A moonlighting metabolic protein influences repair at DNA double-stranded breaks

Catalytically active proteins with divergent dual functions are often described as ‘moonlighting’. In this work we characterize a new, chromatin-based function of Lys20, a moonlighting protein that is well known for its role in metabolism. Lys20 was initially described as homocitrate synthase (HCS), the first enzyme in the lysine biosynthetic pathway in yeast. Its nuclear localization led to the discovery of a key role for Lys20 in DNA damage repair through its interaction with the MYST family histone acetyltransferase Esa1. Overexpression of Lys20 promotes suppression of DNA damage sensitivity of esa1 mutants. In this work, by taking advantage of LYS20 mutants that are active in repair but not in lysine biosynthesis, the mechanism of suppression of esa1 was characterized. First we analyzed the chromatin landscape of esa1 cells, finding impaired histone acetylation and eviction. Lys20 was recruited to sites of DNA damage, and its overexpression promoted enhanced recruitment of the INO80 remodeling complex to restore normal histone eviction at the damage sites. This study improves understanding of the evolutionary, structural and biological relevance of independent activities in a moonlighting protein and links metabolism to DNA damage repair.     

The beta subunit of the Escherichia coli ATP synthase exhibits a tight membrane binding property

We have developed a chromatographic procedure to analyze the association of the subunits of the Escherichia coli F1Fo-ATP synthase with the cytoplasmic membrane. Minicells containing [35S]-labeled ATP synthase subunits are treated with lysozyme, solubilized, and chromatographed on a Sepharose CL-2B column in buffer containing urea and taurodeoxycholate. ATP synthase subunits are resolved into membrane intrinsic and membrane extrinsic subunits. Interestingly, a significant amount (36%) of the F1 subunit beta fractionates with the membrane intrinsic Fo subunits. About half of this amount (19%) of beta is non-specifically bound to the membrane. Interaction of beta with the membrane is not mediated by the amino terminal portion of beta.     

A review on economically adsorbents on heavy metals removal in water and wastewater

Heavy metals contamination in water has been an issue to the environment and human health. The persisting contamination level has been observed and concerned by the public due to continuous deterioration of water quality. On the other hand, conventional treatment system could not completely remove the toxic metals in the water, thus alternative purification methods using inexpensive materials were endeavor to improve the current treatment process. Wide ranges of low cost adsorbents were used to remove heavy metal in aqueous solution and wastewater. The low cost adsorbents were usually collected from agricultural waste, seafood waste, food waste, industrial by-product and soil. These adsorbents are readily available in a copious amount. Besides, the pretreatment are not complicated to be conducted on the raw products, which is economically sound for an alternative treatment. The previous studies have provided much evidence of low cost adsorbents’ efficiency in removing metal ions from aqueous solution or wastewater. In this review, several low cost adsorbents in the recent literature have been studied. The maximum adsorption capacity, affecting factors such as pH, contact times, temperature, initial concentration and modified materials were revised and summarized in this review for further reference. Comparisons of the adsorbent between the modified and natural products were also demonstrated to provide a clear understanding on the kinetic uptake of the selected adsorbents. Some of the natural adsorbents appeared as good heavy metal removal, while some were not and require further modifications and improvements to enhance the adsorption capacity. SWOT analysis (strength, weakness, opportunities, threat) was also performed on the low cost adsorbents to identify the advantages of using low cost adsorbents and solve the weaknesses encountered by the utilization of low cost materials. This tool helps to determine the potential quality of low cost materials in the application for water and wastewater treatment.     

Immunoglobulin structure exhibits control over CDR motion

Motions of the IgG structure are evaluated using normal mode analysis of an elastic network model to detect hinges, the dominance of low frequency modes, and the most important internal motions. One question we seek to answer is whether or not IgG hinge motions facilitate antigen binding. We also evaluate the protein crystal and packing effects on the experimental temperature factors and disorder predictions. We find that the effects of the protein environment on the crystallographic temperature factors may be misleading for evaluating specific functional motions of IgG. The extent of motion of the antigen binding domains is computed to show their large spatial sampling. We conclude that the IgG structure is specifically designed to facilitate large excursions of the antigen binding domains. Normal modes are shown as capable of computationally evaluating the hinge motions and the spatial sampling by the structure. The antigen binding loops and the major hinge appear to behave similarly to the rest of the structure when we consider the dominance of the low frequency modes and the extent of internal motion. The full IgG structure has a lower spectral dimension than individual F(ab) domains, pointing to more efficient information transfer through the antibody than through each domain. This supports the claim that the IgG structure is specifically constructed to facilitate antigen binding by coupling motion of the antigen binding loops with the large scale hinge motions.     

Development of a novel methodology for cryopreservation of melanoma cells applied to CSF470 therapeutic vaccine

CSF470 vaccine is a mixture of four lethally irradiated melanoma cell lines, administered with BCG and GM-CSF, which is currently being tested in a Phase II/III Clinical trial in stage II/III melanoma patients. To prepare vaccine doses, irradiated melanoma cell lines are frozen using dimethyl sulfoxide (Me₂SO) and stored in liquid nitrogen (liqN₂). Prior to inoculation, doses must be thawed, washed to remove Me₂SO and suspended for clinical administration. Avoiding the use of Me₂SO and storage in liqN₂ would allow future freeze-drying of CSF470 vaccine to facilitate pharmaceutical production and distribution. We worked on the development of an alternative cryopreservation methodology while keeping the vaccine’s biological and immunogenic properties. We tested different freezing media containing trehalose suitable to remain as excipients in a freeze-dried product, to cryopreserve melanoma cells either before or after gamma irradiation. Melanoma cells incorporated trehalose after 5 h incubation at 37 °C by fluid-phase endocytosis, reaching an intracellular concentration that varied between 70–140 mM depending on the cell line. Optimal freezing conditions were 0.2 M trehalose and 30 mg/ml human serum albumin, at −84 °C. Vaccine doses could be frozen in trehalose at −84 °C for at least four months keeping their cellular integrity, antigen expression and apoptosis/necrosis profile after gamma-irradiation as compared to Me₂SO control. Non-irradiated melanoma cell lines also showed comparable proliferative capacity after both cryopreservation procedures. Trehalose-freezing medium allowed us to cryopreserve melanoma cells, either alive or after gamma irradiation, at −84 °C avoiding the use of Me₂SO and liqN₂ storage. These cryopreservation conditions could be suitable for future freeze-drying of CSF470 vaccine.     

热带季节性湿润林苦楝(Melia azedarach)径向生长季节动态及其对环境因子的响应

气候变化导致的温度升高和降水格局改变可能会影响到树木的生长速率和季节物候。西双版纳热带季节性湿润林分布在石灰岩山中部,属于热带喀斯特生境。由于土层浅薄,土壤保水能力极差,植物生长更容易遭到受到季节性干旱气候的影响。为探究热带季节性湿润林的树木径向生长季节动态及其对环境因子的响应,利用高精度树木生长仪连续两年监测了云南西双版纳热带季节性湿润林中落叶树种苦楝（Melia azedarach）的树干径向变化,并与同步监测的环境因子进行相关分析。结果表明,苦楝径向生长开始、结束以及持续生长的时间在年际间存在差异。与2018年相比,2019年苦楝生长开始和结束的时间较晚,且年生长量较小,这可能是与2019年雨季开始较晚且在生长季早期经历了严重的高温干旱有关。苦楝的径向日生长量与日降水量和相对湿度呈正相关关系,与光合有效辐射、水汽压亏缺和风速呈负相关关系,表明了在苦楝的径向生长主要受水分条件限制。在干旱年份（2019年）,苦楝的日生长量与降水和相对湿度的相关性更强。研究结果有助于进一步了解热带喀斯特生境树木生长对气候变化的敏感性以及树木适应季节性干旱气候的策略。     

New noninvasive methodology for real-time monitoring of lipid flip

A new methodology for the detection of lipid flip was developed. This methodology relies on the quenching of the fluorescence of the cascade-blue-labeled lipid through complex formation with a membrane-impermeable cyclen-tetranaphthalenethiourea synthetic receptor for this dye. The high affinity of the receptor to cascade-blue label allows the use of micromolar concentrations of this receptor during the experiment. At these low concentrations, the receptor does not interfere with the membrane integrity and, therefore, renders this new methodology less invasive to the model and cell membranes than commonly utilized 7-nitro-1,2,3-benzoxadiazol-4-yl (NBD)-dithionite methodology. Unlike with the NBD-dithionite assay, where the fluorescence quenching of the NBD group is achieved through its chemical modification, this new assay relies on the noncovalent interactions between cascade-blue label and the receptor. Therefore, the quenching can be reverted by either competitive displacement of the lipid-attached label with a water-soluble substrate or by enzymatic degradation of the receptor leading to the label release and fluorescence dequenching. We demonstrate that this new methodology is suitable for the study of lipid flip in both model spherical bilayer membranes and in-vitro experiments.     

Mitochondrial DNA profiling via genomic analysis in mesial temporal lobe epilepsy patients with hippocampal sclerosis

Introduction

Mitochondria have an essential role in neuronal excitability and neuronal survival. In addition to energy production, mitochondria also play a crucial role in the maintenance of intracellular calcium homeostasis, generation of reactive oxygen species and mechanisms of cell death. There is a relative paucity of data about the role of mitochondria in epilepsy. Mitochondrial genome analysis is rarely carried out in the investigation of some diseases. In mesial temporal lobe epilepsies (MTLE) cases, genome analysis has never been used previously. The aim of this study is to show mitochondrial dysfunctions using genome analysis in patients with MTLE-hippocampal sclerosis (HS).

Methods

44 patients with MTLE-HS and 86 matched healthy unrelated controls were included in this study. The patients were divided into four groups according to their clinical presentation as the following: Group 1 consists of patients with intractable epilepsy who refused operation; Group 2 of operated seizure free patients; Group 3 of operated patients with seizures; and Group 4 unoperated seizure free patients with or without antiepileptic drugs. Blood samples were used to isolate DNA. Parallel tagged sequencing was employed to allow pyrosequencing of 130 samples. Complete mtDNA is amplified in two overlapping fragments (11 and 9 kb). The PCR amplicons were pooled in equimolar ratios. Titanium kits were used to produce shotgun libraries according to the manufacturer's protocol.

Results

The average coverage in total was 130 ± 30 and an average of 2365127 bases and 337 bp fragment length was received from all samples. The mean mtDNA heteroplasmy in patients was 26.35 ± 12.3 and in controls 25.03 ± 9.34. Three mutations had prominently high significance in patient samples. The most significantly associated variation was located in the MT-ATP-8 gene (8502 A > T, Asn46Ile) whereas the other two were in the MT-ND4 (11994 C > T, Thr412Ile) and MT-ND5 (13231 A > C, Lys299Gln) genes.

Conclusions

We have observed that three mutations were significantly related to the presence of epilepsy. These mutations were found at the 8502, 11994, and 13231 bp of mtDNA, which resulted in amino acid changes at the MT-ATP-8, MT-ND4 and MT-ND5 genes. Finding mutations can lead us to knowing more about the pathophysiology of the MTLE disease.     

Genetic elimination of field-cage populations of Mediterranean fruit flies

The Mediterranean fruit fly (medfly, Ceratitis capitata Wiedemann) is a pest of over 300 fruits, vegetables and nuts. The sterile insect technique (SIT) is a control measure used to reduce the reproductive potential of populations through the mass release of sterilized male insects that mate with wild females. However, SIT flies can display poor field performance, due to the effects of mass-rearing and of the irradiation process used for sterilization. The development of female-lethal RIDL (release of insects carrying a dominant lethal) strains for medfly can overcome many of the problems of SIT associated with irradiation. Here, we present life-history characterizations for two medfly RIDL strains, OX3864A and OX3647Q. Our results show (i) full functionality of RIDL, (ii) equivalency of RIDL and wild-type strains for life-history characteristics, and (iii) a high level of sexual competitiveness against both wild-type and wild-derived males. We also present the first proof-of-principle experiment on the use of RIDL to eliminate medfly populations. Weekly releases of OX3864A males into stable populations of wild-type medfly caused a successive decline in numbers, leading to eradication. The results show that genetic control can provide an effective alternative to SIT for the control of pest insects.