A novel antimicrobial peptide Scyreptin1-30 from Scylla paramamosain exhibiting potential therapy of Pseudomonas aeruginosa early infection in a mouse burn wound model.

Antimicrobial resistance (AMR) constitutes a significant global threat to human health. In recent years, there has been a concerning surge in infections caused by multidrug-resistant bacteria, highlighting the pressing need to urgently explore novel and effective alternatives to conventional antibiotics. Antimicrobial peptides (AMPs) have emerged as a focal point of research, capturing significant attention as promising antimicrobial agents. In this study, we have identified a novel cationic antimicrobial peptide (AMP) named Scyreptin1-30, derived from the marine invertebrate Scylla paramamosain. The results showed that Scyreptin1-30 exhibits a broad-spectrum antimicrobial activity, demonstrating significant potency against both bacteria and fungi, and even against the clinically isolated multidrug-resistant bacteria Pseudomonas aeruginosa. Moreover, Scyreptin1-30 exhibited rapid bactericidal kinetic. The results of antibacterial mechanism showed that Scyreptin1-30 destroyed the integrity of bacterial membranes, leading to bacterial death and exhibited potent anti-biofilm activity against P. aeruginosa. The activity of Scyreptin1-30 against bacteria had a favorable thermal stability, displayed a certain ion tolerance, and showed no discernible cytotoxicity when assessed against both the mammalian cell line HEK293T and the fish cell lines ZF4. In an In vivo study, Scyreptin1-30 exhibited a remarkably reduction in the bacterial load caused by multidrug-resistant P. aeruginosa at the site of infection, and promoted wound healing in a mouse model of burn infection. This study indicated that Scyreptin1-30 holds promise as an effective antibacterial agent, potentially serving as a topical skin treatment against multidrug-resistant bacterial infections, including those caused by P. aeruginosa.

Evaluation of the Hair Cell Regeneration in Zebrafish Larvae by Measuring and Quantifying the Startle Responses.

The zebrafish has become an established model organism for the study of hearing and balance systems in the past two decades. The classical approach to examine hair cells is to use dye to conduct selective staining, which shows the number and morphology of hair cells but does not reveal their function. Startle response is a behavior closely related to the auditory function of hair cells; therefore it can be used to measure the function of hair cells. In this study, we developed a device to measure the startle response of zebrafish larvae. By applying various levels of stimulus, it showed that the system can discern a 10 dB difference. The hair cell in zebrafish can regenerate after damage due to noise exposure or drug treatment. With this device, we measured the startle response of zebrafish larvae during and after drug treatment. The results show a similar trend to the classical hair cell staining method. The startle response was reduced with drug treatment and recovered after removal of the drug. Together it demonstrated the capability of this behavioral assay in evaluating the hair cell functions of fish larvae and its potential as a high-throughput screening tool for auditory-related gene and drug discovery.

Unilateral Laryngeal Pacing System and Its Functional Evaluation.

Goal. To establish a reliable instrumental system for synchronized reactivation of a unilaterally paralyzed vocal fold and evaluate its functional feasibility. Methods. Unilateral vocal fold paralysis model was induced by destruction of the left recurrent laryngeal nerve (RLN) in anesthetized dogs. With a micro controller-based electronic system, electromyography (EMG) signals from cricothyroid (CT) muscle on the ipsilateral side were recorded and used to trigger pacing of paralyzed vocalis muscles. The dynamic movement of vocal folds was continuously monitored using an endoscope, and the opening and closing of the glottis were quantified with customized imaging processing software. Results. The recorded video images showed that left side vocal fold was obviously paralyzed after destructing the RLN. Using the pacing system with feedback triggering EMG signals from the ipsilateral CT muscle, the paralyzed vocal fold was successfully reactivated, and its movement was shown to be synchronized with the healthy side. Significance. The developed unilateral laryngeal pacing system triggered by EMG from the ipsilateral side CT muscle could be successfully used in unilateral vocal fold paralysis with the advantage of avoiding disturbance to the healthy side muscles.

Filtering of acoustic signals within the hearing organ.

The detection of sound by the mammalian hearing organ involves a complex mechanical interplay among different cell types. The inner hair cells, which are the primary sensory receptors, are stimulated by the structural vibrations of the entire organ of Corti. The outer hair cells are thought to modulate these sound-evoked vibrations to enhance hearing sensitivity and frequency resolution, but it remains unclear whether other structures also contribute to frequency tuning. In the current study, sound-evoked vibrations were measured at the stereociliary side of inner and outer hair cells and their surrounding supporting cells, using optical coherence tomography interferometry in living anesthetized guinea pigs. Our measurements demonstrate the presence of multiple vibration modes as well as significant differences in frequency tuning and response phase among different cell types. In particular, the frequency tuning at the inner hair cells differs from other cell types, causing the locus of maximum inner hair cell activation to be shifted toward the apex of the cochlea compared with the outer hair cells. These observations show that additional processing and filtering of acoustic signals occur within the organ of Corti before inner hair cell excitation, representing a departure from established theories.

In vivo outer hair cell length changes expose the active process in the cochlea.

BACKGROUND: Mammalian hearing is refined by amplification of the sound-evoked vibration of the cochlear partition. This amplification is at least partly due to forces produced by protein motors residing in the cylindrical body of the outer hair cell. To transmit power to the cochlear partition, it is required that the outer hair cells dynamically change their length, in addition to generating force. These length changes, which have not previously been measured in vivo, must be correctly timed with the acoustic stimulus to produce amplification. METHODOLOGY/PRINCIPAL FINDINGS: Using in vivo optical coherence tomography, we demonstrate that outer hair cells in living guinea pigs have length changes with unexpected timing and magnitudes that depend on the stimulus level in the sensitive cochlea. CONCLUSIONS/SIGNIFICANCE: The level-dependent length change is a necessary condition for directly validating that power is expended by the active process presumed to underlie normal hearing.

Modulation and Interaction of Immune-Associated Parameters with Antioxidant in the Immunocytes of Crab Scylla paramamosain Challenged with Lipopolysaccharides.

Invertebrates are dependent on cellular and humoral immune defences against microbial infection. Scylla paramamosain is an important commercial species, but the fundamental knowledge on its immune defense related to the antioxidant and immune-associated reactions is still lacking. The study was to differentiate the responses of immune-associated parameters of haemolymph components in S. paramamosain when challenged with bacterial lipopolysaccharides (LPSs). The immunostimulating effects of LPS in crab by triggering various immune parameters (phagocytosis, lysozyme, antibacterial activity, phenoloxidase, and the generation of superoxide and nitric oxide) were investigated. Results showed that the generation of free radicals, phenoloxidase, lysozyme and antibacterial activities was significantly increased through the exposure periods. Conversely, total hemocyte count and lysosomal membrane stability decreased significantly as the exposure period extended to 96 h. The relationship between the antioxidant enzymes and immune reactions due to LPS was highly significant. In addition, ROS production was positively correlated with antioxidant showing immediate response of antioxidant defense to the oxyradicals generated. Overall, the study indicated that nonspecific immune components in hemocytes of crab showed active response to the LPS stimulation, and their responses suggested that many immune-associated parameters could be modulated and interrelated with the influence of antioxidants in crustaceans.

A differentially amplified motion in the ear for near-threshold sound detection.

The ear is a remarkably sensitive pressure fluctuation detector. In guinea pigs, behavioral measurements indicate a minimum detectable sound pressure of ∼20 µPa at 16 kHz. Such faint sounds produce 0.1-nm basilar membrane displacements, a distance smaller than conformational transitions in ion channels. It seems that noise within the auditory system would swamp such tiny motions, making weak sounds imperceptible. Here we propose a new mechanism contributing to a resolution of this problem and validate it through direct measurement. We hypothesized that vibration at the apical side of hair cells is enhanced compared with that at the commonly measured basilar membrane side. Using in vivo optical coherence tomography, we demonstrated that apical-side vibrations peaked at a higher frequency, had different timing and were enhanced compared with those at the basilar membrane. These effects depend nonlinearly on the stimulus sound pressure level. The timing difference and enhancement of vibrations are important for explaining how the noise problem is circumvented.

Antioxidant enzymes from the crab Scylla paramamosain: gene cloning and gene/protein expression profiles against LPS challenge.

Recent studies revealed that antioxidant enzymes play important roles in antioxidant responses caused by metabolic process or pathogen invasion. Catalase is one of these key enzymes which has been characterized and highly conserved from invertebrates to vertebrates. In the present study, a full-length cDNA sequence of catalase was cloned from the hemocyte suppression subtractive hybridization library of the crab Scylla paramamosain. The Sp-catalase (Sp-CAT) cDNA sequence contained 2551bp with an open reading frame of 1551bp encoding 517 amino acid residues. The conserved catalytic active residues His-71, Asn-144 and Tyr-354 were predicted in the amino acid sequence of Sp-CAT. The deduced Sp-CAT protein had a calculated molecular mass of 59 kDa with an estimated isoelectric point of 6.4. Multiple alignment analysis revealed that the deduced amino acid sequence of Sp-CAT shared high identity (75.4%) with those of other species. The Sp-CAT mRNA transcripts were demonstrated in multiple tissues of normal S. paramamosain. After LPS challenge, the expression level of Sp-CAT gene was increased significantly in hemocyte at 3 and 6 h, and in hepatopancreas at 6 h, respectively, determined by quantitative real-time PCR. Furthermore, the activities of CAT and SOD were also measured in different tissues and serum after LPS challenge. The CAT activity was significantly increased at 3, 6, 24 and 48 h in hemocyte lysate, at 3 h in serum, and at 24 and 48 h in hepatopancreas after LPS challenge. In addition, the SOD activity was significantly induced at 3 and 6 h in hemocyte lysate, 3 and 12 h in serum, 12 and 48 h in hepatopancreas post LPS stimulation, indicating a tissue and time-dependent antioxidant response in the crab. Taken together, these data demonstrated that a strong antioxidant response occurred in the LPS-challenged crab, which might be involved in the protection of host against microbial infections.

Identification of genes differentially expressed in hemocytes of Scylla paramamosain in response to lipopolysaccharide.

Although the crab Scylla paramamosain has been cultured in China for a long time, little knowledge is available on how crabs respond to infection by bacteria. A forward suppression subtractive hybridization (SSH) cDNA library was constructed from their hemocytes and the up-regulated genes were identified in order to isolate differentially expressed genes in S. paramamosain in response to bacterial lipopolysaccharide (LPS). A total of 721 clones on the middle scale in the SSH library were sequenced. Among these genes, 271 potentially functional genes were recognized based on the BLAST searches in NCBI and were categorized into seven groups in association with different biological processes using AmiGO against the Gene Ontology database. Of the 271 genes, 269 translatable DNA sequences were predicted to be proteins, and the putative amino acid sequences were searched for conserved domains and proteins using the CD-Search service and BLASTp. Among 271 genes, 179 (66.1%) were annotated to be involved in different biological processes, while 92 genes (33.9%) were classified as an unknown-function gene group. It was noted that only 18 of the 271 genes (6.6%) had previously been reported in other crustaceans and most of the screened genes showed less similarity to known sequences based on BLASTn results, suggesting that 253 genes were found for the first time in S. paramamosain. Furthermore, two up-regulated genes screened from the SSH library were selected for full-length cDNA sequence cloning and in vivo expression study, including Sp-superoxide dismutase (Sp-Cu-ZnSOD) gene and Sp-serpin gene. The differential expression pattern of the two genes during the time course of LPS challenge was analyzed using real-time PCR. We found that both genes were significantly expressed in LPS-challenged crabs in comparison with control. Taken together, the study primarily provides the data of the up-regulated genes associated with different biological processes in S. paramamosain in response to LPS, by which the interesting genes or proteins potentially involved in the innate immune defense of S. paramamosain will be investigated in future.

In vivo imaging and low-coherence interferometry of organ of Corti vibration.

An optical coherence tomography (OCT) system is built to acquire in vivo both images and vibration measurements of the organ of Corti of the guinea pig. The organ of Corti is viewed through a approximately 300-microm-diam hole in the bony wall of the cochlea at the scala tympani of the first cochlear turn. In imaging mode, the image is acquired as reflectance R(x,z). In vibration mode, the basilar membrane (BM) or reticular lamina (RL) are selected by the investigator interactively from the R(x,z) image. Under software control, the system moves the scanning mirrors to bring the sensing volume of the measurement to the desired membrane location. In vivo images of the organ of Corti are presented, indicating reflectance signals from the BM, RL, tectorial membrane, and Reissner's membrane. The tunnel of Corti and the inner sulcus are also visible in the images. Vibrations of +/-2 and +/-22 nm are recorded in the BM in response to low and high sound levels at 14 kHz above a noise floor of 0.2 nm.