Reinterpreting Long-Term Evolution Experiments: Is Delayed Adaptation an Example of Historical Contingency or a Consequence of Intermittent Selection?

Van Hofwegen et al. demonstrated that Escherichia coli rapidly evolves the ability to use citrate when long selective periods are provided (D. J. Van Hofwegen, C. J. Hovde, and S. A. Minnich, J Bacteriol 198:1022–1034, 2016, http://dx.doi.org/10.1128/JB.00831-15). This contrasts with the extreme delay (15 years of daily transfers) seen in the long-term evolution experiments of Lenski and coworkers. Their idea of “historical contingency” may require reinterpretation. Rapid evolution seems to involve selection for duplications of the whole cit locus that are too unstable to contribute when selection is provided in short pulses.     

Intracellular delivery of an antisense?oligonucleotide via endocytosis of a G protein-coupled receptor

Gastrin-releasing peptide receptor (GRPR), a member of the G protein-coupled receptor superfamily, has been utilized for receptor-mediated targeting of imaging and therapeutic agents; here we extend its use to oligonucleotide delivery. A splice-shifting antisense oligonucleotide was conjugated to a bombesin (BBN) peptide, and its intracellular delivery was tested in GRPR expressing PC3 cells stably transfected with a luciferase gene interrupted by an abnormally spliced intron. The BBN-conjugate produced significantly higher luciferase expression compared to unmodified oligonucleotide, and this increase was reversed by excess BBN peptide. Kinetic studies revealed a combination of saturable, receptor-mediated endocytosis and non-saturable pinocytosis for uptake of the conjugate. The K_m value for saturable uptake was similar to the EC₅₀ value for the pharmacological response, indicating that receptor-mediated endocytosis was a primary contributor to the response. Use of pharmacological and molecular inhibitors of endocytosis showed that the conjugate utilized a clathrin-, actin- and dynamin-dependent pathway to enter PC3 cells. The BBN-conjugate partially localized in endomembrane vesicles that were associated with Rab7 or Rab9, demonstrating that it was transported to late endosomes and the trans-golgi network. These observations suggest that the BBN-oligonucleotide conjugate enters cells via a process of GRPR mediated endocytosis followed by trafficking to deep endomembrane compartments.     

Stacking behavior of an intertidal abalone: an adaptive response or a consequence of space limitation?

Intertidal populations of black abalone Haliotis cracherodii Leach at Santa Cruz Island, California, vary in density among surge channels from < 1 to 126 abalone/m². Dense populations are characterized by high levels of intraspecific secondary substratum use (“stacking”) for attachment surfaces, though it is rare in low density areas. Use of shell surfaces by black abalone appears not to be an evolved adaptive strategy. Individuals in stacks fed significantly more than expected, yet solitary abalone next to stacks shared food trapped by stacks. In the laboratory, starvation did not increase the propensity of an abalone to stack. Stacks were equally common in the breeding and non-breeding seasons, suggesting that stacking behavior was not a mechanism to enhance reproductive success. Stacking did not enhance avoidance of predators or competitors. Laboratory experiments showed that stacking is density dependent and probably the result of limitation of primary attachment space. Removals of significant predators (sea otters and Chumash Indians) of adult abalone during the past two centuries probably led to increased densities of black abalone, which in turn has had an indirect effect on the prevalence of stacking.     

Votive well or refuse tip? Chronicle of an abandonment: taphonomic study of the faunal remains of an iron age well-cistern

Abstract

The well-cistern of the Iberian Fortress of Vilars (Catalonia, Spain) is a monumental feature dating to the late fifth century BCE (Vilars, phases III-IV). Management of water resources is key to interpreting the nature of the fortress, as water was essential not only for human and livestock consumption, but for irrigation, construction, and crafts. Moreover, water was integral to the site in both times of peace and war, as the structures that guaranteed provision and management coincide with facets of military architecture (moat). Yet, why was the well-cistern backfilled with animal remains and other archaeological material only a few years after its construction? To answer this and other questions, this study follows two main lines of research. Firstly, analyses were carried out on the archaeozoological remains collected inside the feature so as to interpret and characterise the nature of the assemblage. Secondly, taphonomic analyses (macro- and microscopic) were undertaken so as to reconstruct the dynamics of accumulation and the premature abandonment of the well-cistern’s primary function: supplying potable water. The results suggest that the abandonment of the well-cistern might be due in part to a drop of the groundwater level during a period of drought.     

Delayed cerebellar ataxia: a new complication of falciparum malaria?

Twelve cases of an unusual phenomenon of ataxia were investigated in otherwise well, conscious patients recovering from a febrile attack of presumed falciparum malaria. The ataxia occurred as the fever was subsiding, usually after an afebrile period of two to four days. The delay between onset of fever and the ataxia was three to four weeks. Peripheral blood of all the patients contained gametocytes of Plasmodium falciparum, and in some cases ring stages. The ataxia was most noticeable in the legs and the clinical picture suggested selective impairment of the cerebellar system. Signs of improvement appeared in a few weeks but complete recovery took one to four months. The most likely pathogenic mechanism of the ataxia in these cases was an immune reaction triggered by the malaria parasite and affecting the cerebellum or its connections, or both.     

Is post-contrast MRI a valuable method for the study of the nutrition of the intervertebral disc?

Decreased nutrition has been proposed as a potential mechanism leading to intervertebral disc degeneration. A method to investigate it in vivo is the MRI evaluation of the transport of a paramagnetic contrast agent, which is assumed to diffuse through the endplate to the disc using the same mechanisms as the cell nutrients. However, previous numerical studies questioned the value of this method as a model to investigate disc nutrition. To assess its validity, a parametric osmoporoelastic finite element model of a lumbar intervertebral disc incorporating diffusion and convection of a solute (representing the contrast agent) was developed. A Taguchi sensitivity analysis was performed in order to assess the relevance of various parameters which influence the solute transport. Subsequently, a full-factorial sensitivity analysis was used to investigate specifically the diffusion coefficients of the contrast agent. The most important parameters in determining the results were the disc height, the diffusion coefficients and the pharmacokinetic of the contrast agent. However, diffusion coefficients values as measured in in vitro studies would lead to insubstantial enhancement of the MRI signal. Thus, transport mechanisms other than pure diffusion should be active in in vivo transport of the contrast agent. In conclusion, the study showed that post-contrast MRI may not be suited for a quantitative analysis, but only for a qualitative examination aimed for example to detect endplate lesions. Open questions remain on the use of post-contrast MRI for the investigation of the relevance of reduced nutrition as a trigger to disc degeneration.     

A systematic study of labelling an α-helix in a protein with a lanthanide using IDA-SH or NTA-SH tags

The previously published IDA-SH and NTA-SH tags are small synthetic lanthanide-binding tags derived from cysteine, which afford site-specific lanthanide labelling by disulfide-bond formation with a cysteine residue of the target protein. Following attachment to a single cysteine in an α-helix, sizeable pseudocontact shifts (PCS) can be observed, if the lanthanide is immobilized by additional coordination to a negatively charged amino-acid side chain that is located in a neighboring turn of the helix. To identify the best labelling strategy for PCS measurements, we performed a systematic study, where IDA-SH or NTA-SH tags were ligated to a cysteine residue in position i of an α-helix, and aspartate or glutamate residues were placed in the positions i ? 4 or i + 4. The largest anisotropy components of the magnetic susceptibility tensor were observed for an NTA-SH tag in position i with a glutamate residue in position i ? 4. While the NTA-SH tag produced sizeable PCSs regardless of the presence of nearby carboxyl groups of the protein, the IDA-SH tag generated a good lanthanide binding site only if an aspartate was placed in position i + 4. The findings provide a firm basis for the design of site-directed mutants that are suitable for the reliable generation of PCSs in proteins with paramagnetic lanthanides.     

Is productivity of mesic savannas light limited or water limited? Results of a simulation study

A soil–plant–atmosphere model was used to estimate gross primary productivity (GPP) and evapotranspiration (ET) of a tropical savanna in Australia. This paper describes model modifications required to simulate the substantial C4 grass understory together with C3 trees. The model was further improved to include a seasonal distribution of leaf area and foliar nitrogen through 10 canopy layers. Model outputs were compared with a 5‐year eddy covariance dataset. Adding the C4 photosynthesis component improved the model efficiency and root‐mean‐squared error (RMSE) for total ecosystem GPP by better emulating annual peaks and troughs in GPP across wet and dry seasons. The C4 photosynthesis component had minimal impact on modelled values of ET. Outputs of GPP from the modified model agreed well with measured values, explaining between 79% and 90% of the variance and having a low RMSE (0.003–0.281 g C m^?2 day^?1). Approximately, 40% of total annual GPP was contributed by C4 grasses. Total (trees and grasses) wet season GPP was approximately 75–80% of total annual GPP. Light‐use efficiency (LUE) was largest for the wet season and smallest in the dry season and C4 LUE was larger than that of the trees. A sensitivity analysis of GPP revealed that daily GPP was most sensitive to changes in leaf area index (LAI) and foliar nitrogen (N_f) and relatively insensitive to changes in maximum carboxylation rate (V_cmax), maximum electron transport rate (J_max) and minimum leaf water potential (ψ_min). The modified model was also able to represent daily and seasonal patterns in ET, (explaining 68–81% of variance) with a low RMSE (0.038–0.19 mm day^?1). Current values of N_f, LAI and other parameters appear to be colimiting for maximizing GPP. By manipulating LAI and soil moisture content inputs, we show that modelled GPP is limited by light interception rather than water availability at this site.     

Delayed repair of radiation induced clustered DNA damage: friend or foe?

A signature of ionizing radiation exposure is the induction of DNA clustered damaged sites, defined as two or more lesions within one to two helical turns of DNA by passage of a single radiation track. Clustered damage is made up of double strand breaks (DSB) with associated base lesions or abasic (AP) sites, and non-DSB clusters comprised of base lesions, AP sites and single strand breaks. This review will concentrate on the experimental findings of the processing of non-DSB clustered damaged sites. It has been shown that non-DSB clustered damaged sites compromise the base excision repair pathway leading to the lifetime extension of the lesions within the cluster, compared to isolated lesions, thus the likelihood that the lesions persist to replication and induce mutation is increased. In addition certain non-DSB clustered damaged sites are processed within the cell to form additional DSB. The use of E. coli to demonstrate that clustering of DNA lesions is the major cause of the detrimental consequences of ionizing radiation is also discussed. The delayed repair of non-DSB clustered damaged sites in humans can be seen as a "friend", leading to cell killing in tumour cells or as a "foe", resulting in the formation of mutations and genetic instability in normal tissue.     

Which is the best method of sterilization of tumour bone for reimplantation? a biomechanical and histopathological study

Introduction  

Sterilization and re-usage of tumour bone for reconstruction after tumour resection is now gaining popularity in the East. This recycle tumour bone needs to be sterilized in order to eradicate the tumour cells before re-implantation for limb salvage procedures. The effect of some of these treatments on the integrity and sterility of the bone after treatment has been published but there has yet been a direct comparison between the various methods of sterilization to determine the one method that gives the best tumour kill without compromising the bone's structural integrity.     

Intracellular killing of bacteria: is Dictyostelium a model macrophage or an alien?

Predation of bacteria by phagocytic cells was first developed during evolution by environmental amoebae. Many of the core mechanisms used by amoebae to sense, ingest and kill bacteria have also been conserved in specialized phagocytic cells in mammalian organisms. Here we focus on recent results revealing how Dictyostelium discoideum senses and kills non‐pathogenic bacteria. In this model, genetic analysis of intracellular killing of bacteria has revealed a surprisingly complex array of specialized mechanisms. These results raise new questions on these processes, and challenge current models based largely on studies in mammalian phagocytes. In addition, recent studies suggest one additional level on complexity by revealing how Dictyostelium recognizes specifically various bacterial species and strains, and adapts its metabolism to process them. It remains to be seen to what extent mechanisms uncovered in Dictyostelium are also used in mammalian phagocytic cells.     

Troponin-tropomyosin: an allosteric switch or a steric blocker?

The interaction of myosin subfragment 1 (S1) with actin-tropomyosin-troponin (regulated actin) is highly nucleotide dependent. The binding of S1 or S1-ADP (but not S1-ATP nor N,N'-rho-phenylenedimaleimide-modified S1-ATP) to regulated actin activates ATP hydrolysis even in the absence of Ca(2+). Investigations with S1 and S1-ADP have led to the idea that some actin sites are directly blocked toward the binding of S1 either by tropomyosin or troponin. The blocked state is thought to occur only at ionic strengths greater than 50 mM. The question is whether nonactivating S1 binding is blocked under the same conditions. We show that troponin inhibits binding of the nonactivating state, N,N'-rho-phenylenedimaleimide-S1-ATP, to actin but only when tropomyosin is absent. A lag in the rate of binding of activating S1 to actin (an indicator of the blocked state) occurs only in the presence of tropomyosin. Thus, tropomyosin inhibits binding of rigor S1 but not S1-ATP-like states. No evidence for an ionic strength-dependent change in the mechanism of regulation was observed either from measurements of the rate of activating S1 binding or from the equilibrium binding of nonactivating S1 to actin. At all conditions examined, N,N'-rho-phenylenedimaleimide-S1-ATP bound to regulated actin in the absence of Ca(2+). These results support the view of regulation in which tropomyosin movement is an allosteric switch that is modulated by activating myosin binding but that does not function solely by regulating myosin binding.     

Non-canonical autophagy: an exception or an underestimated form of autophagy?

Macroautophagy (hereafter called autophagy) is a dynamic and evolutionarily conserved process used to sequester and degrade cytoplasm and entire organelles in a sequestering vesicle with a double membrane, known as the autophagosome, which ultimately fuses with a lysosome to degrade its autophagic cargo. Recently, we have unraveled two distinct forms of autophagy in cancer cells, which we term canonical and non-canonical autophagy. In contrast to classical or canonical autophagy, non-canonical autophagy is a process that does not require the entire set of autophagy-related (Atg) proteins in particular Beclin 1, to form the autophagosome. Non-canonical autophagy is therefore not blocked by the knockdown of Beclin 1 or of its binding partner hVps34. Moreover overexpression of Bcl-2, which is known to block canonical starvation-induced autophagy by binding to Beclin 1, is unable to reverse the non-canonical autophagy triggered by the polyphenol resveratrol in the breast cancer MCF-7 cell line. In MCF-7 cells, at least, non-canonical autophagy is involved in the caspase-independent cell death induced by resveratrol.     

What makes an aquaporin a glycerol channel? A comparative study of AqpZ and GlpF

The recent availability of high-resolution structures of two structurally highly homologous, but functionally distinct aquaporins from the same species, namely Escherichia coli AqpZ, a pure water channel, and GlpF, a glycerol channel, presents a unique opportunity to understand the mechanism of substrate selectivity in these channels. Comparison of the free energy profile of glycerol conduction through AqpZ and GlpF reveals a much larger barrier in AqpZ (22.8 kcal/mol) than in GlpF (7.3 kcal/mol). In either channel, the highest barrier is located at the selectivity filter. Analysis of substrate-protein interactions suggests that steric restriction of AqpZ is the main contribution to this large barrier. Another important difference is the presence of a deep energy well at the periplasmic vestibule of GlpF, which was not found in AqpZ. The latter difference can be attributed to the more pronounced structural asymmetry of GlpF, which may play a role in attracting glycerol.