Identification and characterization of carbapenem binding sites within the RND-transporter AcrB

Understanding the molecular determinants for recognition, binding and transport of antibiotics by multidrug efflux systems is important for basic research and useful for the design of more effective antimicrobial compounds. Imipenem and meropenem are two carbapenems whose antibacterial activity is known to be poorly and strongly affected by MexAB-OprM, the major efflux pump transporter in Pseudomonas aeruginosa. However, not much is known regarding recognition and transport of these compounds by AcrAB-TolC, which is the MexAB-OprM homologue in Escherichia coli and by definition the paradigm model for structural studies on efflux pumps. Prompted by this motivation, we unveiled the molecular details of the interaction of imipenem and meropenem with the transporter AcrB by combining computer simulations with biophysical experiments. Regarding the interaction with the two main substrate binding regions of AcrB, the so-called access and deep binding pockets, molecular dynamics simulations revealed imipenem to be more mobile than meropenem in the former, while comparable mobilities were observed in the latter. This result is in line with isothermal titration calorimetry, differential scanning experiments, and binding free energy calculations, indicating a higher affinity for meropenem than imipenem at the deep binding pocket, while both sharing similar affinities at the access pocket. Our findings rationalize how different physico-chemical properties of compounds reflect on their interactions with AcrB. As such, they constitute precious information to be exploited for the rational design of antibiotics able to evade efflux pumps.     

Toward intensifying design of experiments in upstream bioprocess development: An industrial Escherichia coli feasibility study

In this study, step variations in temperature, pH, and carbon substrate feeding rate were performed within five high cell density Escherichia coli fermentations to assess whether intraexperiment step changes, can principally be used to exploit the process operation space in a design of experiment manner. A dynamic process modeling approach was adopted to determine parameter interactions. A bioreactor model was integrated with an artificial neural network that describes biomass and product formation rates as function of varied fed‐batch fermentation conditions for heterologous protein production. A model reliability measure was introduced to assess in which process region the model can be expected to predict process states accurately. It was found that the model could accurately predict process states of multiple fermentations performed at fixed conditions within the determined validity domain. The results suggest that intraexperimental variations of process conditions could be used to reduce the number of experiments by a factor, which in limit would be equivalent to the number of intraexperimental variations per experiment. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1343–1352, 2016     

REPEATED ASSESSMENT OF THE ENDOGENOUS 24-HOUR PROFILE OF BLOOD PRESSURE UNDER CONSTANT ROUTINE*

The impact of environmental and behavioral factors on the 24-h profile of blood pressure (BP) has been well established. Various attempts have been made to control these exogenous factors, in order to investigate a possible endogenous circadian variation of BP. Recently, we reported the results of the first environmentally and behaviorally controlled laboratory study with 24-h recordings of BP and heart rate (HR) during maintained wakefulness. In this constant-routine study, a pronounced endogenous circadian rhythm of HR was found, but circadian variation of BP was absent. This result suggested that the circadian rhythm of BP observed in earlier controlled studies, with sleep allowed, was evoked by the sleep–wake cycle as opposed to the endogenous circadian pacemaker. In order to verify our previous finding during maintained wakefulness, we repeated the experiment five times with six normotensive, healthy young subjects. Statistical analyses of the hourly measurements of BP and HR confirmed the replicable presence of an endogenous circadian rhythm of HR, as well as the consistent absence of an endogenous circadian variation of BP. Thus, this study provided additional evidence that the 24-h profile of BP—as observed under normal circumstances—is the sole result of environmental and behavioral factors such as the occurrence of sleep, and has no endogenous circadian component. (Chronobiology International, 18(1), 85–98, 2001)     

Auditory assessment of avian predator threat in semi-captive ringtailed lemurs (Lemur catta)

Antiraptor responses from forest-living ringtailed lemurs to advertisement calls of naturally-occurring red-tailed hawks suggested that the lemurs discriminated these calls from other environmental sounds. A series of playback experiments, using real animal sounds and synthetic sound probes, was conducted to investigate the acoustic basis of this putative discrimination. Two semi-captive groups of ringtails served as study subjects: one group had many years of experience living in the forest, whereas the other group had relatively little such experience. Responses to playbacks suggested that both groups used the same acoustic criteria to discriminate “calls of large hawks” from other sounds, but the range of auditory stimuli that evoked antiraptor responses was broader for the experienced group than for the inexperienced group. Although several interpretations of the experimental results are possible, one that seems particularly compatible with the data is the “prototype” concept of stimulus categorization.     

Temperature as a predictor of survival of the pine processionary moth in the Italian Alps

1 The pine processionary moth Thaumetopoea pityocampa is expanding its geographical range in Europe, as a consequence of enhanced winter survival under a warmer climate. A combination of daytime nest temperatures and night air temperatures determines the number of hours larvae are able to feed (hours above realized feeding threshold, RFT). 2 We tested the RFT‐based model for survival across multiple areas of the insect’s range in the Italian Alps over a 2‐year period. In a series of translocation experiments using natural temperature gradients as spatial analogues for global warming, we transferred colonies of T. pityocampa larvae to sites within zones of historical distribution, recent distribution, and outside the present range. The sites included traditional (Pinus nigra, Pinus sylvestris) as well as novel (Pinus mugo, Pinus uncinata, Pinus cembra) hosts. 3 Survival during precold (August to November), cold (December to February) and postcold (March to May) periods were analysed against climatic variables (temperature and rainfall) and predictors developed by the model. 4 Host species did not significantly affect final survival, with the exception of slower larval development, and resulting lower cold tolerance, on P. cembra than on P. mugo at the same site. 5 Across all the sites and hosts, final survival of colonies depended on the number of feeding hours during the cold period (RFT), which explained 82% of the variance in a regression model. We recommend using RFT, or its surrogate daily mean minimum temperature when nest temperature is not available, in predictive models of range expansion of T. pityocampa under climate change scenarios.     

Exploring the linkage between cell culture process parameters and downstream processing utilizing a plackett‐burman design for a model monoclonal antibody

Linkage of upstream cell culture with downstream processing and purification is an aspect of Quality by Design crucial for efficient and consistent production of high quality biopharmaceutical proteins. In a previous Plackett‐Burman screening study of parallel bioreactor cultures we evaluated main effects of 11 process variables, such as agitation, sparge rate, feeding regimens, dissolved oxygen set point, inoculation density, supplement addition, temperature, and pH shifts. In this follow‐up study, we observed linkages between cell culture process parameters and downstream capture chromatography performance and subsequent antibody attributes. In depth analysis of the capture chromatography purification of harvested cell culture fluid yielded significant effects of upstream process parameters on host cell protein abundance and behavior. A variety of methods were used to characterize the antibody both after purification and buffer formulation. This analysis provided insight in to the significant impacts of upstream process parameters on aggregate formation, impurities, and protein structure. This report highlights the utility of linkage studies in identifying how changes in upstream parameters can impact downstream critical quality attributes. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:163–170, 2017     

HYBRIDIZATION,NATURAL SELECTION,AND EVOLUTION OF REPRODUCTIVE ISOLATION: A 25‐YEARS SURVEY OF AN ARTIFICIAL SYMPATRIC AREA BETWEEN TWO MOSQUITO SIBLING SPECIES OF THE Aedes mariae COMPLEX

Natural selection can act against maladaptive hybridization between co‐occurring divergent populations leading to evolution of reproductive isolation among them. A critical unanswered question about this process that provides a basis for the theory of speciation by reinforcement, is whether natural selection can cause hybridization rates to evolve to zero. Here, we investigated this issue in two sibling mosquitoes species, Aedes mariae and Aedes zammitii, that show postmating reproductive isolation (F1 males sterile) and partial premating isolation (different height of mating swarms) that could be reinforced by natural selection against hybridization. In 1986, we created an artificial sympatric area between the two species and sampled about 20,000 individuals over the following 25 years. Between 1986 and 2011, the composition of mating swarms and the hybridization rate between the two species were investigated across time in the sympatric area. Our results showed that A. mariae and A. zammitii have not completed reproductive isolation since their first contact in the artificial sympatric area. We have discussed the relative role of factors such as time of contact, gene flow, strength of natural selection, and biological mechanisms causing prezygotic isolation to explain the observed results.     

PBL (Problem-based learning)教学模式在微生物 制药设计性实验教学中的应用

微生物制药设计性实验是提高学生专业技能和培养创新能力的一种重要途径.为提高教学质量,我们在设计性实验教学中开展了基于问题的学习(PBL)教学模式.PBL教学模式的引入显著地提高了学生的学习积极性,锻炼了学生分析和解决问题的能力,增强了沟通和交流能力.本文报道PBL教学模式在微生物制药设计性实验中的实施情况,并对实施过程中出现的一些问题进行分析和探讨.     

Crossing experiments and behavioral observations reveal reproductive incompatibility among three putative species of the whitefly Bemisia tabaci

Abstract The whitefly Bemisia tabaci has a global distribution and extensive genetic diversity. Recent phylogenetic analyses as well as crossing experiments suggest that B. tabaci is a complex composed of > 20 cryptic species, but more crossing studies are required to examine the reproductive compatibility among the putative species and thus further clarify the systematics of this species complex. We conducted crossing experiments and behavioral observations to investigate the reproductive compatibility between the Mediterranean, Asia II 3, and Asia II 1 putative species of B. tabaci collected from Zhejiang, China. Female progeny were never produced in inter-species crosses, demonstrating a lack of egg fertilization; while 55%–75% females were produced in all the intra-species treatments. Continuous behavioral observations showed that frequent courtship events occurred in both intra-species treatments and inter-putative species crosses. However, copulation events occurred only in the three intra-species treatments with one exception: that one copulation event occurred between Asia II 3 and Mediterranean in the crosses where two cohorts of females and males of different putative species were enclosed together in a small arena but were not allowed access to their intra-specific mates for a long period of time. These data demonstrated complete reproductive isolation between the Mediterranean, Asia II 3, and Asia II 1 putative species, and further showed that the isolation is due to lack of copulation. Demonstration of reproductive isolation between the Mediterranean and two indigenous putative species from China provides further evidence for the existence of cryptic species within the B. tabaci complex.     

A tracked approach for automated NMR assignments in proteins (TATAPRO)

A novel automated approach for the sequence specific NMR assignments of ¹H^N, ¹³C, ¹³C, ¹³C/¹H and ¹⁵N spins in proteins, using triple resonance experimental data, is presented. The algorithm, TATAPRO (Tracked AuTomated Assignments in Proteins) utilizes the protein primary sequence and peak lists from a set of triple resonance spectra which correlate ¹H^N and ¹⁵N chemical shifts with those of ¹³C, ¹³C and ¹³C/¹H. The information derived from such correlations is used to create a `master_list' consisting of all possible sets of ¹H^N _i, ¹⁵N_i, ¹³C _i, ¹³C _i, ¹³C_i/¹H _i, ¹³C _i–1, ¹³C _i–1 and ¹³C_i–1/ ¹H _i–1 chemical shifts. On the basis of an extensive statistical analysis of ¹³C and ¹³C chemical shift data of proteins derived from the BioMagResBank (BMRB), it is shown that the 20 amino acid residues can be grouped into eight distinct categories, each of which is assigned a unique two-digit code. Such a code is used to tag individual sets of chemical shifts in the master_list and also to translate the protein primary sequence into an array called pps_array. The program then uses the master_list to search for neighbouring partners of a given amino acid residue along the polypeptide chain and sequentially assigns a maximum possible stretch of residues on either side. While doing so, each assigned residue is tracked in an array called assig_array, with the two-digit code assigned earlier. The assig_array is then mapped onto the pps_array for sequence specific resonance assignment. The program has been tested using experimental data on a calcium binding protein from Entamoeba histolytica (Eh-CaBP, 15 kDa) having substantial internal sequence homology and using published data on four other proteins in the molecular weight range of 18–42 kDa. In all the cases, nearly complete sequence specific resonance assignments (> 95%) are obtained. Furthermore, the reliability of the program has been tested by deleting sets of chemical shifts randomly from the master_list created for the test proteins.