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Purpose: The inhibition of thrombin has proven to be an efficacious therapeutic approach for managing cardiovascular disease (CVD), with widespread implementation in clinical settings. Oral ingestion of peptides and protein drugs is influenced by gastrointestinal digestive enzymes. We aimed to evaluate the thrombin inhibitory properties of hirudo hydrolysates (HHS) produced by pepsin and propose a comprehensive approach to screen and evaluate thrombin inhibitors. Methods: We evaluated the in vitro inhibitory properties of the hirudo extract, both before and after hydrolysis with pepsin, toward thrombin. We screened for the most potent thrombin inhibitory peptide (TIP) using nano liquid chromatography-tandem mass spectrometry (Nano LC-MS/MS) coupled with in silico analysis. Next, we employed the thrombin inhibition activity IC50 to investigate the interaction between TIP and thrombin, and conducted in vitro evaluations of its anticoagulant effects (APTT, TT, PT), as well as its ability to inhibit platelet aggregation. Furthermore, we utilized UV-Vis spectroscopy to explore structural changes in thrombin upon binding with TIP and employed molecular dynamics simulations to delve deeper into the potential atomic-level interaction modes between thrombin and TIP. Results: The retention rate of thrombin inhibition for HHS was found to be between 60% and 75%. A total of 90 peptides from the HHS were identified using LC-MS/MS combined with de novo sequencing. Asn-Asp-Leu-Trp-Asp-Gln-Gly-Leu-Val-Ser-Gln-Asp-Leu (NDLWDQGLVSQDL, P1) was identified as the most potent thrombin inhibitory peptide after in silico screening (molecular docking and ADMET). Then, the in vitro study revealed that P1 had a high inhibitory effect on thrombin (IC50: 2,425.5 ± 109.7 μM). P1 exhibited a dose-dependent prolongation of the thrombin time (TT) and a reduction in platelet aggregation rate. Both UV-Vis spectroscopy and molecular dynamics simulations demonstrated that P1 binds effectively to thrombin. Conclusion: Overall, the results suggested that HHS provides new insights for searching and evaluating potential antithrombotic compounds. The obtained P1 can be structurally optimized for in-depth evaluation in animal and cellular experiments. [ABSTRACT FROM AUTHOR]

PurposeThe inhibition of thrombin has proven to be an efficacious therapeutic approach for managing cardiovascular disease (CVD), with widespread implementation in clinical settings. Oral ingestion of peptides and protein drugs is influenced by gastrointestinal digestive enzymes. We aimed to evaluate the thrombin inhibitory properties of hirudo hydrolysates (HHS) produced by pepsin and propose a comprehensive approach to screen and evaluate thrombin inhibitors.MethodsWe evaluated the in vitro inhibitory properties of the hirudo extract, both before and after hydrolysis with pepsin, toward thrombin. We screened for the most potent thrombin inhibitory peptide (TIP) using nano liquid chromatography-tandem mass spectrometry (Nano LC-MS/MS) coupled with in silico analysis. Next, we employed the thrombin inhibition activity IC50 to investigate the interaction between TIP and thrombin, and conducted in vitro evaluations of its anticoagulant effects (APTT, TT, PT), as well as its ability to inhibit platelet aggregation. Furthermore, we utilized UV-Vis spectroscopy to explore structural changes in thrombin upon binding with TIP and employed molecular dynamics simulations to delve deeper into the potential atomic-level interaction modes between thrombin and TIP.ResultsThe retention rate of thrombin inhibition for HHS was found to be between 60% and 75%. A total of 90 peptides from the HHS were identified using LC-MS/MS combined with de novo sequencing. Asn-Asp-Leu-Trp-Asp-Gln-Gly-Leu-Val-Ser-Gln-Asp-Leu (NDLWDQGLVSQDL, P1) was identified as the most potent thrombin inhibitory peptide after in silico screening (molecular docking and ADMET). Then, the in vitro study revealed that P1 had a high inhibitory effect on thrombin (IC50: 2,425.5 ± 109.7 μM). P1 exhibited a dose-dependent prolongation of the thrombin time (TT) and a reduction in platelet aggregation rate. Both UV-Vis spectroscopy and molecular dynamics simulations demonstrated that P1 binds effectively to thrombin.ConclusionOverall, the results suggested that HHS provides new insights for searching and evaluating potential antithrombotic compounds. The obtained P1 can be structurally optimized for in-depth evaluation in animal and cellular experiments.

During July -August of 2021, thirty-one leeches were collected from two localities in Erbil and its suburbs for studying the morphological features of jaws, denticles,and salivary gland cells. Leeches were two blood-sucking species; Hirudo orientalis(Utevsky & Trontelj, 2005) (Family, Hirudinidae) and Limnatis paluda(Tennent 1859) (Family, Praobdellidae). The investigations conducted using a stereomicroscope (SM) andscanning electron microscopy (SEM). H. orientalisjaws were white and rigid, bearing sharp teeth, while L. paludajaws were gray and soft bearing fewer blunt teeth with plentiful papilla and both are monostichodont. In the present study, the salivary glands of adult leeches were examined by SEM. They are composed of unicellular glands arranged in grape patterns with spherical, ovoid, and pear shapes in various cell sizes; the cell bunches of gland cells were highly developed and interconnected to one another by tiny channels. A bigger canal that led to the jaws was created by combining channels from each bunch.

The aim of this study is to describe the leech species that are used in some of the alternative medicine clinics in Baghdad Province based on morphometric measurements and colouring pattern of the body. A collection of twenty leeches was provided from some clinics. All specimens were identified and described based on standard available keys. The morphometric characteristics and colouring patterns were recorded and the indicated that these leeches were Hirudo orientalisUtevsky & Trontelj, 2005 and H.verbana Carena, 1820.

Noxious stimuli or injury can trigger long-lasting sensitization to non-nociceptive stimuli (referred to as allodynia in mammals). Long-term potentiation (LTP) of nociceptive synapses has been shown to contribute to nociceptive sensitization (hyperalgesia) and there is even evidence of heterosynaptic spread of LTP contributing to this type of sensitization. This study will focus on how activation of nociceptors elicits heterosynaptic LTP (hetLTP) in non-nociceptive synapses. Previous studies in the medicinal leech (Hirudo verbana) have demonstrated that high-frequency stimulation (HFS) of nociceptors produces both homosynaptic LTP as well as hetLTP in non-nociceptive afferent synapses. This hetLTP involves endocannabinoid-mediated disinhibition of non-nociceptive synapses at the presynaptic level, but it is not clear if there are additional processes contributing to this synaptic potentiation. In this study, we found evidence for the involvement of postsynaptic level change and observed that postsynaptic N-methyl-d-aspartate (NMDA) receptors (NMDARs) were required for this potentiation. Next, Hirudo orthologs for known LTP signaling proteins, CamKII and PKCζ, were identified based on sequences from humans, mice, and the marine mollusk Aplysia. In electrophysiological experiments, inhibitors of CamKII (AIP) and PKCζ (ZIP) were found to interfere with hetLTP. Interestingly, CamKII was found to be necessary for both induction and maintenance of hetLTP, whereas PKCζ was only necessary for maintenance. These findings show that activation of nociceptors can elicit a potentiation of non-nociceptive synapses through a process that involves both endocannabinoid-mediated disinhibition and NMDAR-initiated signaling pathways. NEW & NOTEWORTHY Pain-related sensitization involves increases in signaling by non-nociceptive sensory neurons. This can allow non-nociceptive afferents to have access to nociceptive circuitry. In this study, we examine a form of synaptic potentiation in which nociceptor activity elicits increases in non-nociceptive synapses. This process involves endocannabinoids, "gating" the activation of NMDA receptors, which in turn activate CamKII and PKCζ. This study provides an important link in how nociceptive stimuli can enhance non-nociceptive signaling related to pain. [ABSTRACT FROM AUTHOR]

An analytical method for the simultaneous determination of 73 pesticides in Hirudo using gas chromatography-tandem mass spectrometry(GC-MS/MS) and QuEChERS procedure was developed. After being soaked in water, the samples were extracted with acidic acetonitrile and cleaned up by a mixture of sorbents (150 mg PSA and 150 mg C18). In order to obtain the optimum recovery rate of pesticide analysis, the amounts of the adsorbents were investigated. The analytes were separated by a HP-5 MS column connected to an electron impact ion source(EI) with dynamic multi-reaction monitoring MS mode. Under the optimized conditions, the results showed that the 73 pesticides had good linearity within the range of 10.0-500 μg/L, with correlation coefficients(r) higher than 0.99. The average recoveries ranged from 71%-117%, and the relative standard deviation(RSD) was 1.1%-12% (n=6) at three spiking levels (0.02, 0.04 and 0.2 mg/kg). The LOQs were 0.02 mg/kg and LODs were 0.001-0.008 mg/kg. The method is simple, sensitive, reliable, and can meet the requirements of practical detection for pesticides. [ABSTRACT FROM AUTHOR]

Hirudo is highly effective in promoting blood circulation and removing blood stasis, a vital medicine for breaking blood and removing blood stasis in ophthalmopathy. Presently, Hirudo is commonly used in ophthalmology for age-related macular degeneration, diabetic retinopathy, cataract, retinal vein occlusion and other ophthalmic diseases, with the mechanism thereof possibly being related to anti-VEGF, improvement of microcirculation and anti-apoptosis. Further, although new functions have been found in Hirudo extract, such as transdifferentiation and inhibition of WERI-RB-1, these studies remain in the basic exploratory stage, and the specific molecular mechanism requires further verification. This paper concisely describes the application and research status of Hirudo and the extract thereof in ophthalmic diseases, so as to provide a new direction for further research on the treatment of ophthalmopathy using Hirudo and the extract thereof.

Endocannabinoids are traditionally thought to have an analgesic effect. However, it has been shown that while endocannabinoids can depress nociceptive signaling, they can also enhance non-nociceptive signaling. Therefore, endocannabinoids have the potential to contribute to non-nociceptive sensitization after an injury. Using Hirudo verbana (the medicinal leech), a model of injury-induced sensitization was developed in which a reproducible piercing injury was delivered to the posterior sucker of Hirudo. Injury-induced changes in the non-nociceptive threshold of Hirudo were determined through testing with Von Frey filaments and changes in the response to nociceptive stimuli were tested by measuring the latency to withdraw to a nociceptive thermal stimulus (Hargreaves apparatus). To test the potential role of endocannabinoids in mediating injury-induced sensitization, animals were injected with tetrahydrolipstatin (THL), which inhibits synthesis of the endocannabinoid transmitter 2-arachidonoylglycerol (2-AG). Following injury, a significant decrease in the non-nociceptive response threshold (consistent with non-nociceptive sensitization) and a significant decrease in the response latency to nociceptive stimulation (consistent with nociceptive sensitization) were observed. In animals injected with THL, a decrease in non-nociceptive sensitization in injured animals was observed, but no effect on nociceptive sensitization was observed. [ABSTRACT FROM AUTHOR]

In the present study it was aimed to determine the acute toxicity of zinc on southern medicinal leech Hirudo verbana Carena, 1820 that is used in ecotoxicological studies. The experiment was conducted for 96 hours using the semi -static bioassay method with replicate two times. The leeches were exposed to zinc concentrations of 6.25 mg/L, 12.50 mg/L, 25 mg/L, 50 mg/L, 100 mg/L and 200 mg/L, respectively. As a result of the experiment, the LC50 values for 24, 48, 72, and 96 hours were determined 48.30 mg/L, 19.68 mg/L, 15.48 mg/L, and 14.12 mg/L, respectively. The LT50 values for 25 mg/L, 50 mg/L, 100 mg/L and 200 mg/L zinc concentrations were determined 38.63 hours, 24.55 hours, 14.62 hours, and 12.78 hours, respectively. Defecation, intensive mucus output, uncontrolled swimming, attempt to escape from the experiment media, reducing the cling power, coiling (buckling), vomiting the digestive blood, the body deformations, penile prolapse, hemorrhage, and abrasion in the wall of the body were the main physiological and behavioral responses in the leeches which were exposed to the different zinc solutions. The data obtained revealed that the zinc sensitivity of H. verbana was moderate when compared to other organisms. It is expected that the data of the present study will contribute to the management of endangered medicinal leech populations. [ABSTRACT FROM AUTHOR]

In this paper, the resource distribution, pharmacological action and culture status of hirudos were reviewed. Hirudo resources are widely distributed and have a variety of pharmacological effects, but culture is difficult, so the scientific development of hirudos culture and solving the problem of hirudos breeding can alleviate the contradiction in the medicine market and contribute to the sustainable development of hirudo medicine. [ABSTRACT FROM AUTHOR]

The renewal and shedding of hard ectodermal structures are widespread in invertebrates. The most impressive example of these processes in Trochozoa is the cuticle shedding observed in Hirudo medicinalis, originally studied over a century ago. Here we investigate cuticle shedding across three leech species: Hirudo sp., Haemopis cf. sanguisuga, and Erpobdella cf. octoculata. According to the ultrastructural data, all three species have a typical leech cuticle, consisting of basi‐ and epicuticles. Using in vivo observations and morphological investigations, we confirm cuticle shedding in Hirudo sp., and for the first time, report this process in Haemopis cf. sanguisuga. The successive stages of shedding of the old cuticle and formation of the new cuticle are ultrastructurally described for both species. Interestingly, individuals of Erpobdella cf. octoculata did not shed their cuticle during the entire observation period, neither as adults nor as juveniles. The semi‐terrestrial lifestyle and the consumption of large food volumes may possibly be a prerequisite driving the development of regular cuticle shedding in Hirudo sp. and Haemopis cf. sanguisuga. [ABSTRACT FROM AUTHOR]

Leeches are found in terrestrial, aquatic, and marine habitats on all continents. Sanguivorous leeches have been used in medicine for millennia. Modern scientific uses include studies of neurons, anticoagulants, and gut microbial symbioses. Hirudo verbana, the European medicinal leech, maintains a gut community dominated by two bacterial symbionts, Aeromonas veronii and Mucinivorans hirudinis, which sometimes account for as much as 97% of the total crop microbiota. The highly simplified gut anatomy and microbiome of H. verbana make it an excellent model organism for studying gut microbial dynamics. The North American medicinal leech, Macrobdella decora, is a hirudinid leech native to Canada and the northern United States. In this study, we show that M. decora symbiont communities are very similar to those in H. verbana. We performed an extensive study using field-caught M. decora and purchased H. verbana from two suppliers. Deep sequencing of the V4 region of the 16S rRNA gene allowed us to determine that the core microbiome of M. decora consists of Bacteroides, Aeromonas, Proteocatella, and Butyricicoccus. The analysis revealed that the compositions of the gut microbiomes of the two leech species were significantly different at all taxonomic levels. The R2 value was highest at the genus and amplicon sequence variant (ASV) levels and much lower at the phylum, class, and order levels. The gut and bladder microbial communities were distinct. We propose that M. decora is an alternative to H. verbana for studies of wild-caught animals and provide evidence for the conservation of digestive-tract and bladder symbionts in annelid models. IMPORTANCE Building evidence implicates the gut microbiome in critical animal functions such as regulating digestion, nutrition, immune regulation, and development. Simplified, phylogenetically diverse models for hypothesis testing are necessary because of the difficulty of assigning causative relationships in complex gut microbiomes. Previous research used Hirudo verbana as a tractable animal model of digestive-tract symbioses. Our data show that Macrobdella decora may work just as well without the drawback of being an endangered organism and with the added advantage of easy access to field-caught specimens. The similarity of the microbial community structures of species from two different continents reveals the highly conserved nature of the microbial symbionts in sanguivorous leeches. [ABSTRACT FROM AUTHOR]

We characterized the behavioral responses of two leech species, Hirudo verbana and Erpobdella obscura, to mechanical skin stimulation and examined the interactions between the pressure mechanosensory neurons (P cells) that innervate the skin. To quantify behavioral responses, we stimulated both intact leeches and isolated body wall preparations from the two species. In response to mechanical stimulation, Hirudo showed local bending behavior, in which the body wall shortened only on the side of the stimulation. Erpobdella, in contrast, contracted both sides of the body in response to touch. To investigate the neuronal basis for this behavioral difference, we studied the interactions between P cells. Each midbody ganglion has four P cells; each cell innervates a different quadrant of the body wall. Consistent with local bending, activating any one P cell in Hirudo elicited polysynaptic inhibitory potentials in the other P cells. In contrast, the P cells in Erpobdella had excitatory polysynaptic connections, consistent with the segment-wide contraction observed in this species. In addition, activating individual P cells caused asymmetrical body wall contractions in Hirudo and symmetrical body wall contractions in Erpobdella. These results suggest that the different behavioral responses in Erpobdella and Hirudo are partly mediated by interactions among mechanosensory cells.

In the animal kingdom, behavioral traits encompass a broad spectrum of biological phenotypes that have critical roles in adaptive evolution, but an EvoDevo approach has not been broadly used to study behavior evolution. Here, we propose that, by integrating two leech model systems, each of which has already attained some success in its respective field, it is possible to take on behavioral traits with an EvoDevo approach. We first identify the developmental changes that may theoretically lead to behavioral evolution and explain why an EvoDevo study of behavior is challenging. Next, we discuss the pros and cons of the two leech model species, Hirudo, a classic model for invertebrate neurobiology, and Helobdella, an emerging model for clitellate developmental biology, as models for behavioral EvoDevo research. Given the limitations of each leech system, neither is particularly strong for behavioral EvoDevo. However, the two leech systems are complementary in their technical accessibilities, and they do exhibit some behavioral similarities and differences. By studying them in parallel and together with additional leech species such as Haementeria, it is possible to explore the different levels of behavioral development and evolution. Research Highlights: Behavior is a biological phenotype that can be studied by using an EvoDevo approach.Changes in the neural circuits can result in behavioral change.The leech Hirudo has been used to study neural circuits in the past half‐century.Helobdella has been used to study leech development.By comparing the different leeches, it is possible to characterize the molecular changes underlying changes in neural circuits and behavior.As a direct developer, the entire course of development is experimentally tractable in the leech. This would provide a unique opportunity to study the EvoDevo of behaviors. [ABSTRACT FROM AUTHOR]

Given the key role of innate immunity in both defense against pathogens and tissue regeneration, innovative studies are becoming crucial to provide further information on how both processes are linked together and to clarify how immune cells perform the coordinated regulation of the aforementioned processes. The present review is mainly focused on two proteins that have been recently found to carry out critical functions in innate immune system regulation, i. e. the Allograft inflammatory factor-1 (AIF-1) and RNASET2, a protein belonging to the T2 ribonuclease family. Their crucial role in both the activation and modulation of the inflammatory response and in the remodeling of connective tissue during grafts and wound repair have been thoroughly investigated in the medicinal leech and will pave the way for novel therapeutic approaches to control immune and systemic responses to disease, injury, and bacterial infection, based on the functionalities of these biomolecules. [ABSTRACT FROM AUTHOR]

The digestive tract of medicinal leeches from commercial suppliers has been investigated previously and comprises of a relatively simple bacterial community. However, the microbiome of medicinal leeches collected directly from the natural habitat has not been examined. In this study, we characterized the bacterial community in the digestive tract (anterior crop, posterior crop, and intestine) of the European medicinal leech, Hirudo verbana, collected from the Danube river using culture-independent and culture-dependent approaches. Culture-independent approach confirmed that the digestive tract of H. verbana carries a relatively simple bacterial community with species richness in the individual samples ranging from 43 to164. The dominant bacterial taxon was Mucinivorans sp. (49.7% of total reads), followed by Aeromonas sp. (18.7% of total reads). Several low abundance taxa, new for H. verbana, such as Phreatobacter, Taibaiella, Fluviicola, Aquabacterium, Burkholderia, Hydrogenophaga, Wolinella, and unidentified Chitinophagia, were also detected. The aerobic culturing approach showed Aeromonas veronii (Proteobacteria), the known leech symbiont, as the most dominant taxon followed by several Pseudomonas and Acidovorax spp. No significant differences in the bacterial community composition were detected among different parts of the digestive tract of individual leeches. However, the overall composition of the bacterial community among individual specimen varied significantly and this is possibly due to differences in leech age, feeding status, and blood source. Our results showed that the core bacterial community of H. verbana collected from the natural habitat is similar to that reported from the digestive tract of commercially supplied leeches maintained in the laboratory. [ABSTRACT FROM AUTHOR]

Thic article reviews the species, identification, chemical composition and pharmacological effects oO hirudo. Hirudo ic widely sourced, difficult te identify, contains a variete of active ingredients, and has a variete of pharmacologicaO effects. In-depth research on hirudo ic of great sienificanco te the qualita control and clinical application of hirudo. [ABSTRACT FROM AUTHOR]

Animal toxins and venoms have recently been developed as cancer treatments possessing tumor cell growth-inhibitory, antiangiogenesis, and proapoptotic effects. Endometriosis is a common benign gynecological disorder in reproductive-age women, and no definite treatment for this disorder is without severe side effects. As endometriosis and malignant tumors share similar characteristics (progressive, invasive, estrogen-dependent growth, and recurrence), animal toxins and venoms are thought to be effective against endometriosis. The objective of this study was to outline studies using toxic animal-based medicinal materials (TMM) as endometriosis treatment and to explore its clinical applicability. Preclinical and clinical studies using TMM were searched for in four databases from inception to October 2020. A total of 20 studies of TMM on endometriosis were included. In eight clinical studies, herbal medicines containing TMM were effective in relieving symptoms of endometriosis, with no side effects. In twelve experimental studies, the main therapeutic mechanisms of TMM against endometriosis were proapoptotic, antiangiogenesis, estrogen level-reducing, and possible anti-inflammatory effects. TMM are thus considered promising sources for the development of an effective treatment method for endometriosis. Further studies are needed to clarify the therapeutic mechanism of TMM against endometriosis and to provide sufficient grounds for clinical application.

With the evolution for several millions of years, leeches have developed a perfect capability to resist mechanical loads, which provides many inspirations to engineer new materials and new devices. To uncover the mechanism of its strong survival ability, several mechanical approaches, such as compression, tension, adhesion, impact and blood suction experiments were tried. Our experimental results show that a leech ( Hirudo nipponia ) can surprisingly withstand a compressive force of nearly 10 6 times its body weight. In tension, this animal demonstrates large deformation and its strain can reach a value bigger than 3. To avoid being removed from the host skin, it produces an adhesion force superior to 118 times its body weight, and it can endure an impact force at least 1500 times its weight. Also the leech skin can bear an internal fluid pressure of around 6 times the atmospheric pressure. These data show that the leech cannot be killed easily through normal mechanical loading approaches. All these amazing performances lie in hierarchical structures and ductility of the skin with highly developed and compact annuluses, and this feature is beneficial to leech's survival. [ABSTRACT FROM AUTHOR]

Leech saliva extract (LSE) in the liposome-based gel was used as a supplementary treatment to relief the signs and symptoms of osteoarthritis (OA). The saliva of medical leech was extracted and nano liposomes were used to formulate the supplement to enhance skin absorption. A clinical trial was designed to evaluate the therapeutic effects of LSE liposomal gel. Lenquesne and VAS questionnaires were used as indexes of this supplement therapy efficacy for 30 days. Questionnaires analysis showed that after one-month administration of LSE liposomal gel, patients' pain was relieved approximately up to 50%; also, due to reduction in joint inflammation and stiffness, the range of motion was increased and the patients' quality of life was enhanced (p < 0.001). LSE nano scaled liposomal gel as an innovative supplement therapy in OA patients, makes desirable therapeutic approach, which seems to make a significant impact on patient's quality of life and self-care capability. [ABSTRACT FROM AUTHOR]

Sensitivity to water waves is a key modality by which aquatic predators can detect and localize their prey. For one such predator -- the medicinal leech, Hirudo verbana -- behavioral responses to visual and mechanical cues from water waves are well documented. Here, we quantitatively characterized the response patterns of a multisensory interneuron, the S cell, to mechanically and visually cued water waves. As a function of frequency, the response profile of the S cell replicated key features of the behavioral prey localization profile in both visual and mechanical modalities. In terms of overall firing rate, the S cell responsewas not direction selective, and although the direction of spike propagation within the S cell system did follow the direction of wave propagation under certain circumstances, it is unlikely that downstream neuronal targets can use this information. Accordingly, we propose a role for the S cell in the detection of waves but not in the localization of their source. We demonstrated that neither the head brain nor the tail brain are required for the S cell to respond to visually cued water waves. [ABSTRACT FROM AUTHOR]

In recent years, several studies have demonstrated that the RNASET2 gene is involved in the control of tumorigenicity in ovarian cancer cells. Furthermore, a role in establishing a functional cross-talk between cancer cells and the surrounding tumor microenvironment has been unveiled for this gene, based on its ability to act as an inducer of the innate immune response. Although several studies have reported on the molecular features of RNASET2, the details on the mechanisms by which this evolutionarily conserved ribonuclease regulates the immune system are still poorly defined. In the effort to clarify this aspect, we report here the effect of recombinant human RNASET2 injection and its role in regulating the innate immune response after bacterial challenge in an invertebrate model, the medicinal leech. We found that recombinant RNASET2 injection induces fibroplasias, connective tissue remodeling and the recruitment of numerous infiltrating cells expressing the specific macrophage markers CD68 and HmAIF1. The RNASET2-mediated chemotactic activity for macrophages has been further confirmed by using a consolidated experimental approach based on injection of the Matrigel biomatrice (MG) supplemented with recombinant RNASET2 in the leech body wall. One week after injection, a large number of CD68 and HmAIF-1 macrophages massively infiltrated MG sponges. Finally, in leeches challenged with lipopolysaccharides (LPS) or with the environmental bacteria pathogen Micrococcus nishinomiyaensis, numerous macrophages migrating to the site of inoculation expressed high levels of endogenous RNASET2. Taken together, these results suggest that RNASET2 is likely involved in the initial phase of the inflammatory response in leeches. [ABSTRACT FROM AUTHOR]

For many animals processing of tactile information is a crucial task in behavioral contexts like exploration, foraging and stimulus avoidance. The leech, having infrequent access to food, developed an energy efficient reaction to tactile stimuli, avoiding unnecessary muscle movements: The local bend behavior moves only a small part of the body wall away from an object touching the skin, while the rest of the animal remains stationary. Amazingly, the precision of this localized behavioral response is similar to the spatial discrimination threshold of the human fingertip, although the leech skin is innervated by an order of magnitude fewer mechanoreceptors and each midbody ganglion contains only 400 individually identified neurons in total. Prior studies suggested that this behavior is controlled by a three-layered feed-forward network, consisting of four mechanoreceptors (P cells), approximately 20 interneurons and 10 individually characterized motor neurons, all of which encode tactile stimulus location by overlapping, symmetrical tuning curves. Additionally, encoding of mechanical force was attributed to three types of mechanoreceptors reacting to distinct intensity ranges: T cells for touch, P cells for pressure and N cells for strong, noxious skin stimulation. In this study, we provide evidences that tactile stimulus encoding in the leech is more complex than previously thought. Combined electrophysiological, anatomical and voltage sensitive dye approaches indicate that P and T cells both play a major role in tactile information processing resulting in local bending. Our results indicate that tactile encoding neither relies on distinct force intensity ranges of different cell types, nor location encoding is restricted to spike count tuning. Instead, we propose that P and T cells form a mixed type population, which simultaneously employs temporal response features and spike counts for multiplexed encoding of touch location and force intensity. This hypothesis is supported by our finding that previously identified local bend interneurons receive input from both P and T cells. Some of these interneurons seem to integrate mechanoreceptor inputs, while others appear to use temporal response cues, presumably acting as coincidence detectors. Further voltage sensitive dye studies can test these hypotheses how a tiny nervous system performs highly precise stimulus processing.

For many animals processing of tactile information is a crucial task in behavioral contexts like exploration, foraging, and stimulus avoidance. The leech, having infrequent access to food, developed an energy efficient reaction to tactile stimuli, avoiding unnecessary muscle movements: The local bend behavior moves only a small part of the body wall away from an object touching the skin, while the rest of the animal remains stationary. Amazingly, the precision of this localized behavioral response is similar to the spatial discrimination threshold of the human fingertip, although the leech skin is innervated by an order of magnitude fewer mechanoreceptors and each midbody ganglion contains only 400 individually identified neurons in total. Prior studies suggested that this behavior is controlled by a three-layered feed-forward network, consisting of four mechanoreceptors (P cells), approximately 20 interneurons and 10 individually characterized motor neurons, all of which encode tactile stimulus location by overlapping, symmetrical tuning curves. Additionally, encoding of mechanical force was attributed to three types of mechanoreceptors reacting to distinct intensity ranges: T cells for touch, P cells for pressure, and N cells for strong, noxious skin stimulation. In this study, we provide evidences that tactile stimulus encoding in the leech is more complex than previously thought. Combined electrophysiological, anatomical, and voltage sensitive dye approaches indicate that P and T cells both play a major role in tactile information processing resulting in local bending. Our results indicate that tactile encoding neither relies on distinct force intensity ranges of different cell types, nor location encoding is restricted to spike count tuning. Instead, we propose that P and T cells form a mixed type population, which simultaneously employs temporal response features and spike counts for multiplexed encoding of touch location and force intensity. This hypothesis is supported by our finding that previously identified local bend interneurons receive input from both P and T cells. Some of these interneurons seem to integrate mechanoreceptor inputs, while others appear to use temporal response cues, presumably acting as coincidence detectors. Further voltage sensitive dye studies can test these hypotheses how a tiny nervous system performs highly precise stimulus processing. [ABSTRACT FROM AUTHOR]

Digestive-tract microbiota exert tremendous influence over host health. Host-symbiont model systems are studied to investigate how symbioses are initiated and maintained, as well as to identify host processes affected by resident microbiota. The medicinal leech, Hirudo verbana, is an excellent model to address such questions owing to a microbiome that is consistently dominated by two species, Aeromonas veronii and Mucinivorans hirudinis, both of which are cultivable and have sequenced genomes. This review outlines current knowledge about the dynamics of the H. verbana microbiome. We discuss in depth the factors required for A. veronii colonization and proliferation in the leech crop and summarize the current understanding of interactions between A. veronii and its annelid host. Lastly, we discuss leech usage in modern medicine and highlight how leech-therapy associated infections, often attributable to Aeromonas spp., are of growing clinical concern due in part to an increased prevalence of fluoroquinolone resistant strains. [ABSTRACT FROM AUTHOR]

Endocannabinoids can elicit persistent depression of excitatory and inhibitory synapses, reducing or enhancing (disinhibiting) neural circuit output, respectively. In this study, we examined whether differences in Cl- gradients can regulate which synapses undergo endocannabinoid-mediated synaptic depression vs. disinhibition using the well-characterized central nervous system (CNS) of the medicinal leech, Hirudo verbana. Exogenous application of endocannabinoids or capsaicin elicits potentiation of pressure (P) cell synapses and depression of both polymodal (Npoly) and mechanical (Nmech) nociceptive synapses. In P synapses, blocking Cl- export prevented endocannabinoid-mediated potentiation, consistent with a disinhibition process that has been indicated by previous experiments. In Nmech neurons, which are depolarized by GABA due to an elevated Cl- equilibrium potentials (ECl), endocannabinoidmediated depression was prevented by blocking Cl- import, indicating that this decrease in synaptic signaling was due to depression of excitatory GABAergic input (disexcitation). Npoly neurons are also depolarized by GABA, but endocannabinoids elicit depression in these synapses directly and were only weakly affected by disruption of Cl- import. Consequently, the primary role of elevated ECl may be to protect Npoly synapses from disinhibition. All forms of endocannabinoid-mediated plasticity required activation of transient potential receptor vanilloid (TRPV) channels. Endocannabinoid/TRPV-dependent synaptic plasticity could also be elicited by distinct patterns of afferent stimulation with low-frequency stimulation (LFS) eliciting endocannabinoid-mediated depression of Npoly synapses and highfrequency stimulus (HFS) eliciting endocannabinoid-mediated potentiation of P synapses and depression of Nmech synapses. These findings demonstrate a critical role of differences in Cl- gradients between neurons in determining the sign, potentiation vs. depression, of synaptic modulation under normal physiological conditions. [ABSTRACT FROM AUTHOR]

Safe evacuation of people from building and outdoor environments, and search and rescue operations, always will remain actual in course of all socio-technological developments. Modern facilities offer a range of automated systems to guide residents towards emergency exists. The systems are assumed to be infallible. But what if they fail? How occupants not familiar with a building layout will be looking for exits in case of very limited visibility where tactile sensing is the only way to assess the environment? Analogous models of human behaviour, and socio-dynamics in general, are provided to be fruitful ways to explore alternative, or would-be scenarios. Crowd, or a single person, dynamics could be imitated using particle systems, reaction–diffusion chemical medium, electro-magnetic fields, or social insects. Each type of analogous model offer unique insights on behavioural patterns of natural systems in constrained geometries. In this particular paper we have chosen leeches to analyse patterns of exploration. Reasons are two-fold. First, when deprived from other stimuli leeches change their behavioural modes in an automated regime in response to mechanical stimulation. Therefore leeches can give us invaluable information on how human beings might behave under stress and limited visibility. Second, leeches are ideal blueprints of future soft-bodied rescue robots. Leeches have modular nervous circuitry with a rich behavioral spectrum. Leeches are multi-functional, fault-tolerant with autonomous inter-segment coordination and adaptive decision-making. We aim to answer the question: how efficiently a real building can be explored and whether there any dependencies on the pathways of exploration and geometrical complexity of the building. In our case studies we use templates made on the floor plan of real building. [ABSTRACT FROM AUTHOR]

Background: Dried Whitmania pigra is used for the treatment of cardiovascular and cerebrovascular diseases in traditional Chinese medicine and hot water and alcohol extracts also have anticogulant activity. However, a lower molecular weight and more stable anticogulant is needed. Objective: The objective of the following study is to purify and characterize of an anticoagulant oligopeptide from Hirudo (Whitmania pigra Whitman). Materials and Methods: Gel filtration on Sephadex G-50, ion exchange on diethylaminoethyl-cellulose, and semi-prepared high-performance liquid chromatography were used to purify Hirudo. Automated coagulation analyzer was used for evaluating anticoagulant activity. Molecular weight was measured by Matrix-assisted laser desorption ionization time of flight mass spectrometry. Amino acid sequence of the oligopeptide was measured by amino acid sequence analyzer. Results: A new anticoagulant, named whitide, isolated from Hirudo was purified, with a molecular weight 1997.1 Da. Amino acid sequence of the oligopeptide was identified as Gly-Pro-ALa-Gly-Hyp-Val-Gly-Ala-Hyp-Gly-Gly-Hyp-Gly-Val-Arg-Gly-Leu-Hyp-Gly-Asp-Arg-Gly. The results revealed that its amino acid sequence had strong homology to various types of collagen. Conclusion: Whitide might be an orally anticoagulant for its hot and trypsin stable. [ABSTRACT FROM AUTHOR]

This study aimed to evaluate the efficacy and safety of Chinese patent medicines containing Hirudo in the treatment of atherosclerosis(AS) by network Meta-analysis, and to provide evidence-based reference for clinical treatment of AS. The clinical randomized controlled trial(RCT) on the treatment of atherosclerosis with Chinese patent medicines containing Hirudo were searched in CNKI, Wanfang, VIP, SinoMed, PubMed and EMbase from the establishment of the databases to July 1, 2022. And data extraction and quality assessment of the included RCT was performed according to the Cochrane standards. Stata 17 and ADDIS 1.16.5 were then used for Bayesian model network Meta-analysis. Finally, 67 RCTs with a total sample size of 6 826 cases were included, 3 569 cases in the experimental group and 3 257 cases in the control group, involving three oral Chinese patent medicines. Network Meta-analysis showed that in terms of reducing intima-media thickness(IMT), the top three Chinese patent medicines were Tongxinluo Capsules+sta-tins&gt;Maixuekang Capsules+statins&gt;Maixuekang Capsules. In terms of reducing plaque area, the top one was Maixuekang Capsules+sta-tins, and the other Chinese patent medicines had similar efficacy. For lowering AS Crouse scores, the top three were Maixuekang Capsules&gt;Tongxinluo Capsules+statins&gt;Naoxintong Capsules. For decreasing plaque number, the top three were Naoxintong Capsules+sta-tins&gt;Tongxinluo Capsules+statins&gt;Tongxinluo Capsules. With regard to adverse reactions/events, Naoxintong Capsules+statins had the lo-west incidence. In conclusion, in Chinese patent medicines containing Hirudo for the treatment of AS, Tongxinluo Capsules+statins, Maixuekang Capsules, Maixuekang Capsules+statins, and Naoxintong Capsules+statins were the primary choices to reduce IMT, AS Crouse scores, plaque area, and plaque number, respectively. The efficacy of Chinese patent medicines containing Hirudo with or without statins was more significant than that of statins alone in the four outcome indexes. Additionally, the treatment of AS should be evaluated comprehensively, and attention should be paid to Chinese patent medicines or their combination with western medicine, to optimize the treatment effect and minimize adverse reactions as the benchmark.

The "local bend response" of the medicinal leech (Hirudo verbana) is a stimulus-response pathway that enables the animal to bend away from a pressure stimulus applied anywhere along its body. The neuronal circuitry that supports this behavior has been well described, and its responses to individual stimuli are understood in quantitative detail. We probed the local bend system with pairs of electrical stimuli to sensory neurons that could not logically be interpreted as a single touch to the body wall and used multiple suction electrodes to record simultaneously the responses in large numbers of motor neurons. In all cases, responses lasted much longer than the stimuli that triggered them, implying the presence of some form of positive feedback loop to sustain the response. When stimuli were delivered simultaneously, the resulting motor neuron output could be described as an evenly weighted linear combination of the responses to the constituent stimuli. However, when stimuli were delivered sequentially, the second stimulus had greater impact on the motor neuron output, implying that the positive feedback in the system is not strong enough to render it immune to further input. [ABSTRACT FROM AUTHOR]

Medicinal leeches are predatory annelids that exhibit countershading and reside in aquatic environments where light levels might be variable. They also leave the water and must contend with terrestrial environments. Yet, leeches generally maintain a dorsal upward position despite lacking statocysts. Leeches respond visually to both green and near-ultraviolet (UV) light. I used LEDs to test the hypothesis that ventral, but not dorsal UV would evoke compensatory movements to orient the body. Untethered leeches were tested using LEDs emitting at red (632 nm), green (513 nm), blue (455 nm) and UV (372 nm). UV light evoked responses in 100 % of trials and the leeches often rotated the ventral surface away from it. Visible light evoked no or modest responses (12-15 % of trials) and no body rotation. Electrophysiological recordings showed that ventral sensilla responded best to UV, dorsal sensilla to green. Additionally, a higher order interneuron that is engaged in a variety of parallel networks responded vigorously to UV presented ventrally, and both the visible and UV responses exhibited pronounced light adaptation. These results strongly support the suggestion that a dorsal light reflex in the leech uses spectral comparisons across the dorsal-ventral axis rather than, or in addition to, luminance. [ABSTRACT FROM AUTHOR]

Leeches have four mechanosensory pressure neurons (P cells) in each midbody ganglion. Within a ganglion, P cells show complex electrical and chemical connections that vary between species. In Hirudo verbana, stimulating one P cell causes a weak depolarization followed by a strong hyperpolarization in the other P cells; however, stimulating a P cell in Erpobdella obscura produces strong depolarizations in the other P cells. In this study, we examined interactions between P cells in the American medicinal leech Macrobdella decora. Not only is Macrobdella more closely related to Hirudo than to Erpobdella, but Hirudo and Macrobdella also have very similar behavioral responses to mechanical stimulation. Despite the phylogenetic relationship and behavioral similarities between the two species, we found that intracellular stimulation of one P cell in Macrobdella causes a depolarization in the other P cells, rather than the hyperpolarization seen in Hirudo. Experiments performed in a high Mg, 0 Ca saline solution and a high Mg, high Ca saline solution suggest that the P cells in Macrobdella have a monosynaptic excitatory connection, a polysynaptic inhibitory connection, and a weak electrical coupling, similar to the connections between P cells in Hirudo. The difference in net response of P cells between these two species seems to be based on differences in the strengths of the chemical connections. These results demonstrate that even when behavioral patterns are conserved in closely related species, the underlying neural circuitry is not necessarily tightly constrained. [ABSTRACT FROM AUTHOR]