Iron regulatory protein from the hard tick Haemaphysalis longicornis: characterization, function and assessment as a protective antigen.

BACKGROUND: Ticks are blood-feeding ectoparasites with different host specificities and are capable of pathogen transmission. Iron regulatory proteins (IRPs) play crucial roles in iron homeostasis in vertebrates. However, their functions in ticks remain poorly understood. The aim of the present study was to investigate the characteristics, functions, molecular mechanisms, and the vaccine efficacy of IRP in the hard tick Haemaphysalis longicornis. RESULTS: The full-length complementary DNA of IRP from Haemaphysalis longicornis (HlIRP) was 2973 bp, including a 2772 bp open reading frame. It is expressed throughout three developmental stages (larvae, nymphs, and adult females) and in various tissues (salivary glands, ovaries, midgut, and Malpighian tubules). Recombinant Haemaphysalis longicornis IRP (rHlIRP) was obtained via a prokaryotic expression system and exhibited aconitase, iron chelation, radical-scavenging, and hemolytic activities in vitro. RNA interference-mediated IRP knockdown reduced tick engorgement weight, ovary weight, egg mass weight, egg hatching rate, and ovary vitellin content, as well as prolonging the egg incubation period. Proteomics revealed that IRP may affect tick reproduction and development through proteasome pathway-associated, ribosomal, reproduction-related, and iron metabolism-related proteins. A trial on rabbits against adult Haemaphysalis longicornis infestation demonstrated that rHlIRP vaccine could significantly decrease engorged weight (by 10%), egg mass weight (by 16%) and eggs hatching rate (by 22%) of ticks. The overall immunization efficacy using rHlIRP against adult females was 41%. CONCLUSION: IRP could limit reproduction and development in Haemaphysalis longicornis, and HlIRP was confirmed as a candidate vaccine antigen to impair tick iron metabolism and protect the host against tick infestation. © 2024 Society of Chemical Industry.

A generalized AI method for pathology cancer diagnosis and prognosis prediction based on transfer learning and hierarchical split.

Objective.This study aims to propose a generalized AI method for pathology cancer diagnosis and prognosis prediction based on transfer learning and hierarchical split.Approach.We present a neural network framework for cancer diagnosis and prognosis prediction in pathological images. To enhance the network's depth and width, we employ a hierarchical split block (HS-Block) to create an AI-aided diagnosis system suitable for semi-supervised clinical settings with limited labeled samples and cross-domain tasks. By incorporating a lightweight convolution unit based on the HS-Block, we improve the feature information extraction capabilities of a regular network (RegNet). Additionally, we integrate a Convolutional Block Attention Module into the first and last convolutions to optimize the extraction of global features and local details. To address limited sample labels, we employ a dual-transfer learning (DTL) mechanism named DTL-HS-Regnet, enabling semi-supervised learning in clinical settings.Main results.Our proposed DTL-HS-Regnet model outperforms other advanced deep-learning models in three different types of cancer diagnosis tasks. It demonstrates superior feature extraction ability, achieving an average sensitivity, specificity, accuracy, and F1 score of 0.9987, 1.0000, 1.0000 and 0.9992, respectively. Furthermore, we evaluate the model's capability to directly extract prognosis prediction information from pathological images by constructing patient cohorts. The results show that the correlation between DTL-HS-Regnet predictions and the presence of cancer-associated fibroblasts is comparable to that of pathologists.Significance.Our proposed AI method offers a generalized approach for cancer diagnosis and prognosis prediction in pathology. The outstanding performance of the DTL-HS-Regnet model demonstrates its potential for improving current practices in image digital pathology, expanding the boundaries of cancer treatment in two critical areas.

Drug-Embedded Nanovesicles Assembled from Peptide-Decorated Hyaluronic Acid for Rheumatoid Arthritis Synergistic Therapy.

Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease that causes endless pain and poor quality of life in patients. Usage of a lubricant combined with anti-inflammatory therapy is considered a reasonable and effective approach for the treatment of RA. Herein, inspired by glycopeptides, a peptide-decorated hyaluronic acid was synthesized, and the grafted Fmoc-phenylalanine-phenylalanine-COOH (FmocFF) peptide self-assembled with ß-sheet conformations could induce the folding of polymer molecular chains to form a vesicle structure in aqueous solution. The hydrophobic anti-inflammatory drug curcumin (Cur) could be embedded in the vesicle walls through π-π interactions with the FmocFF peptide. Furthermore, the inflammation suppression function of the Cur-loaded vesicles both in vitro and in vivo was demonstrated to be an effective treatment for RA therapy. This work proposes new insights into the folding and hierarchical assembly of glycopeptide mimics, providing an efficient approach for constructing intelligent platforms for drug delivery, disease therapy, and diagnostic applications.

Glycoside combinations of Buyang Huanwu decoction ameliorate atherosclerosis via STAT3, HIF-1, and VEGF.

Buyang Huanwu decoction (BYHWD) is a classical traditional prescription. Glycosides are effective extracts of BYHWD, which have been proven to protect blood vessels and prevent atherosclerosis (AS). However, the mechanism of glycosides in inhibiting abnormal angiogenesis in atherosclerosis is still unclear. The specific amygdalin (AG), paeoniflorin (PF), and astragaloside IV (ASV) contents in the BYHWD-containing serum were detected using mass spectrometry. Network pharmacology and molecular docking are used to screen the targets of glycosides for treating atherosclerosis. The predicted targets were validated in an AS model of rat thoracic aortic endothelial cells (RTAEC) induced by oxidized low-density lipoprotein (ox-LDL). According to the mass spectrometry data, the specific contents of AG, PF, and ASV in the serum were 24.11 ng/ml, 20.94 ng/ml, and 69.87 ng/ml, respectively. Results of bioinformatics analysis show that signal transducer and activator of transcription (STAT)-3, hypoxia-inducible factor (HIF)-1, and vascular endothelial-derived growth factor (VEGF) may be involved in the treatment of AS with glycosides. The results of cell experiments revealed that glycoside combinations could treat atherosclerosis by inhibiting STAT3, HIF-1, and VEGF. AG, PF, and ASV are the effective ingredients of BYHWD. Glycoside combinations significantly ameliorate atherosclerosis by inhibiting STAT3, HIF-1, and VEGF.

Protein regulation mechanism of cold tolerance in Haemaphysalis longicornis.

Ticks are external parasitic arthropods that can transmit a variety of pathogens by sucking blood. Low-temperature tolerance is essential for ticks to survive during the cold winter. Exploring the protein regulation mechanism of low-temperature tolerance of Haemaphysalis longicornis could help to explain how ticks survive in winter. In this study, the quantitative proteomics of several tissues of H. longicornis exposed to low temperature were studied by data independent acquisition technology. Totals of 3 699, 3 422, and 1 958 proteins were identified in the salivary gland, midgut, and ovary, respectively. The proteins involved in energy metabolism, cell signal transduction, protein synthesis and repair, and cytoskeleton synthesis changed under low-temperature stress. The comprehensive analysis of the protein regulation of multiple tissues of female ticks exposed to low temperature showed that maintaining cell homeostasis, maintaining cell viability, and enhancing cell tolerance were the most important means for ticks to maintain vital signs under low temperature. The expression of proteins involved in and regulating the above cell activities was the key to the survival of ticks under low temperatures. Through the analysis of a large amount of data, we found that the expression levels of arylamine N-acetyltransferase, inositol polyphosphate multikinase, and dual-specificity phosphatase were up-regulated under low temperature. We speculated that they might have important significance in low-temperature tolerance. Then, we performed RNA interference on the mRNA of these 3 proteins, and the results showed that the ability of female ticks to tolerate low temperatures decreased significantly.

Proteomic changes in various organs of Haemaphysalis longicornis under long-term starvation.

Haemaphysalis longicornis (Neumann), a tick of public health and veterinary importance, spend the major part of their life cycle off-host, especially the adult host-seeking period. Thus, they have to contend with prolonged starvation. Here, we investigated the underlying molecular mechanism of tick starvation endurance in the salivary glands, midguts, ovaries, and Malpighian tubules of starved H. longicornis ticks using the data-independent acquisition quantitative proteomic approach to study the proteome changes. Essential synthases such as glutamate synthase, citrate synthase, and ATP synthase were up-regulated probably due to increased proteolysis and amino acid catabolism during starvation. The up-regulation of succinate dehydrogenase, ATP synthase, cytochrome c oxidase, and ADP/ATP translocase closely fits with an increased oxidative phosphorylation function during starvation. The differential expression of superoxide dismutase, glutathione reductase, glutathione S-transferase, thioredoxin, and peroxiredoxin indicated fasting-induced oxidative stress. The up-regulation of heat shock proteins could imply the activation of a protective mechanism that checks excessive protein breakdown during starvation stress. The results of this study could provide useful information about the vulnerabilities of ticks that could aid in tick control efforts.

Proteomic analysis suggests that monoterpenes in lemongrass disrupt Ca2+ homeostasis in Haemaphysalis longicornis leading to mitochondrial depolarization and cytotoxicity.

Complex mixtures of bioactive ingredients in plant essential oils present complex chemistries which involve different modes of action. An increasing body of scientific reports has recently focused on the acaricidal activities of plant essential oils attributed to their monoterpene components, but information about their underlying molecular mechanism of action is scarce. Here, after the chemical analysis of lemongrass oil, a proteomic analysis of the ovary, salivary gland, and midgut of Haemaphysalis longicornis exposed to Cymbopogon citratus (lemongrass) essential oil was performed via data-independent acquisition mass spectrometry (DIA-MS) technology to further elucidate the molecular mechanisms involved. Pathway analysis reveals the activation of metabolic pathways mediated by oxidoreductases and transferases. Furthermore, the upregulation of various calcium-associated proteins and the upregulation of cytochrome c1, cytochrome c oxidase polypeptide IV, and programmed cell death protein 6-like isoform X1 suggest a cytotoxic mode of action via the formation of reactive oxygen species (ROS), mitochondrial Ca2+ overload, mitochondrial uncoupling, and depolarization, and ATP depletion leading to either apoptotic or necrotic death. Morphological alterations observed after the RNAi of a major detoxification enzyme (glutathione S-transferase) merit further investigation. Hence, the cytotoxic mode of action exhibited by C. citratus oil could be vital for the development of eco-friendly acaricide.

Transcriptome profile of Haemaphysalis longicornis (Acari: Ixodidae) exposed to Cymbopogon citratus essential oil and citronellal suggest a cytotoxic mode of action involving mitochondrial Ca2+ overload and depolarization.

Haemaphysalis longicornis is an ixodid tick species of medical and veterinary importance. Investigation into the acaricidal activities of botanicals have increased recently but information about their molecular mechanism of action is scarce. Here, RNA-seq analysis of the ticks exposed to Cymbopogon citratus essential oil and citronellal was performed and the responsive genes were identified. More than 6.39 G clean reads with Q20 ≥ 94.88% were obtained for each H. longicornis sample, with an average GC content of 50.94%. Using the Trinity method, 166,710 unigenes with a mean length of 869 bp and a maximum contig length of 29,156 bp were obtained. The upregulation of genes was concentration-dependent in most of the treated groups. Many genes responsive to C. citratus oil and citronellal were stress-related and they include genes associated with adrenergic signaling/calcium channels, cGMP-PKG signaling, apoptosis, focal adhesion, ECM-receptor interaction, ubiquitin-mediated proteolysis, mTOR signaling pathway, and longevity regulating pathway. The upregulation of genes (CACNAID, ADCY9, TPM1, and MYH6) associated with calcium channels suggests a neurotoxic mode of action, whereas, the upregulation of apoptosis-associated genes (CYC, DRONC, CASP7, CASP9, BCL2L1, bcl-xL, etc.) suggests a cytotoxic mode of action. The metabolism of C. citratus essential oil generates oxidative stress which increases the intra-mitochondrial free Ca2+ and triggers the formation of reactive oxygen species (ROS) that culminates to mitochondrial depolarization, ATP depletion, and either necrotic or apoptotic death. The neurotoxic and cytotoxic effects exhibited by the monoterpenes in H. longicornis is vital and could be exploited for the advancement of acaricide development and eco-friendly tick control.

Comprehensive dynamic analysis of proteins in the spermatheca of female Haemaphysalis longicornis after copulation.

Ticks are obligate blood-sucking parasitic arthropods. When sucking the blood of hosts, they can also transmit a variety of pathogens to hosts that severely endanger the health of humans and animals. The spermatheca is an organ for the storage and protection of sperm and an important component of the reproductive system of female ticks. The spermatheca content changes dramatically over time after copulation. In particular, some proteins and polypeptide substances can influence the physiological functions of female ticks and promote blood feeding and egg laying by female ticks. To investigate the molecular mechanisms underlying the productive process of Haemaphysalis longicornis, data-independent acquisition (DIA) quantitative proteomics technology was used to perform in-depth research of the dynamic changes in all proteins in the spermatheca of ticks within a short time after copulation to look for key proteins in the spermatheca contents after copulation that affect the reproduction of female ticks in order to provide meaningful information for the comprehensive prevention and control of ticks.

Protein regulation strategies of the mouse spleen in response to Babesia microti infection.

BACKGROUND: Babesia is a protozoan parasite that infects red blood cells in some vertebrates. Some species of Babesia can induce zoonoses and cause considerable harm. As the largest immune organ in mammals, the spleen plays an important role in defending against Babesia infection. When infected with Babesia, the spleen is seriously injured but still actively initiates immunomodulatory responses. METHODS: To explore the molecular mechanisms underlying the immune regulation and self-repair of the spleen in response to infection, this study used data-independent acquisition (DIA) quantitative proteomics to analyse changes in expression levels of global proteins and in phosphorylation modification in spleen tissue after Babesia microti infection in mice. RESULTS: After mice were infected with B. microti, their spleens were seriously damaged. Using bioinformatics methods to analyse dynamic changes in a large number of proteins, we found that the spleen still initiated immune responses to combat the infection, with immune-related proteins playing an important role, including cathepsin D (CTSD), interferon-induced protein 44 (IFI44), interleukin-2 enhancer-binding factor 2 (ILF2), interleukin enhancer-binding factor 3 (ILF3) and signal transducer and activator of transcription 5A (STAT5A). In addition, some proteins related to iron metabolism were also involved in the repair of the spleen after B. microti infection, including serotransferrin, lactoferrin, transferrin receptor protein 1 (TfR1) and glutamate-cysteine ligase (GCL). At the same time, the expression and phosphorylation of proteins related to the growth and development of the spleen also changed, including protein kinase C-δ (PKC-δ), mitogen-activated protein kinase (MAPK) 3/1, growth factor receptor-bound protein 2 (Grb2) and P21-activated kinase 2 (PAK2). CONCLUSIONS: Immune-related proteins, iron metabolism-related proteins and growth and development-related proteins play an important role in the regulation of spleen injury and maintenance of homeostasis. This study provides an important basis for the diagnosis and treatment of babesiosis.

Effects of the essential oil from Cymbopogon citratus on mortality and morphology of the tick Haemaphysalis longicornis (Acari: Ixodidae).

Haemaphysalis longicornis is one of the most prevalent tick species across eastern Asia, Australia, and New Zealand, and has been implicated as a vector of several pathogenic agents. This study evaluated the in vitro acaricidal efficacy of Cymbopogon citratus (lemongrass) essential oil on unfed H. longicornis using the adult and nymph immersion test, and the larval packet test. Six concentrations with three replications each of 10, 20, 30, 40, 50 and 60 mg/mL (adults and nymphs) were used, and 2.5, 5, 10, 20, 40 and 80 mg/mL (larvae), with control group (50% ethanol). The adult and nymph mortality rates were 98 and 100% at 50 mg/mL, and 95 and 100% at 60 mg/mL, respectively, whereas the larval mortality rate was 94 and 96% at 40 and 80 mg/mL, respectively. Mortality of adult, nymph and larva increased significantly in a dose-dependent manner. The LC50 for adult, nymph, and larva, were 29.21 (95% confidence interval 25.90-32.58), 28.18 (23.78-32.25), and 28.06 (25.57-30.90) mg/mL, respectively. Scanning electron microscopy and light microscopy revealed a disjointed sensilla base from the sockets, cuticular cracks, blocked aeropyles, and shrinking of the midgut. These results showed that C. citratus essential oil could be a good eco-friendly alternative control strategy against ectoparasites like ticks due to its high acaricidal efficacy.

Toll-interacting protein deficiency promotes neurodegeneration via impeding autophagy completion in high-fat diet-fed ApoE-/- mouse model.

The excessive accumulation of specific cellular proteins or autophagic vacuoles (AVs) within neurons is a pathologic hallmark of neurodegenerative diseases. Constitutive autophagy in neurons prevents abnormal intracellular protein aggregation and is critical for maintaining cell survival. Since our previous study showed that Toll-interacting protein (Tollip)-deficient macrophages had constitutive disruption of endosome-lysosome fusion, we hypothesize that Tollip deficiency may also promote neuron death via blockage of autophagy completion. Indeed, we observed significantly higher levels of neuron death in the brain regions of cerebral cortex, hippocampus, and cerebellum from ApoE-/-/Tollip-/- mice as compared to ApoE-/- mice fed with high fat diet (HFD). We further documented diminished density of neurons and increased ratios of TUNEL positive cells in the hippocampus of ApoE-/-/Tollip-/- mice. The ultrastructural electron microscopy analyses revealed neuron cell shrinkage as well as loss of intracellular structure in brain tissues from ApoE-/-/Tollip-/- mice. There was dramatic accumulation of autophagosomes in the cytoplasm, elevated accumulation of ß-amyloid and α-synuclein, and increased levels of p62 and Parkin in the brain tissues from ApoE-/-/Tollip-/- mice as compared to ApoE-/- mice. Our data suggest that Tollip may play a crucial role in sustaining neuron health by facilitating the completion of autophagy, and that Tollip-deficiency may accelerate neuron death related to neurological disease such as Alzheimer's disease.

A Novel Toxin from Haplopelma lividum Selectively Inhibits the NaV1.8 Channel and Possesses Potent Analgesic Efficacy.

Spider venoms are a complex mixture of peptides with a large number of neurotoxins targeting ion channels. Although thousands of peptide toxins have been identified from venoms of numerous species of spiders, many unknown species urgently need to be investigated. In this study, a novel sodium channel inhibitor, µ-TRTX-Hl1a, was identified from the venom of Haplopelma lividum. It contained eight cysteines and formed a conserved cysteine pattern of ICK motif. µ-TRTX-Hl1a inhibited the TTX-resistant (TTX-r) sodium channel current rather than the TTX-sensitive (TTX-s) sodium channel current. Meanwhile, µ-TRTX-Hl1a selectively inhibited NaV1.8 with an IC50 value of 2.19 µM. Intraperitoneal injection of µ-TRTX-Hl1a dose-dependently reduced inflammatory and neuropathic pain in rodent models of formalin-induced paw licking, tail-flicking, acetic acid-induced writhing, and hot plate test. It showed a better analgesic effect than morphine in inflammatory pain and equipotent effect to morphine in neuropathic pain. These findings demonstrate that µ-TRTX-Hl1a might be a valuable tool for physiology studies on NaV1.8 and a promising lead molecule for pain therapeutics.

Genome-scale deletion screening of human long non-coding RNAs using a paired-guide RNA CRISPR-Cas9 library.

CRISPR-Cas9 screens have been widely adopted to analyze coding-gene functions, but high-throughput screening of non-coding elements using this method is more challenging because indels caused by a single cut in non-coding regions are unlikely to produce a functional knockout. A high-throughput method to produce deletions of non-coding DNA is needed. We report a high-throughput genomic deletion strategy to screen for functional long non-coding RNAs (lncRNAs) that is based on a lentiviral paired-guide RNA (pgRNA) library. Applying our screening method, we identified 51 lncRNAs that can positively or negatively regulate human cancer cell growth. We validated 9 of 51 lncRNA hits using CRISPR-Cas9-mediated genomic deletion, functional rescue, CRISPR activation or inhibition and gene-expression profiling. Our high-throughput pgRNA genome deletion method will enable rapid identification of functional mammalian non-coding elements.

Curcumin combined with oxaliplatin effectively suppress colorectal carcinoma in vivo through inducing apoptosis.

Studies have shown chemopreventive and/or chemotherapeutic effects of several curcumin-based combinatorial treatments on colorectal cancer cells. However, their in vivo effects remain unclear. This study has demonstrated the therapeutic effect of curcumin and oxaliplatin, alone or in combination, on subcutaneously xenografted LoVo human colorectal cancer cells in immunodeficient (nu/nu) mice in vivo. Combinatorial administration of curcumin and oxaliplatin evidently inhibited the growth of colorectal cancer in nude mice, which was significantly more effective than either agent alone. Curcumin combined with oxaliplatin treatment induced apoptosis, accompanied by ultrastructural changes and cell cycle arrest in S and G2/M phases. Further mechanism analysis indicated that while the number of apoptotic tumor cells and the expression of Bax, caspase-3, and poly (ADP-ribose) polymerase (PARP) increased significantly, the expression of Bcl-2, survivin, HSP70, pro-caspase-3, and pro-PARP were dramatically suppressed in tumor cells after the treatment with combinatorial curcumin and oxaliplatin for 22 days. Taken together, the present study has demonstrated that administration of combined curcumin and oxaliplatin effectively suppressed colorectal carcinoma in vivo through inducing apoptosis and thus may provide an effective treatment for colorectal carcinoma.

Purification of vitellin and dynamics of vitellogenesis in the parthenogenetic tick Haemaphysalis longicornis (Acari: Ixodidae).

Vitellin (Vt) was purified from eggs of parthenogenetic bush tick Haemaphysalis longicornis by gel filtration and ion exchange chromatography. Our results revealed that only one single Vt existed in parthenogenetic bush tick, and the purified Vt was proved to be a hemoglycolipoprotein consisting of nine polypeptides with molecular weights of 203, 147, 126, 82, 74, 70, 61, 47 and 31 kDa, respectively. Polyclonal antibody and monoclonal antibody against Vt were produced using the purified Vt. The change in vitellogenin (Vg) and Vt levels over time of the parthenogenetic H. longicornis was established, and the Vg content in haemolymph and Vt in ovary at different feeding or engorgement statuses was also determined using a double antibody sandwich enzyme-linked immunosorbent assay. The Vg level in haemolymph was distinctly increased on the day of engorgement (1.785 mg/mL) and continued to increase until 2nd day post-engorgement (5.611 mg/mL). There was a slight decrease in Vg level after 4 days of engorgement, and a second peak was observed on day 2 post-oviposition (10.774 mg/mL). Subsequently, Vg content continuously decreased and reached a low level on the 10th day post-oviposition. The Vt content in ovary continuously increased once the female reached its critical weight (0.024 mg per female), and reached the maximum level on day 2 post-oviposition (1.942 mg per female). Afterwards, Vt content rapidly decreased.

Identification of multiple peptides with antioxidant and antimicrobial activities from skin and its secretions of Hylarana taipehensis, Amolops lifanensis, and Amolops granulosus.

Amphibian skin and its secretions contain many kinds of peptides with different bioactivities. In this study, a large number of peptides including antioxidant and antimicrobial peptides were identified from three East Asian frog species Hylarana taipehensis, Amolops lifanensis, and Amolops granulosus. The majority of these peptides were antimicrobial peptides, while eight antioxidant peptides were identified, which included two novel peptides taipehensin-1TP1 (TLIWEFYHQILDEYNKENKG) and taipehensin-2TP1 (CLMARPNYRCKIFKQC). These antioxidant peptides exhibited the ability to scavenge ABTS and/or DPPH free radicals. Moreover, six out of eight antioxidant peptides temporin-TP1, brevinin-1TP1, brevinin-1TP2, brevinin-1TP3, brevinin-1LF1, and palustrin-2GN1 also showed antimicrobial activity.

Diverse families of antimicrobial peptides isolated from skin secretions of three species of East Asian frogs, Babina daunchina, Babina adenopleura, and Rana omeimontis (Ranidae).

Twenty-two novel cDNAs encoding 22 peptide precursors for 19 mature peptides including antimicrobial peptides (AMPs) were identified from East Asian frog species Babina daunchina, Babina adenopleura, and Rana omeimontis skin-derived cDNA libraries. Two atypical members of the brevinin-1 family AMPs, named brevinin-1AN1 (FLTGVLKLASKIPSVLCAVLKTC) and brevinin-1DN1(FLKGVINLASKIPSMLCAVLKTC), were purified from the skin secretions of B. adenopleura and B. daunchina, respectively. A member of the ranatuerin-2 family AMP named ranatuerin-2DN1 (GLFDSITQGLKDTAVKLLDKIKCKLSACPPA) was also purified from the skin secretion of B. daunchina. One AMP named japonicin-2OM1 (FIVPSIFLLKKAFCIALKKNC) was purified from the skin secretion of R. omeimontis. The antimicrobial tests showed that brevinin-1DN1, brevinin-1DN2, brevinin-1AN1, and japonicin-2OM1 possess higher antimicrobial activity against Gram-positive bacteria than Gram-negative bacteria.

Novel analgesic peptides from the tree frog of Hyla japonica.

Two novel analgesic peptides (Analgesin-HJ, FWPVI-NH2 and Analgesin-HJ(I5T), FWPVT-NH2) were identified from the skin of the tree frog, Hyla japonica. There are 171 amino acid residues in the precursor encoding analgesin-HJs. The precursor contains 10 copies of mature peptide, which include 9 copies of analgesin-HJ and one copy of analgesin-HJ(I5T). Results from analgesic experiments using mice models including abdominal writhing induced by acetic acid, formalin-induced paw licking, and thermal pain test indicated that this two peptides exerted comparable analgesic activities with morphine. In addition, they had ability to inhibit inflammatory factor secretion induced by lipopolysaccharides (LPS). Considering their easy production, storage, transfer and potential analgesic activity, analgesin-HJs might be exciting leading compounds or templates for the development of novel analgesic agent. In addition, this study might facilitate to understand skin defensive mechanism of amphibians.

[Study on functions and mechanism of curcumin in inducing colorectal carcinoma cells LoVo apoptosis].

OBJECTIVE: To discuss the biological function and regulation mechanism of curcumin in promoting human colorectal carcinoma (LoVo) cells apoptosis. METHOD: Conventional in vitro culture in human colorectal carcinoma cells LoVo, When 80%-90% confluence was reached, cells were treated with curcumin at different concentrations (0-20 mg x L(-1)). Curcumin's effect on cell proliferation level was examined by MTT colorimetry. The ultrastructure of curcumin-treated LoVo cells were observed with transmission electron microscope (TEM). The amount of PI-positive LoVo cells after the curcumin treatment were determined by flow cytometry. The cell apoptosis rate was detected by Annexin V-FITC/PI double staining. The mRNA level of Bax, Bcl-2, Caspase-3 and Bcl-xL were tested by means of RT-PCR. RESULT: MTT test indicates curcumin could inhibite the growth and proliferation of LoVo cells in a time- and concentration-dependent manner. TEM examination showed that curcumin can make LoVo cell morphological changes, showing the typical characteristics of apoptotic cells. Flow cytometry instrument analysis showed that curcumin can arrest cell cycle at S phase, and induce apoptosis of LoVo cells. RT-PCR test showed that curcumin can activate the expression of Bax and Caspase-3, inhibit the expression of Bcl-2 and Bcl-xL at the mRNA level. CONCLUSION: Curcumin can significantly inhibit the proliferation and induce the apoptosis of human colorectal carcinoma cells LoVo. Such biological effect may be associated with activating Caspase-3 signal channel by activating Bax expression and inhibiting Bcl-2 and Bcl-xL expression. This study lays an important foundation for further discussing the mechanism of curcumin in inducing human colorectal carcinoma LoVo apoptosis.