new λ Red recombineering method (Direct and Inverted Repeat stimulated excision; DIRex) for fast and easy generation of single point mutations, small insertions or replacements as well as deletions of any size, in bacterial genes. The method does not leave any resistance marker or scar sequence and requires only one transformation to generate a semi-stable intermediate insertion mutant. Spontaneous excision of the intermediate efficiently and accurately generates the final mutant. In addition, the intermediate is transferable between strains by generalized transductions, enabling transfer of the mutation into multiple strains without repeating the recombineering step. Existing methods that can be used to accomplish similar results are either (i) more complicated to design, (ii) more limited in what mutation types can be made, or (iii) require expression of extrinsic factors in addition to λ Red. I demonstrate the utility of the method by generating several deletions, small insertions/replacements, and single nucleotide exchanges in Escherichia coli and Salmonella enterica. Furthermore, the design parameters that influence the excision frequency and the success rate of generating desired point mutations have been examined to determine design guidelines for optimal efficiency. [ABSTRACT FROM AUTHOR]

Giant reed ( Arundo donax L.) is a C3 perennial, warm-season, rhizomatous grass of emerging interest for bioenergy and biomass derivatives production, and for phytoremediation. It only propagates vegetatively and very little genetic variation is found among ecotypes, basically precluding breeding efforts. With the objective to increase the genetic variation in this species, we developed and applied a mutagenesis protocol based on γ-irradiation of in vitro cell cultures from which regenerants were obtained. Based on a radiosensitivity test, the irradiation dose reducing to 50% the number of regenerants per callus (RD50) was estimated at 35 Gy. A large mutagenic experiment was carried out by irradiating a total of 3120 calli with approx. 1×, 1.5× and 2× RD50. A total of 1004 regenerants from irradiated calli were hardened in pots and transplanted to the field. Initial phenotypic characterization of the collection showed correlated responses of biomass-related quantitative traits to irradiation doses. Approx. 10% of field-grown clones showed remarkable morphological aberrations including dwarfism, altered tillering, abnormal inflorescence, leaf variegation and others, which were tested for stability over generations. Clone lethality reached 0.4%. Our results show for the first time that physical mutagenesis can efficiently induce new genetic and phenotypic variation of agronomic and prospective industrial value in giant reed. The methodology and the plant materials described here may contribute to the domestication and the genetic improvement of this important biomass species. [ABSTRACT FROM AUTHOR]

The epitome of any genome research is to identify all the existing genes in a genome and investigate their roles. Various techniques have been applied to unveil the functions either by silencing or over-expressing the genes by targeted expression or random mutagenesis. Rice is the most appropriate model crop for generating a mutant resource for functional genomic studies because of the availability of high-quality genome sequence and relatively smaller genome size. Rice has syntenic relationships with members of other cereals. Hence, characterization of functionally unknown genes in rice will possibly provide key genetic insights and can lead to comparative genomics involving other cereals. The current review attempts to discuss the available gain-of-function mutagenesis techniques for functional genomics, emphasizing the contemporary approach, activation tagging and alterations to this method for the enhancement of yield and productivity of rice. [ABSTRACT FROM AUTHOR]

Background: Radiogenetic therapy is a novel approach in the treatment of cancer, which employs genetic modification to alter the sensitivity of tumor cells to the effect of applied radiation. Aim: To select a potent radiation inducible promoter in the context of brain tumors and to investigate if CArG radio responsive motifs or other elements in the promoter nucleotide sequences can correlate to its response to radiation. Methods: To select initial candidates for promoter inducible elements, the levels of mRNA expression of six different promoters were assessed using Quantitative RTPCR in D54 MG cells before and after radiation exposure. Recombinant Ad/reporter genes driven by five different promoters; CMV, VEGF, FLT-1, DR5 and survivin were constructed. Glioma cell lines were infected with different multiplicity of infection of the (promoter) Ad or CMV Ad. Cells were then exposed to a range of radiation (0-12 Gy) at single fraction. Fluorescent microscopy, Luc assay and X-gal staining was used to detect the level of expression of related genes. Different glioma cell lines and normal astrocytes were infected with Ad survivin and exposed to radiation. The promoters were analyzed for presence of CArG radio-responsive motifs and CCAAT box consensus using NCBI blast bioinformatics software. Results: Radiotherapy increases the expression of gene expression by 1.25-2.5 fold in different promoters other than survivin after 2 h of radiation. RNA analysis was done and has shown an increase in copy number of tenfold for survivin. Most importantly cells treated with RT and Ad Luc driven by survivin promoter showed a fivefold increase in expression after 2 Gy of radiation in comparison to non-irradiated cells. Presence or absence of CArG motifs did not correlate with promoter response to radiation. Survivin with the best response to radiation had the lowest number of CCAAT box. Conclusion: Survivin is a selective potent radiation inducible promoter for glioblastoma viral gene therapy and this response to radiation could be independent of CArG motifs. [ABSTRACT FROM AUTHOR]

The article presents an analysis on the effects of low level ionizing radiation exposure to the population. The analyses seek to clarify several matters such as the environmental and biological risks of a certain development, a comparison of the risks with the benefits to be gained, a feasibility on the cost of reducing environmental and biological risks, and the net benefit of a certain development compared to alternative options. It also presents the four ways in estimating the genetic risks of radiation to the population.

The translation elongation factor eIF5A is conserved through evolution and is necessary to rescue the ribosome during translation elongation of polyproline-containing proteins. Although the site of eIF5A binding to the ribosome is known, no systematic analysis has been performed so far to determine the important residues on the surface of eIF5A required for ribosome binding. In this study, we used clustered charged-to-alanine mutagenesis and structural modeling to address this question. We generated four new mutants of yeast eIF5A: tif51A- 4, tif51A- 6, tif51A- 7 and tif51A- 11, and complementation analysis revealed that tif51A- 4 and tif51A- 7 could not sustain cell growth in a strain lacking wild-type eIF5A. Moreover, the allele tif51A- 4 also displayed negative dominance over wild-type eIF5A. Both in vivo GST-pulldowns and in vitro fluorescence anisotropy demonstrated that eIF5A from mutant tif51A- 7 exhibited an importantly reduced affinity for the ribosome, implicating the charged residues in cluster 7 as determinant features on the eIF5A surface for contacting the ribosome. Notably, modified eIF5A from mutant tif51A- 4, despite exhibiting the most severe growth phenotype, did not abolish ribosome interactions as with mutant tif51A- 7. Taking into account the modeling eIF5A + 80S + P-tRNA complex, our data suggest that interactions of eIF5A with ribosomal protein L1 are more important to stabilize the interaction with the ribosome as a whole than the contacts with P-tRNA. Finally, the ability of eIF5A from tif51A- 4 to bind to the ribosome while potentially blocking physical interaction with P-tRNA could explain its dominant negative phenotype. [ABSTRACT FROM AUTHOR]

Objectives: Analysis of a mass burial of seven males at CA-ALA-554, a prehistoric site in the Amador Valley, CA, was undertaken to determine if the individuals were "locals" or "non-locals," and how they were genetically related to one another. Methods: The study includes osteological, genetic (mtDNA), and stable (C, N, O, S) and radiogenic (Sr) isotope analyses of bone and tooth (first and third molars) samples. Results: Isotopes in first molars, third molars, and bone show they spent the majority of their lives living together. They are not locals to the Amador Valley, but were recently living to the east in the San Joaquin Valley, suggesting intergroup warfare as the cause of death. The men were not maternally related, but represent at least four different matrilines. The men also changed residence as a group between age 16 and adult years. Conclusions: Isotope data suggest intergroup warfare accounts for the mass burial. Genetic data suggest the raiding party included sets of unrelated men, perhaps from different households. Generalizing from this case and others like it, we hypothesize that competition over territory was a major factor behind ancient warfare in Central California. We present a testable model of demographic expansion, wherein villages in high-population-density areas frequently fissioned, with groups of individuals moving to lower-population-density areas to establish new villages. This model is consistent with previous models of linguistic expansion. [ABSTRACT FROM AUTHOR]

Most nucleoside kinases, besides the catalytic domain, feature an allosteric domain which modulates their activity. Generally, non-substrate analogs, interacting with allosteric sites, represent a major opportunity for developing more selective and safer therapeutics. We recently developed a series of nonnucleoside non-competitive inhibitors of human adenosine kinase (hAK), based on a pyrrolobenzoxa(thia) zepinone scaffold. Based on computational analysis, we hypothesized the existence of a novel allosteric site on hAK, topographically distinct from the catalytic site. In this study, we have adopted a multidisciplinary approach including molecular modeling, biochemical studies, and site-directed mutagenesis to validate our hypothesis. Based on a three-dimensional model of interaction between hAK and our molecules, we designed, cloned, and expressed specific, single and double point mutants of hAK (Q74A, Q78A, H107A, K341A, F338A, and Q74A-F338A). Kinetic characterization of recombinant enzymes indicated that these mutations did not affect enzyme functioning; conversely, mutated enzymes are endowed of reduced susceptibility to our non-nucleoside inhibitors, while maintaining comparable affinity for nucleoside inhibitors to the wild-type enzyme. This study represents the first characterization and validation of a novel allosteric site in hAK and may pave the way to the development of novel selective and potent non-nucleoside inhibitors of hAK endowed with therapeutic potential. [ABSTRACT FROM AUTHOR]

Purpose: Promoters developed for radiogene therapy always show non-negligible transcriptional activities, even when cells are not irradiated. This study developed a tightly radiation-controlled molecular switch based on radiation responsive element (CArG) repeats for in vivo molecular imaging using the Cre/loxP system.Procedures: Different numbers of CArG repeats were cloned as a basal promoter directly, and its pre- and postirradiation transcriptional activities were analyzed by luciferase assay. Nine CArG repeats (E9) were chosen for use as a radiation-controlled molecular switch for the Cre/loxP system, and the feasibility of the switch in vitro and in vivo was demonstrated by luciferase assay and bioluminescence imaging, respectively.Results: The E9 promoter, which exhibits extremely low transcriptional activity, showed a 1.8-fold enhancement after irradiation with a clinical dose of 2 Gy. Both in vitro and in vivo results indicated that E9 is relatively inert but sufficient to trigger the Cre/loxP system. The luciferase activity of stable H1299/pSTOP-FLuc cells transfected with pE9-NLSCre and exposed to 2-Gy radiation can reach 44 % of that of the same cells transfected with pCMV-NLSCre and not subjected to irradiation. By contrast, no appreciable difference was observed in reporter gene expression in both H1299/pSTOPFluc cells and tumors transfected with pE4Pcmv-NLSCre before and after irradiation, because the strong basal transcriptional activity of the CMV promoter, which acts as a copartner of E4, masked the response of E4 to radiation.Conclusions: Our results provide detailed insight into CArG elements as a radiation-controlled molecular switch that can facilitate the development of radiogene therapy. [ABSTRACT FROM AUTHOR]

Heat generation produced by radioactive decay of long-lived isotopes in the Earth's crust (radiogenic heat flow) within the Paleozoic provinces of the Central Asian Fold Belt is considered. Heat flow from the mantle is calculated as the difference between the observed heat flow and the radiogenic flow. The major cause of this heat flow is the transition of potential energy of gravity differentiation into heat; in this respect, mantle heat flow is called gravigenic. Calculation shows that the fractions of radiogenic and gravigenic heat flows in the Paleozoides studied are nearly equal. [ABSTRACT FROM AUTHOR]

A major unsolved question in evolutionary biology concerns the relationship between natural and sexual selection. Sexual selection might augment natural selection, for example if mutations that harm female fecundity also reduce male mating success. Conversely, sexual selection might favour traits that impair naturally selected fitness components. We induced detrimental mutations in Callosobruchus maculatus beetles using X-ray irradiation and then experimentally measured the effect of precopulatory sexual selection on offspring number and survival rate. Sexual selection treatment had a negative effect on egg-to-adult survivorship, although the number of progeny reaching adulthood was unaffected, perhaps because eggs and juveniles that failed to develop lessened competition on the survivors. We hypothesize that the negative effect of sexual selection arose because sexually competitive males transmitted a smaller nuptial gift or carried alleles that conferred reduced survival. Although we found no evidence that sexual selection on males can purge alleles that are detrimental to naturally selected fitness components, such benefits might exist in other environmental or genetic contexts. [ABSTRACT FROM AUTHOR]

The dynamic mutability of microsatellite repeats is implicated in the modification of gene function and disease phenotype. Studies of the enhanced instability of long trinucleotide repeats (TNRs)-- the cause of multiple human diseases--have revealed a remarkable complexity of mutagenic mechanisms. Here, we show that cold, heat, hypoxic, and oxidative stresses induce mutagenesis of a long CAG repeat tract in human cells. We show that stress-response factors mediate the stress-induced mutagenesis (SIM) of CAG repeats. We show further that SIM of CAG repeats does not involve mismatch repair, nucleotide excision repair, or transcription, processes that are known to promote TNR mutagenesis in other pathways of instability. Instead, we find that these stresses stimulate DNA rereplication, increasing the proportion of cells with >4 C-value (C) DNA content. Knockdown of the replication originlicensing factor CDT1 eliminates both stress-induced rereplication and CAG repeat mutagenesis. In addition, direct induction of rereplication in the absence of stress also increases the proportion of cells with >4C DNA content and promotes repeat mutagenesis. Thus, environmental stress triggers a unique pathway for TNR mutagenesis that likely is mediated by DNA rereplication. This pathway may impact normal cells as they encounter stresses in their environment or during development or abnormal cells as they evolve metastatic potential. [ABSTRACT FROM AUTHOR]

Genotype profiling of patients before radiotherapy may help to identify individuals who will not respond to a given treatment or who are likely to experience severe side effects. Radiogenomics will help us achieve personalized therapy for safer and more effective outcomes.Radiogenomics is the whole genome application of radiogenetics, which focuses on uncovering the underlying genetic causes of individual variation in sensitivity to radiation. There is a growing consensus that radiosensitivity is a complex, inherited polygenic trait, dependent on the interaction of many genes involved in multiple cell processes. An understanding of the genes involved in processes such as DNA damage response and oxidative stress response, has evolved toward examination of how genetic variants, most often, single nucleotide polymorphisms (SNPs), may influence interindividual radioresponse. Many experimental approaches, such as candidate SNP association studies, genome-wide association studies and massively parallel sequencing are being proposed to address these questions. We present a review focusing on recent advances in association studies of SNPs to radiotherapy response and discuss challenges and opportunities for further studies. We also highlight the clinical perspective of radiogenomics in the future of personalized treatment in radiation oncology. [ABSTRACT FROM AUTHOR]

Cyprinid herpesvirus 3 (CyHV 3) is causing severe economic losses worldwide in common and koi carp industries, and a safe and efficacious attenuated vaccine compatible with mass vaccination is needed. We produced single deleted recombinants using prokaryotic mutagenesis. When producing a recombinant lacking open reading frame 134 (ORF134), we unexpectedly obtained a clone with additional deletion of ORF56 and ORF57. This triple deleted recombinant replicated efficiently in vitro and expressed an in vivo safety/efficacy profile compatible with use as an attenuated vaccine. To determine the role of the double ORF56-57 deletion in the phenotype and to improve further the quality of the vaccine candidate, a series of deleted recombinants was produced and tested in vivo. These experiments led to the selection of a double deleted recombinant lacking ORF56 and ORF57 as a vaccine candidate. The safety and efficacy of this strain were studied using an in vivo bioluminescent imaging system (IVIS), qPCR, and histopathological examination, which demonstrated that it enters fish via skin infection similar to the wild type strain. However, compared to the parental wild type strain, the vaccine candidate replicated at lower levels and spread less efficiently to secondary sites of infection. Transmission experiments allowing water contamination with or without additional physical contact between fish demonstrated that the vaccine candidate has a reduced ability to spread from vaccinated fish to naïve sentinel cohabitants. Finally, IVIS analyses demonstrated that the vaccine candidate induces a protective mucosal immune response at the portal of entry. Thus, the present study is the first to report the rational development of a recombinant attenuated vaccine against CyHV 3 for mass vaccination of carp. We also demonstrated the relevance of the CyHV 3 carp model for studying alloherpesvirus transmission and mucosal immunity in teleost skin. [ABSTRACT FROM AUTHOR]

A novel toluene monooxygenase (TMO) six-gene cluster from Bradyrhizobium sp. BTAi1 having an overall 35, 36, and 38 % protein similarity with toluene o-xylene monooxygenase (ToMO) of Pseudomonas sp. OX1, toluene 4-monooxygenase (T4MO) of Pseudomonas mendocina KR1, and toluene- para-monooxygenase (T pMO) of Ralstonia pickettii PKO1, respectively, was cloned and expressed in Escherichia coli TG1, and its potential activity was investigated for aromatic hydroxylation and trichloroethylene (TCE) degradation. The natural substrate toluene was hydroxylated to p-cresol, indicating that the new toluene monooxygenase (T4MO·BTAi1) acts as a para hydroxylating enzyme, similar to T4MO and T pMO. Some shifts in regiospecific hydroxylations were observed compared to the other wild-type TMOs. For example, wild-type T4MO·BTAi1 formed catechol (88 %) and hydroquinone (12 %) from phenol, whereas all the other wild-type TMOs were reported to form only catechol. Furthermore, it was discovered that TG1 cells expressing wild-type T4MO·BTAi1 mineralized TCE at a rate of 0.67 ± 0.10 nmol Cl/h/mg protein. Saturation and site directed mutagenesis were used to generate eight variants of T4MO·BTAi1 at alpha-subunit positions P101, P103, and H214: P101T/P103A, P101S, P101N/P103T, P101V, P103T, P101V/P103T, H214G, and H214G/D278N; by testing the substrates phenol, nitrobenzene, and naphthalene, positions P101 and P103 were found to influence the regiospecific oxidation of aromatics. For example, compared to wild type, variant P103T produced four fold more m-nitrophenol from nitrobenzene as well as produced mainly resorcinol (60 %) from phenol whereas wild-type T4MO·BTAi1 did not. Similarly, variants P101T/P103A and P101S synthesized more 2-naphthol and 2.3-fold and 1.6-fold less 1-naphthol from naphthalene, respectively. [ABSTRACT FROM AUTHOR]

?18OPvalues and87Sr/86Sr ratios were determined on disarticulated xenacanthiform, hybodontid and ctenacanthid shark tooth material from several Early Permian (Sakmarian–Kungurian) continental bone beds of northern Texas and southern Oklahoma as well as from the marine Middle Permian (Roadian) of northern Arizona. The ?18OPvalues derived from the teeth of bone beds are in the range of 17.6–23.5‰ VSMOW, and are mostly depleted in18O by 0.5–5‰ relative to proposed coeval marine ?18OPvalues. This indicates an adaptation to freshwater habitats on the Early Permian coastal plain by several sharks. Distinctly higher ?18OPvalues from two bone beds are attributed to significant evaporative enrichment in18O in flood plain ponds.87Sr/86Sr ratios of around 0.71077 are notably more radiogenic than87Sr/86Sr of contemporaneous seawater. In contrast, the isotopic composition of teeth from the marine Kaibab Formation is characterised by low ?18OPvalues in the range of 13.4–15.6‰ VSMOW while87Sr/86Sr ratios of around 0.70821 are closer to the Roadian seawater value. The distinctly depleted ?18OPvalues cannot be readily explained by fluvially affected freshening in a nearshore marine environment, so a diagenetic alteration of the Kaibab material seems to be more likely, excluding it from further interpretation. [ABSTRACT FROM AUTHOR]

By combining targeted mutagenesis, computational refinement, and directed evolution, amodestly active, computationally designed Diels- Alderase was converted into the most proficient biocatalyst for [4+2] cycloadditions known. The high stereoselectivity and minimal product inhibition of the evolved enzyme enabled preparative scale synthesis of a single product diastereomer. X-ray crystallography of the enzyme- product complex shows that the molecular changes introduced over the course of optimization, including addition of a lid structure, gradually reshaped the pocket for more effective substrate preorganization and transition state stabilization. The good overall agreement between the experimental structure and the original design model with respect to the orientations of both the bound product and the catalytic side chains contrasts with other computationally designed enzymes. Because design accuracy appears to correlate with scaffold rigidity, improved control over backbone conformation will likely be the key to future efforts to design more efficient enzymes for diverse chemical reactions. [ABSTRACT FROM AUTHOR]

Background Recent efforts in HIV-1 vaccine design have focused on immunogens that evoke potent neutralizing antibody responses to a broad spectrum of viruses circulating worldwide. However, the development of effective vaccines will depend on the identification and characterization of the neutralizing antibodies and their epitopes. We developed bioinformatics methods to predict epitope networks and antigenic determinants using structural information, as well as corresponding genotypes and phenotypes generated by a highly sensitive and reproducible neutralization assay. 282 clonal envelope sequences from a multiclade panel of HIV-1 viruses were tested in viral neutralization assays with an array of broadly neutralizing monoclonal antibodies (mAbs: b12, PG9,16, PGT121 - 128, PGT130 - 131, PGT135 - 137, PGT141 - 145, and PGV04). We correlated IC50 titers with the envelope sequences, and used this information to predict antibody epitope networks. Structural patches were defined as amino acid groups based on solvent-accessibility, radius, atomic depth, and interaction networks within 3D envelope models. We applied a boosted algorithm consisting of multiple machine-learning and statistical models to evaluate these patches as possible antibody epitope regions, evidenced by strong correlations with the neutralization response for each antibody. Results We identified patch clusters with significant correlation to IC50 titers as sites that impact neutralization sensitivity and therefore are potentially part of the antibody binding sites. Predicted epitope networks were mostly located within the variable loops of the envelope glycoprotein (gp120), particularly in V1/V2. Site-directed mutagenesis experiments involving residues identified as epitope networks across multiple mAbs confirmed association of these residues with loss or gain of neutralization sensitivity. Conclusions Computational methods were implemented to rapidly survey protein structures and predict epitope networks associated with response to individual monoclonal antibodies, which resulted in the identification and deeper understanding of immunological hotspots targeted by broadly neutralizing HIV-1 antibodies. [ABSTRACT FROM AUTHOR]

Background The L-type calcium channel Cav1.2 is important for brain and heart function. The ubiquitous calcium sensing protein calmodulin (CaM) regulates calcium dependent gating of Cav1.2 channels by reducing calcium influx, a process known as calcium-dependent inactivation (CDI). Dissecting the calcium-dependence of CaM in this process has benefited greatly from the use of mutant CaM molecules which are unable to bind calcium to their low affinity (Nlobe) and high affinity (C-lobe) binding sites. Unlike CDI, it is unknown whether CaM can modulate the activation gating of Cav1.2 channels. Results We examined a Cav1.2 point mutant in the N-terminus region of the channel (A39V) that has been previously linked to Brugada syndrome. Using mutant CaM constructs in which the Nand/ or C-lobe calcium binding sites were ablated, we were able to show that this Brugada syndrome mutation disrupts N-lobe CDI of the channel. In the course of these experiments, we discovered that all mutant CaM molecules were able to alter the kinetics of channel activation even in the absence of calcium for WT-Cav1.2, but not A39V-Cav1.2 channels. Moreover, CaM mutants differentially shifted the voltage-dependence of activation for WT and A39V-Cav1.2 channels to hyperpolarized potentials. Our data therefore suggest that structural changes in CaM that arise directly from site directed mutagenesis of calcium binding domains alter activation gating of Cav1.2 channels independently of their effects on calcium binding, and that the N-terminus of the channel contributes to this CaM dependent process. Conclusions Our data indicate that caution must be exercised when interpreting the effects of CaM mutants on ion channel gating. [ABSTRACT FROM AUTHOR]