The Alarm Pheromone and Alarm Response of the Clonal Raider Ant.

Ants communicate via an arsenal of different pheromones produced in a variety of exocrine glands. For example, ants release alarm pheromones in response to danger to alert their nestmates and to trigger behavioral alarm responses. Here we characterize the alarm pheromone and the alarm response of the clonal raider ant Ooceraea biroi, a species that is amenable to laboratory studies but for which no pheromones have been identified. During an alarm response, ants quickly become unsettled, leave their nest pile, and are sometimes initially attracted to the source of alarm, but ultimately move away from it. We find that the alarm pheromone is released from the head of the ant and identify the putative alarm pheromone as a blend of two compounds found in the head, 4-methyl-3-heptanone and 4-methyl-3-heptanol. These compounds are sufficient to induce alarm behavior alone and in combination. They elicit similar, though slightly different behavioral features of the alarm response, with 4-methyl-3-heptanone being immediately repulsive and 4-methyl-3-heptanol being initially attractive before causing ants to move away. The behavioral response to these compounds in combination is dose-dependent, with ants becoming unsettled and attracted to the source of alarm pheromone at low concentrations and repulsed at high concentrations. While 4-methyl-3-heptanone and 4-methyl-3-heptanol are known alarm pheromones in other more distantly related ant species, this is the first report of the chemical identity of a pheromone in O. biroi, and the first alarm pheromone identified in the genus Ooceraea. Identification of a pheromone that triggers a robust, consistent, and conserved behavior, like the alarm pheromone, provides an avenue to dissect the behavioral and neuronal mechanisms underpinning chemical communication.

Cx43-Delivered miR-181b Negatively Regulates Sirt1/FOXO3a Signalling Pathway-Mediated Apoptosis on Intestinal Injury in Sepsis.

INTRODUCTION: Gap junctions can transmit signals between cells, including miRNAs, leading to the amplification of adjacent cell damage. No previous study has addressed gap junctions and miRNAs in sepsis because the internal mechanism of sepsis-induced intestinal injury is complex. Therefore, we studied the relationship between connexin43 (Cx43) and miR-181b and provided a research direction for further study of sepsis. METHODS: A mouse caecal ligation and puncture method was used to construct a mouse sepsis model. Firstly, damage to intestinal tissues at different time points was analysed. The levels of Cx43, miR-181b, Sirt1, and FOXO3a in intestinal tissues and the transcription and translation of the apoptosis-related genes Bim and puma, which are downstream of FOXO3a were analysed. Secondly, the effect of Cx43 levels on miR-181b and Sirt1/FOXO3a signalling pathway activity was explored by using the Cx43 inhibitor heptanol. Finally, luciferase assays were used to determine miR-181b binding to the predicted target sequence. RESULTS: The results show that during sepsis, intestinal injury becomes increasingly worse with time, and the expression of Cx43 and miR-181b increase. In addition, we found that heptanol could significantly reduce intestinal injury. This finding indicates that inhibiting Cx43 regulates the transfer of miR-181b between adjacent cells, thereby reducing the activity of the Sirt1/FOXO3a signalling pathway and reducing the degree of intestinal injury during sepsis. CONCLUSIONS: In sepsis, the enhancement of Cx43 gap junctions leads to an increase in miR-181b intercellular transfer, affects the downstream SIRT1/FOXO3a signalling pathway and causes cell and tissue damage.

Virus-Induced Plant Volatiles Promote Virus Acquisition and Transmission by Insect Vectors.

Rice dwarf virus (RDV) is transmitted by insect vectors Nephotettix virescens and Nephotettix cincticeps (Hemiptera: Cicadellidae) that threatens rice yield and results in substantial economic losses. RDV induces two volatiles ((E)-ß-caryophyllene (EBC) and 2-heptanol) to emit from RDV-infected rice plants. However, the effects of the two volatiles on the olfactory behavior of both non-viruliferous and viruliferous N. virescens are unknown, and whether the two volatiles could facilitate the spread and dispersal of RDV remains elusive. Combining the methods of insect behavior, chemical ecology, and molecular biology, we found that EBC and 2-heptanol influenced the olfactory behavior of non-viruliferous and viruliferous N. virescens, independently. EBC attracted non-viruliferous N. virescens towards RDV-infected rice plants, promoting virus acquisition by non-viruliferous vectors. The effect was confirmed by using oscas1 mutant rice plants (repressed EBC synthesis), but EBC had no effects on viruliferous N. virescens. 2-heptanol did not attract or repel non-viruliferous N. virescens. However, spraying experiments showed that 2-heptanol repelled viruliferous N. virescens to prefer RDV-free rice plants, which would be conducive to the transmission of the virus. These novel results reveal that rice plant volatiles modify the behavior of N. virescens vectors to promote RDV acquisition and transmission. They will provide new insights into virus-vector-plant interactions, and promote the development of new prevention and control strategies for disease management.

Heptanol-mediated phase separation determines phase preference of molecules in live cell membranes.

The localization of many membrane proteins within cholesterol- and sphingolipid-containing microdomains is essential for proper cell signaling and function. These membrane domains, however, are too small and dynamic to be recorded, even with modern super-resolution techniques. Therefore, the association of membrane proteins with these domains can only be detected with biochemical assays that destroy the integrity of cells require pooling of many cells and take a long time to perform. Here, we present a simple membrane fluidizer-induced clustering approach to identify the phase-preference of membrane-associated molecules in individual live cells within 10-15 min. Experiments in phase-separated bilayers and live cells on molecules with known phase preference show that heptanol hyperfluidizes the membrane and stabilizes phase separation. This results in a transition from nanosized to micronsized clusters of associated molecules allowing their identification using routine microscopy techniques. Membrane fluidizer-induced clustering is an inexpensive and easy to implement method that can be conducted at large-scale and allows easy identification of protein partitioning in live cell membranes.

One-Pot Bioelectrocatalytic Conversion of Chemically Inert Hydrocarbons to Imines.

Petroleum hydrocarbons are our major energy source and an important feedstock for the chemical industry. With the exception of combustion, the deep conversion of chemically inert hydrocarbons to more valuable chemicals is of considerable interest. However, two challenges hinder this conversion. One is the regioselective activation of inert carbon-hydrogen (C-H) bonds. The other is designing a pathway to realize this complicated conversion. In response to the two challenges, a multistep bioelectrocatalytic system was developed to realize the one-pot deep conversion from heptane to N-heptylhepan-1-imine under mild conditions. First, in this enzymatic cascade, a bioelectrocatalytic C-H bond oxyfunctionalization step based on alkane hydroxylase (alkB) was applied to regioselectively convert heptane to 1-heptanol. By integrating subsequent alcohol oxidation and bioelectrocatalytic reductive amination steps based on an engineered choline oxidase (AcCO6) and a reductive aminase (NfRedAm), the generated 1-heptanol was successfully converted to N-heptylhepan-1-imine. The electrochemical architecture provided sufficient electrons to drive the bioelectrocatalytic C-H bond oxyfunctionalization and reductive amination steps with neutral red (NR) as electron mediator. The highest concentration of N-heptylhepan-1-imine achieved was 0.67 mM with a Faradaic efficiency of 45% for C-H bond oxyfunctionalization and 70% for reductive amination. Hexane, octane, and ethylbenzene were also successfully converted to the corresponding imines. Via regioselective C-H bond oxyfunctionalization, intermediate oxidation, and reductive amination, the bioelectrocatalytic hydrocarbon deep conversion system successfully realized the challenging conversion from inert hydrocarbons to imines that would have been impossible by using organic synthesis methods and provided a new methodology for the comprehensive conversion and utilization of inert hydrocarbons.

The lipoxygenase gene OsRCI-1 is involved in the biosynthesis of herbivore-induced JAs and regulates plant defense and growth in rice.

The pathway mediated by jasmonic acid (JA), biosynthesized via 13-lipoxygenases (LOX), plays a central role in both plant development and defense. In rice, there are at least fourteen 13-LOXs. Yet, only two 13-LOXs have been known to be involved in the biosynthesis of JA and plant defenses in rice. Here we cloned a chloroplast-localized 13-LOX gene from rice, OsRCI-1, whose transcripts were upregulated following infestation by brown planthopper (BPH, Nilaparvata lugens), one of the most important pests in rice. Overexpression of OsRCI-1 (oeRCI lines) increased levels of BPH-induced JA, jasmonate-isoleucine, trypsin protease inhibitors and three volatile compounds, 2-heptanone, 2-heptanol and α-thujene. BPHs showed a decreased colonization, fecundity and mass, and developed slowly on oeRCI plants compared with wild-type (WT) plants. Moreover, BPH-infested oeRCI plants were more attractive to the egg parasitoid of BPH, Anagrus nilaparvatae than equally treated WT plants. The decreased attractiveness to BPH and enhanced attractiveness to the parasitoid of oeRCI plants correlated with higher levels of BPH-induced 2-heptanone and 2-heptanol, and 2-heptanone, respectively. Compared with oeRCI plants, WT plants had higher plant height and 1000-grain weight. These results indicate that OsRCI-1 is involved in herbivore-induced JA bursts and plays a role in plant defense and growth.

Anesthetics and Cell-Cell Communication: Potential Ca2+-Calmodulin Role in Gap Junction Channel Gating by Heptanol, Halothane and Isoflurane.

Cell-cell communication via gap junction channels is known to be inhibited by the anesthetics heptanol, halothane and isoflurane; however, despite numerous studies, the mechanism of gap junction channel gating by anesthetics is still poorly understood. In the early nineties, we reported that gating by anesthetics is strongly potentiated by caffeine and theophylline and inhibited by 4-Aminopyridine. Neither Ca2+ channel blockers nor 3-isobutyl-1-methylxanthine (IBMX), forskolin, CPT-cAMP, 8Br-cGMP, adenosine, phorbol ester or H7 had significant effects on gating by anesthetics. In our publication, we concluded that neither cytosolic Ca2+i nor pHi were involved, and suggested a direct effect of anesthetics on gap junction channel proteins. However, while a direct effect cannot be excluded, based on the potentiating effect of caffeine and theophylline added to anesthetics and data published over the past three decades, we are now reconsidering our earlier interpretation and propose an alternative hypothesis that uncoupling by heptanol, halothane and isoflurane may actually result from a rise in cytosolic Ca2+ concentration ([Ca2+]i) and consequential activation of calmodulin linked to gap junction proteins.

Virus-induced plant volatiles mediate the olfactory behaviour of its insect vectors.

Plant viruses can manipulate their hosts to release odours that are attractive or repellent to their insect vectors. However, the volatile organic compounds (VOCs), either individually or as mixtures, which play a key role in the olfactory behaviour of insect vectors remains largely unknown. Our study focused on green rice leafhoppers (GRLHs) vectoring rice dwarf virus (RDV) revealed that RDV infection significantly induced the emission of (E)-ß-caryophyllene and 2-heptanol by rice plants, which influenced the olfactory behaviour of both non-viruliferous and viruliferous GRLHs. (E)-ß-caryophyllene attracted non-viruliferous GRLHs to settle on RDV-infected plants, but neither attracted nor repelled viruliferous GRLHs. In contrast, 2-heptanol repelled viruliferous GRLHs to settle on RDV-infected plants, but neither repelled nor attracted non-viruliferous GRLHs. Suppression of (E)-ß-caryophyllene synthase OsCAS via CRISPR-Cas9 to generate oscas-1 plants enabled us to confirm the important role played by (E)-ß-caryophyllene in modulating the virus-vector-host plant interaction. These novel results reveal the role of these virus-induced VOCs in modulating the behaviour of its GRLH insect vector and may facilitate the design of new strategies for disease control through manipulation of plant volatile emissions.

Bioactive Male-Produced Volatiles from Anastrepha obliqua and their Role in Attraction of Conspecific Females.

The study of insect semiochemicals, especially pheromones, is of fundamental importance for the development of strategies for controlling agricultural pests. In this study, volatile compounds involved in the communication between males and females of the fruit fly, Anastrepha obliqua (Diptera: Tephritidae), for mating purposes were characterized to develop attractant formulations for females of this species. Extracts containing volatile compounds released by males of A. obliqua were obtained by the dynamic headspace technique and analyzed by gas chromatography coupled with an electroantennographic detector (GC-EAD) and gas chromatography coupled with mass spectrometry (GC-MS). Twenty-one volatile compounds were identified in the aeration extracts of males. Five of them caused EAD responses from the antennae of females: 1-heptanol, linalool, (Z)-3-nonen-1-ol, (E,Z)-3,6-nonadien-1-ol, and (Z,E)-α-farnesene. Six synthetic mixtures of these compounds, including the five-component blend and all possible four-component blends, were formulated in a biopolymer and used in behavioral bioassays conducted in the laboratory arena with conspecific virgin females. One blend of 1-heptanol, linalool, (Z)-3-nonen-1-ol, and (Z,E)-α-farnesene attracted more females than the collection of volatiles from virgin males used as control. The other mixtures were as attractive to A. obliqua females as the control treatment. This study indicates potential for use of these compounds in monitoring and control strategies for this pest.

A robust and economical pulse-chase protocol to measure the turnover of HaloTag fusion proteins.

The self-labeling protein HaloTag has been used extensively to determine the localization and turnover of proteins of interest at the single-cell level. To this end, halogen-substituted alkanes attached to diverse fluorophores are commercially available that allow specific, irreversible labeling of HaloTag fusion proteins; however, measurement of protein of interest half-life by pulse-chase of HaloTag ligands is not widely employed because residual unbound ligand continues to label newly synthesized HaloTag fusions even after extensive washing. Excess unlabeled HaloTag ligand can be used as a blocker of undesired labeling, but this is not economical. In this study, we screened several inexpensive, low-molecular-weight haloalkanes as blocking agents in pulse-chase labeling experiments with the cell-permeable tetramethylrhodamine HaloTag ligand. We identified 7-bromoheptanol as a high-affinity, low-toxicity HaloTag-blocking agent that permits protein turnover measurements at both the cell population (by blotting) and single-cell (by imaging) levels. We show that the HaloTag pulse-chase approach is a nontoxic alternative to inhibition of protein synthesis with cycloheximide and extend protein turnover assays to long-lived proteins.

Olfactory Detection Thresholds for Primary Aliphatic Alcohols in Mice.

Probing the neural mechanisms that underlie each sensory system requires the presentation of perceptually appropriate stimulus concentrations. This is particularly relevant in the olfactory system as additional odorant receptors typically respond with increasing stimulus concentrations. Thus, perceptual measures of olfactory sensitivity provide an important guide for functional experiments. This study focuses on aliphatic alcohols because they are commonly used to survey neural activity in a variety of olfactory regions, probe the behavioral limits of odor discrimination, and assess odor-structure activity relationships in mice. However, despite their frequent use, a systematic study of the relative sensitivity of these odorants in mice is not available. Thus, we assayed the ability of C57BL/6J mice to detect a homologous series of primary aliphatic alcohols (1-propanol to 1-heptanol) using a head-fixed Go/No-Go operant conditioning assay combined with highly reproducible stimulus delivery. To aid in the accessibility of our data, we report the animal's threshold to each odorant according to the 1) ideal gas condition, 2) nonideal gas condition (factoring in the activity of the odorant in the solvent), and 3) the liquid dilution of the odorant in the olfactometer. Of the odorants tested, mice were most sensitive to 1-hexanol and least sensitive to 1-butanol. These updated measures of murine sensitivity will hopefully guide experimenters in choosing appropriate stimulus concentrations for experiments using these odorants.

Gap junction mediated signaling between satellite glia and neurons in trigeminal ganglia.

Peripheral sensory ganglia contain the somata of neurons mediating mechanical, thermal, and painful sensations from somatic, visceral, and oro-facial organs. Each neuronal cell body is closely surrounded by satellite glial cells (SGCs) that have properties and functions similar to those of central astrocytes, including expression of gap junction proteins and functional dye coupling. As shown in other pain models, after systemic pain induction by intra-peritoneal injection of lipopolysaccharide, dye coupling among SGCs in intact trigeminal ganglion was enhanced. Moreover, neuron-neuron and neuron-SGC coupling was also detected. To verify the presence of gap junction-mediated coupling between SGCs and sensory neurons, we performed dual whole cell patch clamp recordings from both freshly isolated and short term cultured cell pairs dissociated from mouse trigeminal ganglia. Bidirectional gap junction mediated electrical responses were frequently recorded between SGCs, between neurons and between neurons and SGCs. Polarization of SGC altered neuronal excitability, providing evidence that gap junction-mediated interactions between neurons and glia within sensory ganglia may contribute to integration of peripheral sensory responses, and to the modulation and coordinaton of neuronal activity.

Involvement of sphingosine-1-phosphate receptors 2/3 in IR-induced sudden cardiac death.

It has been demonstrated that S1P receptors affect heart ischaemia-reperfusion (IR) induced injury. However, whether S1P receptors affect IR-induced cardiac death has not been investigated. The aim of this paper is to demonstrate the role of S1P receptors in IR-induced cardiac death. Healthy adult male Sprague-Dawley rats were assigned to the following groups: non-operation control group, sham operation group, IR group, IR group pretreated with DMSO, IR group pretreated with S1P3 agonist, IR group pretreated with an antagonist of S1P3, IR group pretreated with S1P2 and S1P3 antagonists, IR group pretreated with heptanol and antagonists of S1P2/3, and IR group pretreated with Gap26 and antagonists of S1P2/3 (heptanol acts as a Cx43 uncoupler and the mimic peptide Gap26 as Cx43 blocker). The groups with S1P2 or S1P3 agonist application before reperfusion were used to assess whether these can be used for therapy of IR. The haemodynamics, electrocardiograms (ECG), infarction area, and mortality rates were recorded. Immunohistological connexin 43 (Cx43) expression in the heart was detected in each group. Blocking S1P2/3 receptors with specific antagonists resulted in an increment of IR-induced mortality, increased infarction size, redistribution of Cx43 expression, as well as affecting the heart function. The infarction size, heart function, and mortality were totally or partially restored in the S1P2, S1P3 agonist-pretreated IR group, and the heptanol/Gap26-treated S1P2/3-blocked IR group. The S1P receptor S1P2/3 and Cx43 are involved in the IR-induced cardiac death.

Electromembrane extraction of verapamil and riluzole from urine and wastewater samples using a mixture of organic solvents as a supported liquid membrane: Study on electric current variations.

In this study, the application of a mixture of organic solvents as a supported liquid membrane for improving the efficiency of the electromembrane extraction procedure was investigated. The extraction process was followed by high-performance liquid chromatography analysis of two model drugs (verapamil and riluzole). In this research, four organic solvents, including 1-heptanol, 1-octanol, 2-nitrophenyl octyl ether, and 2-ethyl hexanol, were selected as model solvents and different binary mixtures (v/v 2:1, 1:1 and 1:2) were used as the supported liquid membrane. The mixture of 2-ethyl hexanol and 1-otanol (v/v, 2:1) improved the extraction efficiency of model drugs by 1.5 to 12 times. It was found that extraction efficiency is greatly influenced by the level of electric current. In this study, for various mixtures of organic solvents, the electric current fluctuated between 50 and 2500 µA, and the highest extraction efficiencies were obtained with low and stable electric currents. Finally, the optimized extraction condition was validated and applied for the determination of model drugs in urine and wastewater samples.

Dose-related ethanol intake, Cx43 and Nav1.5 remodeling: Exploring insights of altered ventricular conduction and QRS fragmentation in excessive alcohol users.

BACKGROUND: Chronic, excessive ethanol intake has been linked with various electrical instabilities, conduction disturbances, and even sudden cardiac death, but the underlying cause for the latter is insufficiently delineated. METHODS: We studied surface electrocardiography (ECG) in a community-dwelling cohort with moderate-to-heavy daily alcohol intake (grouped as >90g/day, ≤90g/day, and nonintake). RESULTS: Compared with nonintake, heavier alcohol users showed markedly widened QRS duration and higher prevalence of QRS fragmentation (64.3%, 50.9%, and 33.7%, respectively, χ2 12.0, both p<0.05) on surface ECG across the 3 groups. These findings were successfully recapitulated in 14-week-old C57BL/6 mice that were chronically given a 4% or 6% alcohol diet and showed dose-related slower action potential upstroke, reduced resting membrane potential, and disorganized or decreased intraventricular conduction (all p<0.05). Immunodetection further revealed increased ventricular collagen I depots with Cx43 downregulation and remodeling, together with clustered and diminished membrane Nav1.5 distribution. Administration of Cx43 blocker (heptanol) and Nav1.5 inhibitor (tetrodotoxin) in the mice each attenuated the suppression ventricular conduction compared with nonintake mice (p<0.05). CONCLUSIONS: Chronic excessive alcohol ingestion is associated with dose-related phenotypic intraventricular conduction disturbances and QRS fragmentation that can be recapitulated in mice. The mechanisms may involve suppressed gap junction and sodium channel functions, together with enhanced cardiac fibrosis that may contribute to arrhythmogenesis.

Antinociceptive effect of Aristolochia trilobata stem essential oil and 6-methyl-5-hepten-2yl acetate, its main compound, in rodents.

Aristolochia trilobata L. is an aromatic plant, popularly known as "mil-homens", and its essential oil (EO) is generally used to treat colic, diarrhea and dysentery disorders. We evaluated the antinociceptive effect of A. trilobata stem EO and of its major compound, the (R)-(-)-6-methyl-5-hepten-2-yl acetate (sulcatyl acetate: SA), using acetic acid (0.85%)-induced writhing response and formalin-induced (20 µL of 1%) nociceptive behavior in mice. We also evaluated the EO and SA effect on motor coordination, using the rota-rod apparatus. EO (25, 50 and 100 mg/kg) or SA (25 and 50 mg/kg) reduced nociceptive behavior in the writhing test (p<0.001). EO (100 mg/kg) and SA (25 and 50 mg/kg) decreased the nociception on the first phase of the formalin test (p<0.05). On the second phase, EO (25: p<0.01; 50: p<0.05 and 100 mg/kg: p<0.001) and SA (25 and 50 mg/kg; p<0.001) reduced the nociceptive response induced by formalin. EO and SA were not able to cause changes in the motor coordination of animals. Together, our results suggest that EO has an analgesic profile and SA seems to be one of the active compounds in this effect.

Preparation and structural characterization of corn starch-aroma compound inclusion complexes.

BACKGROUND: Six corn starch inclusion complexes were synthesized using small nonpolar or weak polar aroma compounds (heptanolide, carvone and menthone) and small polar aroma compounds (linalool, heptanol and menthol). The objectives of this study were to (a) investigate the ability of corn starch to form inclusion complexes with these aroma compounds and (b) characterize the structure of the corn starch inclusion complexes. RESULTS: The resulting inclusion ratios were 75.6, 36.9, 43.8, 91.9, 67.2 and 54.7% for heptanolide, carvone, menthone, linalool, heptanol and menthol respectively. The inclusion complexes had laminated structures with a certain amount of holes or blocky constructions. Compared with gelatinized corn starch, the transition temperatures, peak temperatures and enthalpies of the inclusion complexes were significantly different. The major peak of CO at 1771 cm-1 and significant peak shifts revealed the formation of inclusion complexes. X-ray diffractometry (XRD) analyses revealed that the crystallinity of corn starch-polar aroma compound inclusion complexes increased. Based on cross-polarization magic angle spinning 13 C nuclear magnetic resonance (CP-MAS 13 C NMR) results, novel peaks and chemical shifts were attributed to the presence of small aroma compounds, thereby confirming the formation of corn starch inclusion complexes. CONCLUSION: Small nonpolar and polar aroma compounds can be complexed to corn starch. © 2016 Society of Chemical Industry.

Effects of gap junction inhibition on contraction waves in the murine small intestine in relation to coupled oscillator theory.

Waves of contraction in the small intestine correlate with slow waves generated by the myenteric network of interstitial cells of Cajal. Coupled oscillator theory has been used to explain steplike gradients in the frequency (frequency plateaux) of contraction waves along the length of the small intestine. Inhibition of gap junction coupling between oscillators should lead to predictable effects on these plateaux and the wave dislocation (wave drop) phenomena associated with their boundaries. It is these predictions that we wished to test. We used a novel multicamera diameter-mapping system to measure contraction along 25- to 30-cm lengths of murine small intestine. There were typically two to three plateaux per length of intestine. Dislocations could be limited to the wavefronts immediately about the terminated wave, giving the appearance of a three-pronged fork, i.e., a fork dislocation; additionally, localized decreases in velocity developed across a number of wavefronts, ending with the terminated wave, which could appear as a fork, i.e., slip dislocations. The gap junction inhibitor carbenoxolone increased the number of plateaux and dislocations and decreased contraction wave velocity. In some cases, the usual frequency gradient was reversed, with a plateau at a higher frequency than its proximal neighbor; thus fork dislocations were inverted, and the direction of propagation was reversed. Heptanol had no effect on the frequency or velocity of contractions but did reduce their amplitude. To understand intestinal motor patterns, the pacemaker network of the interstitial cells of Cajal is best evaluated as a system of coupled oscillators.

Hydrogen-bonding-driven enantioselective resolution against the Kazlauskas rule to afford γ-amino alcohols by Candida rugosa lipase.

Most lipases resolve secondary alcohols in accordance with the "Kazlauskas rule" to give the R enantiomers. In a similar manner to other lipases, Candida rugosa lipase (CRL) exhibits R enantioselectivity towards heptan-2-ol, although the enantiomeric ratio (E) is low (E=1.6). However, unexpected enantioselectivity (i.e., S enantioselectivity, E=58) of CRL towards 4-(tert-butoxycarbonylamino)butan-2-ol, which has a similar chain length to heptan-2-ol, has been observed. To develop a deeper understanding of the molecular basis for this unusual enantioselectivity, we have conducted a series of molecular modeling and substrate engineering experiments. The results of these computational and experimental analyses indicated that a hydrogen bond between the Ser450 residue and the nitrogen atom of the carbamate group is critical to stabilize the transition state of the S enantiomer.

Three amino acid residues of an odorant-binding protein are involved in binding odours in Loxostege sticticalis L.

Odorant-binding proteins (OBPs) play an important role in insect olfactory processes and are thought to be responsible for the transport of pheromones and other semiochemicals across the sensillum lymph to the olfactory receptors within the antennal sensilla. As an important general odorant binding protein in the process of olfactory recognition, LstiGOBP1 of Loxostege sticticalis L. has been shown to have good affinity to various plant volatiles. However, the binding specificity of LstiGOBP1 should be further explored in order to better understand the olfactory recognition mechanism of L. sticticalis. In this study, real-time PCR experiments indicated that LstiGOBP1 was expressed primarily in adult antennae. Homology modelling and molecular docking were then conducted on the interactions between LstiGOBP1 and 1-heptanol to understand the interactions between LstiGOBP1 and their ligands. Hydrogen bonds formed by amino acid residues might be crucial for the ligand-binding specificity on molecular docking, a hypothesis that was tested by site-directed mutagenesis. As predicted binding sites for LstiGOBP1, Thr15, Trp43 and Val14 were replaced by alanine to determine the changes in binding affinity. Finally, fluorescence assays revealed that the mutants Thr15 and Trp43 had significantly decreased binding affinity to most odours; in mutants that had two-site mutations, the binding to the six odours that were tested was completely abolished. This result indicates that Thr15 and Trp43 were involved in binding these compounds, possibly by forming multiple hydrogen bonds with the functional groups of the ligands. These results provide new insights into the detailed chemistry of odours' interactions with proteins.