A model of restriction endonuclease MvaI in complex with DNA: a template for interpretation of experimental data and a guide for specificity engineering

R.MvaI is a Type II restriction enzyme (REase), which specifically recognizes the pentanucleotide DNA sequence 5'-CCWGG-3' (W indicates A or T). It belongs to a family of enzymes, which recognize related sequences, including 5'-CCSGG-3' (S indicates G or C) in the case of R.BcnI, or 5'-CCNGG-3' (where N indicates any nucleoside) in the case of R.ScrFI. REases from this family hydrolyze the phosphodiester bond in the DNA between the 2nd and 3rd base in both strands, thereby generating a double strand break with 5'-protruding single nucleotides. So far, no crystal structures of REases with similar cleavage patterns have been solved. Characterization of sequence-structure-function relationships in this family would facilitate understanding of evolution of sequence specificity among REases and could aid in engineering of enzymes with new specificities. However, sequences of R.MvaI or its homologs show no significant similarity to any proteins with known structures, thus precluding straightforward comparative modeling. We used a fold recognition approach to identify a remote relationship between R.MvaI and the structure of DNA repair enzyme MutH, which belongs to the PD-(D/E)XK superfamily together with many other REases. We constructed a homology model of R.MvaI and used it to predict functionally important amino acid residues and the mode of interaction with the DNA. In particular, we predict that only one active site of R.MvaI interacts with the DNA target at a time, and the cleavage of both strands (5'-CCAGG-3' and 5'-CCTGG-3') is achieved by two independent catalytic events. The model is in good agreement with the available experimental data and will serve as a template for further analyses of R.MvaI, R.BcnI, R.ScrFI and other related enzymes.     

A narrowing gyre? The Lega Nord and the shifting balance of Italian immigration policy

This article reflects on the relations between xenophobic parties and immigration policy in Italy. It argues that the salience of the Northern League (Lega Nord), the instability of the Italian party system and the recent transformation of Italy from an emigration country to an immigration country all contribute to Italy's difficulty in achieving a coherent immigration policy. Since the first comprehensive immigration policy emerged in 1998 with the Turco-Napolitano law, there have been six changes of government, with frequent oscillation between centre-right and centre-left coalitions. While the effects of these oscillations, particularly of the centre-right's effort to restrict immigrants’ rights and entry possibilities, have been dampened by the Italian courts and the European Union, it is still problematic to speak of an Italian model. Drawing on Carvalho's book, this article will address the effects of the Lega on creating and maintaining this policy imbalance.     

Systemic lupus erythematosus: molecular cloning and analysis of 22 individual recombinant monoclonal kappa light chains specifically hydrolyzing human myelin basic protein

Antibodies hydrolyzing myelin basic protein (MBP) can play an important role in the pathogenesis of multiple sclerosis (MS) and systemic lupus erythematosus (SLE). An immunoglobulin light chain phagemid library derived from peripheral blood lymphocytes of patients with SLE was used. Small pools of phage particles displaying light chains with different affinities for MBP were isolated by affinity chromatography on MBP‐Sepharose, and the fraction eluted with 0.5 M NaCl was used for preparation of individual monoclonal light chains (MLChs, 26–27 kDa). Seventy‐two of 440 individual colonies were randomly chosen, expressed in Escherichia coli in a soluble form, and MLChs were purified by metal chelating chromatography. Twenty‐two of 72 MLChs have high affinity and efficiently hydrolyze only MBP (not other control proteins) demonstrating various pH optima in a 5.7–9.0 range and different substrate specificity in the hydrolysis of four different MBP oligopeptides. Four MLChs demonstrated serine protease‐like and three thiol protease‐like activities, while 11 MLChs were metalloproteases. The activity of three MLChs was inhibited by both phenylmethylsulfonyl fluoride (PMSF) and Ethylenediaminetetraacetic acid (EDTA), two other by EDTA and iodoacetamide, and one by PMSF, EDTA, and iodoacetamide. The ratio of relative activity in the presence of Ca²⁺, Mg²⁺_, Mn²⁺, Ni²⁺, Zn²⁺, Cu²⁺, and Co²⁺ was individual for each of 22 MLCh preparations. It is the first examples of human MLChs, which probably can possess two or even three different proteolytic activities. These observations suggest an extreme diversity of anti‐MBP abzymes in SLE patients. The immune systems of individual SLE patients can generate a variety of anti‐MBP abzymes, which can attack MBP of myelin‐proteolipid sheath of axons and play an important role in MS and SLE pathogenesis. Copyright © 2015 John Wiley & Sons, Ltd.     

Environmental variation and population responses to global change

Species' responses to environmental changes such as global warming are affected not only by trends in mean conditions, but also by natural and human‐induced environmental fluctuations. Methods are needed to predict how such environmental variation affects ecological and evolutionary processes, in order to design effective strategies to conserve biodiversity under global change. Here, we review recent theoretical and empirical studies to assess: (1) how populations respond to changes in environmental variance, and (2) how environmental variance affects population responses to changes in mean conditions. Contrary to frequent claims, empirical studies show that increases in environmental variance can increase as well as decrease long‐term population growth rates. Moreover, environmental variance can alter and even reverse the effects of changes in the mean environment, such that even if environmental variance remains constant, omitting it from population models compromises their ability to predict species' responses to changes in mean conditions. Drawing on theory relating these effects of environmental variance to the curvatures of population growth responses to the environment, we outline how species' traits such as phylogenetic history and body mass could be used to predict their responses to global change under future environmental variability.     

Cold truths: how winter drives responses of terrestrial organisms to climate change

Winter is a key driver of individual performance, community composition, and ecological interactions in terrestrial habitats. Although climate change research tends to focus on performance in the growing season, climate change is also modifying winter conditions rapidly. Changes to winter temperatures, the variability of winter conditions, and winter snow cover can interact to induce cold injury, alter energy and water balance, advance or retard phenology, and modify community interactions. Species vary in their susceptibility to these winter drivers, hampering efforts to predict biological responses to climate change. Existing frameworks for predicting the impacts of climate change do not incorporate the complexity of organismal responses to winter. Here, we synthesise organismal responses to winter climate change, and use this synthesis to build a framework to predict exposure and sensitivity to negative impacts. This framework can be used to estimate the vulnerability of species to winter climate change. We describe the importance of relationships between winter conditions and performance during the growing season in determining fitness, and demonstrate how summer and winter processes are linked. Incorporating winter into current models will require concerted effort from theoreticians and empiricists, and the expansion of current growing‐season studies to incorporate winter.     

Engineering of 2-Cys peroxiredoxin for enhanced stress-tolerance

A typical 2-cysteine peroxiredoxin (2-Cys Prx)-like protein (PpPrx) that alternatively acts as a peroxidase or a molecular chaperone in Pseudomonas putida KT2440 was previously characterized. The dual functions of PpPrx are regulated by the existence of an additional Cys(112) between the active Cys(51) and Cys(171) residues. In the present study, additional Cys residues (Cys(31), Cys(112), and Cys(192)) were added to PpPrx variants to improve their enzymatic function. The optimal position of the additional Cys residues for the dual functionality was assessed. The peroxidase activities of the S31C and Y192C mutants were increased 3- to 4-fold compared to the wild-type, while the chaperone activity was maintained at > 66% of PpPrx. To investigate whether optimization of the dual functions could enhance stress-tolerance in vivo, a complementation study was performed. The S31C and Y192C mutants showed a much greater tolerance than other variants under a complex condition of heat and oxidative stresses. The optimized dual functions of PpPrx could be adapted for use in bioengineering systems and industries, such as to develop organisms that are more resistant to extreme environments.     

Behavioral thermoregulation in a tropical gastropod: links to climate change scenarios

Tropical species are vulnerable to global warming because they live at, or near to, their upper thermal threshold limits. Therefore, the predicted increase in the frequency of warming events in the tropics is expected to be critical for the survival of local species. This study explored the major environmental variables which were thought to be correlated with body temperatures (BTs) of the tropical snail Littoraria scabra at the niche level. A correlation between BT and substrate temperature (ST) was detected from field observations which suggests a possible causal relationship between both substrate and BTs. In contrast, there was no correlation between BT and air temperature. Field observations suggest that 33.4 °C may be L. scabra upper limit of substrate surface temperature, although further experiments are needed to assess if the upper limit of physiological tolerance is actually different. As L. scabra individuals were free to choose their substrata, the observed distribution pattern at the niche level is related to L. scabra's behavior. Additionally, substrate surface temperatures were very heterogeneous at centimeter scale (i.e. from 22.5 to 53.1 °C) and L. scabra was shown to select specific STs (i.e. between 22.5 and 33.4 °C) rather than microhabitat type. Therefore, L. scabra did not seem to behaviorally thermoregulate through microhabitat selection nor aggregation. In contrast, behavioral experiments showed that L. scabra has the ability to actively select a thermally favorable site over short temporal scale (i.e. individual average speed of 1.26 cm min^?1) following exposure to high temperatures above 33.4 °C. Hence, this study supports the crucial need to integrate intertidal invertebrate behavioral responses to thermal constraints in climate change studies.     

浅谈对医学生临床思维与临床技能提高的培养

为适应目前医学事业发展的需要,医学生的临床思维与临床技能的培养至关重要。在教学中,应不断提高教师自身的业务水平和自身素质,给予学生适当引导和启发,采取灵活多样的教学方式,并鼓励学生学会独立思考能力,激发学生的主动性和创造性,同时理论与实践相结合,推出模拟教学,由此激发学生的学习兴趣;并通过病例的采集归纳和整理,加强基本功的训练。从而提高学生临床思维能力与标准实践技能。     

引种保护植物对西双版纳极端冷年的春季物候响应

气温被普遍认为是春季物候期最主要的控制因子之一,然而低温对植物物候的影响效应一直都存在不同的观点。西双版纳由于地处热带地区的北缘,其气温相对于赤道附近的热带地区较低。自1959年以来,西双版纳热带植物园引入了来自世界各个热带地区的4万余种植物进行保护,之前的研究证明西双版纳的低温对这些引种植物的生长有很大影响。因此,1974年西双版纳出现的极端低温势必对引种植物造成极大威胁,同时也是对这些植物低温适应能力的一个考验。通过对比43种引种植物物候期（生长抽梢期与开花期）在1974年与常年的差异情况,分析不同来源（热带亚洲、热带美洲与热带非洲）引种植物对西双版纳低温的适应性。结果表明,经历西双版纳1974年初的极端低温之后,使81%的引种植物生长抽梢期提前,同时也造成35%的引种植物在该年没有开花;而植物生长抽梢提前的主要原因则是极端低温以及低温过后气温迅速回升。引种植物均能顺利度过1974年的最冷时期,并出现生长抽梢物候,这意味着引种植物在经历极端低温之后都能够进行正常的生长活动,但极端低温对引种植物繁殖活动的不利影响大于其对生长活动的影响;引种植物对西双版纳极端低温的适应能力由大到小顺序依次为：亚洲来源植物〉美洲来源植物〉非洲来源植物。因此在迁地保护植物的选择过程中,应多选择亚洲热带植物,其次为美洲热带植物,而对非洲热带植物的引入则需谨慎考察。