Leucokinin and Associated Neuropeptides Regulate Multiple Aspects of Physiology and Behavior in Drosophila

Leucokinins (LKs) constitute a family of neuropeptides identified in numerous insects and many other invertebrates. LKs act on G-protein-coupled receptors that display only distant relations to other known receptors. In adult Drosophila, 26 neurons/neurosecretory cells of three main types express LK. The four brain interneurons are of two types, and these are implicated in several important functions in the fly’s behavior and physiology, including feeding, sleep–metabolism interactions, state-dependent memory formation, as well as modulation of gustatory sensitivity and nociception. The 22 neurosecretory cells (abdominal LK neurons, ABLKs) of the abdominal neuromeres co-express LK and a diuretic hormone (DH44), and together, these regulate water and ion homeostasis and associated stress as well as food intake. In Drosophila larvae, LK neurons modulate locomotion, escape responses and aspects of ecdysis behavior. A set of lateral neurosecretory cells, ALKs (anterior LK neurons), in the brain express LK in larvae, but inconsistently so in adults. These ALKs co-express three other neuropeptides and regulate water and ion homeostasis, feeding, and drinking, but the specific role of LK is not yet known. This review summarizes Drosophila data on embryonic lineages of LK neurons, functional roles of individual LK neuron types, interactions with other peptidergic systems, and orchestrating functions of LK.     

Structural Aspects and Prediction of Calmodulin-Binding Proteins

Calmodulin (CaM) is an important intracellular protein that binds Ca²⁺ and functions as a critical second messenger involved in numerous biological activities through extensive interactions with proteins and peptides. CaM’s ability to adapt to binding targets with different structures is related to the flexible central helix separating the N- and C-terminal lobes, which allows for conformational changes between extended and collapsed forms of the protein. CaM-binding targets are most often identified using prediction algorithms that utilize sequence and structural data to predict regions of peptides and proteins that can interact with CaM. In this review, we provide an overview of different CaM-binding proteins, the motifs through which they interact with CaM, and shared properties that make them good binding partners for CaM. Additionally, we discuss the historical and current methods for predicting CaM binding, and the similarities and differences between these methods and their relative success at prediction. As new CaM-binding proteins are identified and classified, we will gain a broader understanding of the biological processes regulated through changes in Ca²⁺ concentration through interactions with CaM.     

Contribution of Invariant Natural Killer T Cells to the Clearance of Pseudomonas aeruginosa from Skin Wounds

Chronic infections are considered one of the most severe problems in skin wounds, and bacteria are present in over 90% of chronic wounds. Pseudomonas aeruginosa is frequently isolated from chronic wounds and is thought to be a cause of delayed wound healing. Invariant natural killer T (iNKT) cells, unique lymphocytes with a potent regulatory ability in various inflammatory responses, accelerate the wound healing process. In the present study, we investigated the contribution of iNKT cells in the host defense against P. aeruginosa inoculation at the wound sites. We analyzed the re-epithelialization, bacterial load, accumulation of leukocytes, and production of cytokines and antimicrobial peptides. In iNKT cell–deficient (Jα18KO) mice, re-epithelialization was significantly decreased, and the number of live colonies was significantly increased, when compared with those in wild-type (WT) mice on day 7. IL-17A, and IL-22 production was significantly lower in Jα18KO mice than in WT mice on day 5. Furthermore, the administration of α-galactosylceramide (α-GalCer), a specific activator of iNKT cells, led to enhanced host protection, as shown by reduced bacterial load, and to increased production of IL-22, IL-23, and S100A9 compared that of with WT mice. These results suggest that iNKT cells promote P. aeruginosa clearance during skin wound healing.     

In Vivo Electrophysiology of Peptidergic Neurons in Deep Layers of the Lumbar Spinal Cord after Optogenetic Stimulation of Hypothalamic Paraventricular Oxytocin Neurons in Rats

The spinal ejaculation generator (SEG) is located in the central gray (lamina X) of the rat lumbar spinal cord and plays a pivotal role in the ejaculatory reflex. We recently reported that SEG neurons express the oxytocin receptor and are activated by oxytocin projections from the paraventricular nucleus of hypothalamus (PVH). However, it is unknown whether the SEG responds to oxytocin in vivo. In this study, we analyzed the characteristics of the brain–spinal cord neural circuit that controls male sexual function using a newly developed in vivo electrophysiological technique. Optogenetic stimulation of the PVH of rats expressing channel rhodopsin under the oxytocin receptor promoter increased the spontaneous firing of most lamina X SEG neurons. This is the first demonstration of the in vivo electrical response from the deeper (lamina X) neurons in the spinal cord. Furthermore, we succeeded in the in vivo whole-cell recordings of lamina X neurons. In vivo whole-cell recordings may reveal the features of lamina X SEG neurons, including differences in neurotransmitters and response to stimulation. Taken together, these results suggest that in vivo electrophysiological stimulation can elucidate the neurophysiological response of a variety of spinal neurons during male sexual behavior.     

Characteristic tryptic peptides and gelling properties of porcine skin gelatin affected by thermal action

Thermal action in extraction process had effects on characteristic tryptic peptides identification and gelling properties of porcine gelatin. SDS-PAGE, HPLC-LTQ/Orbitrap high-resolution mass spectrometry, texture analyser and rheometer were used to evaluate collagen depolymerisation degree, characteristic tryptic peptides and gelling properties of gelatins prepared in various thermal actions. Results showed that with increasing temperature and time, depolymerisation degree enlarged, while gel strength, gelling and melting temperature decreased. Mass spectra showed that 47 and 49 common characteristic tryptic peptides were identified in gelatins extracted at 50 °C and 100 °C with various times, respectively. Moreover, 34 common characteristic tryptic peptides were identified in all gelatin samples. Further comparison between this work and our previous investigations yielded 20 common characteristic tryptic peptides, which stably exist in various thermal actions. These common characteristic tryptic peptides may be very helpful for the accurate authentication of porcine gelatin.     

Predictive formulas for different measures of cheese yield using milk composition from individual goat samples

The objective of this study was to develop formulas based on milk composition of individual goat samples for predicting cheese yield (%CY) traits (fresh curd, milk solids, and water retained in the curd). The specific aims were to assess and quantify (1) the contribution of major milk components (fat, protein, and casein) and udder health indicators (lactose, somatic cell count, pH, and bacterial count) on %CY traits (fresh curd, milk solids, and water retained in the curd); (2) the cheese-making method; and (3) goat breed effects on prediction accuracy of the %CY formulas. The %CY traits were analyzed in duplicate from 600 goats, using an individual laboratory cheese-making procedure (9-MilCA method; 9 mL of milk per observation) for a total of 1,200 observations. Goats were reared in 36 herds and belonged to 6 breeds (Saanen, Murciano-Granadina, Camosciata delle Alpi, Maltese, Sarda, and Sarda Primitiva). Fresh %CY (%CY_CURD), total solids (%CY_SOLIDS), and water retained (%CY_WATER) in the curd were used as response variables. Single and multiple linear regression models were tested via different combinations of standard milk components (fat, protein, casein) and indirect udder health indicators (UHI; lactose, somatic cell count, pH, and bacterial count). The 2 %CY observations within animal were averaged, and a cross-validation (CrV) scheme was adopted, in which 80% of observations were randomly assigned to the calibration (CAL) set and 20% to the validation (VAL) set. The procedure was repeated 10 times to account for sampling variability. Further, the model presenting the best prediction accuracy in CrV (i.e., comprehensive formula) was used in a secondary analysis to assess the accuracy of the %CY predictive formulas as part of the laboratory cheese-making procedure (within-animal validation, WAV), in which the first %CY observation within animal was assigned to CAL, and the second to the VAL set. Finally, a stratified CrV (SCrV) was adopted to assess the %CY traits prediction accuracy across goat breeds, again using the best model, in which 5 breeds were included in CAL and the remaining one in the VAL set. Fitting statistics of the formulas were assessed by coefficient of determination of validation (R²_VAL) and the root mean square error of validation (RMSE_VAL). In CrV, the formula with the best prediction accuracy for all %CY traits included fat, casein, and UHI (R²_VAL = 0.65, 0.96, and 0.23 for %CY_CURD, %CY_SOLIDS, and %CY_WATER, respectively). The WAV procedure showed R²_VAL higher than those obtained in CrV, evidencing a low effect of the 9-MilCA method and, indirectly, its high repeatability. In the SCrV, large differences for %CY_CURD and %CY_WATER among breeds evidenced that the breed is a fundamental factor to consider in %CY predictive formulas. These results may be useful to monitor milk composition and quantify the influence of milk traits in the composite selection indices of specific breeds, and for the direct genetic improvement of cheese production.     

微波辅助孵育法富集麦胚多肽的工艺优化

目的 麦胚孵育过程中蛋白酶将蛋白质水解成肽和氨基酸，多肽具有明确的生理活性和营养调节作用。本研究以麦胚为试验原料，采用微波辅助预处理的方法，研究孵育的温度、时间、pH及料液比4种因素对孵育后的蛋白酶活力及多肽含量的影响。 方法 通过单因素和响应曲面试验进行工艺条件优化。 结果 与未进行微波辅助预处理的样品相比，微波辅助处理（600 W, 10 s）能显著提高孵育后样品中的蛋白酶酶活力（约9.4倍）和肽含量（约3.1倍）。经过微波辅助处理后，孵育温度为51.5℃、pH为4.0、时间为6.33 h、料液比为1:7时，蛋白酶活力达最高为3826.24 U/g；孵育温度为45.0 ℃、pH为4.8、时间为8 h、料液比为1:7时，肽含量达最高为262.63 mg/g。 结论 微波辅助处理能有效的激活麦胚孵育液中的内源性蛋白酶活性，促进蛋白水解反应，显著提高孵育后的肽含量。该研究结果为麦胚多肽的制备新工艺开发提供了研究基础。     

Cheese ripening in nonconventional conditions: A multiparameter study applied to Protected Geographical Indication Canestrato di Moliterno cheese

A multiparameter study was performed to evaluate the effect of fondaco, a traditional ripening cellar without any artificial temperature and relative humidity control, on the chemical, microbiological, and sensory characteristics of Protected Geographical Indication Canestrato di Moliterno cheese. Ripening in such a nonconventional environment was associated with lower counts of lactococci, lactobacilli, and total viable bacteria, and higher presence of enterococci, in comparison with ripening in a controlled maturation room. Moreover, fondaco cheese underwent accelerated maturation, as demonstrated by faster casein degradation, greater accumulation of free AA, and higher formation of volatile organic compounds. Secondary proteolysis, as assessed by liquid chromatography-mass spectrometry of free AA and low molecular weight peptides, did not show any qualitative difference among cheeses, but fondaco samples evidenced an advanced level of peptidolysis. On the other hand, significant qualitative differences were observed in the free fatty acid profiles and in the sensory characteristics. Principal component analysis showed a clear separation of the fondaco and control cheeses, indicating that ripening in the natural room conferred unique sensory features to the product.     

Facile synthesis of a carbon-rich SiAlCN precursor and investigation of its structural evolution during the polymer-ceramic conversion process

A liquid carbon-rich SiAlCN precursor is facilely synthetized by hydrosilylation between liquid polyaluminocarbosilane (LPACS) and 1,3,5,7-tetravinyl- 1,3,5,7-tetramethylcyclotetrasilazane {[CH₃(CH₂CH₂)SiNH]₄} (TeVSZ). The structural evolution during the polymer-ceramic conversion process is investigated by various methods. The results show that the main cured mechanism is β-addition on hydrosilylation, although α-addition on hydrosilylation, polymerization of vinyl groups and dehydrocoupling reaction between N–H bonds also occur during the cured process. During the pyrolysis process, dehydrogenation and dehydrocarbonation condensation reactions, transamination reactions occur, leading to formation of a three-dimensional network inorganic structure at 400–800 °C, where part of Al–O bonds convert to Al–N bonds. Then the network inorganic structure undergoes demixing and separation into amorphous SiAlCN(O) phase, where the amorphous turbostratic free carbon phase also form at 800–1200 °C. With demixing and decomposition of the amorphous carbon-rich SiAlCN(O) phase, the crystalline β-SiC and graphitic carbon start to form at about 1400 °C, the crystalline sizes of them both enlarge with increasing temperature. However, the crystal growth of β-SiC is distinctly inhibited due to existence of the rich carbon phase, tiny amounts of Al₂O₃ and AlN. In addition, a small amount of AlN can promote the formation of α-SiC at 1800 °C.     

Influence of applied voltage and conductive material in DIET promotion for methane generation

The utilization of biological-, electrode- and conductive material-mediated direct interspecies electron transfer (DIET) between exoelectrogenic bacteria and methanogenic archaea for enhancing methane productivity is widely reported in the literature. However, two cardinal questions are still controversial, i.e., which applied voltage value would be more recommended to enhance methane generation? and how the DIET over IIET has the upper hand in enhancing methane productivity? Herein, the influence of different applied voltages to promote biological-, conductive- and electrode-mediated DIET was investigated in MEC-AD reactors with conductive material. Polarized bioelectrodes induced electrode-mediated DIET (eDIET) and biological DIET (bDIET), in addition to cDIET (conductive material-mediated DIET), improved the methane yield to 315.40 mL/g COD_r with an applied voltage of 0.9 V. Whereas further increase of applied voltage 1.2 V, lessened methane production efficiency due to high-voltage inhibition and adverse effect on DIET promotion. The anaerobic digestion coupled microbial electrolysis cells with optimal electric potential selectively promotes the DIET through polarized electrodes were confirmed through microbial analysis. As the contribution of DIET increased to 80%, the methane yield increased, and the substrate residue decreased, resulting in a significant improvement in methane production.