Excretory Canal Research Articles

MRCK-1 activates non-muscle myosin for outgrowth of a unicellular tube in Caenorhabditis elegans.

The formation and patterning of unicellular biological tubes is essential for metazoan development. It is well established that vascular tubes and neurons use similar guidance cues to direct their development, but the downstream mechanisms that promote the outgrowth of biological tubes are not well characterized. We show that the conserved kinase MRCK-1 and its substrate the regulatory light chain of non-muscle myosin, MLC-4, are required for outgrowth of the unicellular excretory canal in C. elegans. Ablation of MRCK-1 or MLC-4 in the canal causes severe truncations with unlumenized projections of the basal membrane. Structure-function analysis of MRCK-1 indicates that the kinase domain, but not the small GTPase-binding CRIB domain, is required for canal outgrowth. Expression of a phosphomimetic form of MLC-4 rescues canal truncations in mrck-1 mutants and shows enrichment at the growing canal tip. Moreover, our work reveals a novel function for non-muscle myosin downstream of MRCK-1 in excretory canal outgrowth that may be conserved in the development of seamless tubes in other organisms.

Caenorhabditis elegans SEL-5/AAK1 regulates cell migration and cell outgrowth independently of its kinase activity.

During Caenorhabditis elegans development, multiple cells migrate long distances or extend processes to reach their final position and/or attain proper shape. The Wnt signalling pathway stands out as one of the major coordinators of cell migration or cell outgrowth along the anterior-posterior body axis. The outcome of Wnt signalling is fine-tuned by various mechanisms including endocytosis. In this study, we show that SEL-5, the C. elegans orthologue of mammalian AP2-associated kinase AAK1, acts together with the retromer complex as a positive regulator of EGL-20/Wnt signalling during the migration of QL neuroblast daughter cells. At the same time, SEL-5 in cooperation with the retromer complex is also required during excretory canal cell outgrowth. Importantly, SEL-5 kinase activity is not required for its role in neuronal migration or excretory cell outgrowth, and neither of these processes is dependent on DPY-23/AP2M1 phosphorylation. We further establish that the Wnt proteins CWN-1 and CWN-2, together with the Frizzled receptor CFZ-2, positively regulate excretory cell outgrowth, while LIN-44/Wnt and LIN-17/Frizzled together generate a stop signal inhibiting its extension.

A lineage-resolved cartography of microRNA promoter activity in C. elegans empowers multidimensional developmental analysis

Elucidating the expression of microRNAs in developing single cells is critical for functional discovery. Here, we construct scCAMERA (single-cell cartography of microRNA expression based on reporter assay), utilizing promoter-driven fluorescent reporters in conjunction with imaging and lineage tracing. The cartography delineates the transcriptional activity of 54 conserved microRNAs in lineage-resolved single cells throughout C. elegans embryogenesis. The combinatorial expression of microRNAs partitions cells into fine clusters reflecting their function and anatomy. Notably, the expression of individual microRNAs exhibits high cell specificity and divergence among family members. Guided by cellular expression patterns, we identify developmental functions of specific microRNAs, including miR-1 in pharynx development and physiology, miR-232 in excretory canal morphogenesis by repressing NHR-25/NR5A, and a functional synergy between miR-232 and miR-234 in canal development, demonstrating the broad utility of scCAMERA. Furthermore, integrative analysis reveals that tissue-specific fate determinants activate microRNAs to repress protein production from leaky transcripts associated with alternative, especially neuronal, fates, thereby enhancing the fidelity of developmental fate differentiation. Collectively, our study offers rich opportunities for multidimensional expression-informed analysis of microRNA biology in metazoans.

Chloride intracellular channel (CLIC) proteins function as fusogens

Chloride Intracellular Channel (CLIC) family members uniquely transition between soluble and membrane-associated conformations. Despite decades of extensive functional and structural studies, CLICs’ function as ion channels remains debated, rendering our understanding of their physiological role incomplete. Here, we expose the function of CLIC5 as a fusogen. We demonstrate that purified CLIC5 directly interacts with the membrane and induces fusion, as reflected by increased liposomal diameter and lipid and content mixing between liposomes. Moreover, we show that this activity is facilitated by acidic pH, a known trigger for CLICs’ transition to a membrane-associated conformation, and that increased exposure of the hydrophobic inter-domain interface is crucial for this process. Finally, mutation of a conserved hydrophobic interfacial residue diminishes the fusogenic activity of CLIC5 in vitro and impairs excretory canal extension in C. elegans in vivo. Together, our results unravel the long-sought physiological role of these enigmatic proteins.

РОЛЬ ОЗОНОТЕРАПИИ В ЭФФЕКТИВНОСТИ ЛЕЧЕНИЯ ПАЦИЕНТОК С ВОСПАЛИТЕЛЬНЫМИ ЗАБОЛЕВАНИЯМИ РЕПРОДУКТИВНОЙ СИСТЕМЫ

The examination and treatment of 162 women aged 21 to 38 years with subacute and chronic inflammatory diseases of the genitals was carried out. All patients underwent a comprehensive clinical and laboratory examination, including a general clinical examination, assessment of gynecological status with ultrasound, serological examination for chlamydia, viral infections, as well as bacteriological and bacterioscopic examination of the vaginal flora and the cervical canal, as well as PCR diagnostics. The patients were prescribed specific anti-inflammatory treatment taking into account the etiology of the pathogen. As an additional treatment, ozone therapy was used in patients (intravenous infusion of ozonated saline solution of 200 ml, at a concentration of 4-6 mg / l per course – 10 procedures). After treatment, an examination was repeated, including an assessment of clinical symptoms, a laboratory study, including an examination of the vaginal flora and the excretory cervical canal, as well as PCR. Marked clinical effect of ozone therapy in all patients. After the laboratory study, a significant positive dynamic of the following indicators was revealed (a decrease in the content of low-differentiated forms of leukocytes, an increase in the percentage of lymphocytes, normalization of the protein fraction of blood serum). This indicated the relief of the acute phase of the inflammatory process of the female reproductive system. Bacteriological and bacterioscopic, as well as PCR control after the treatment showed the absence of specific and non-specific pathogens in the cervical canal and normalization of the vaginal microflora in 148 patients. In 14 patients, there were no signs of dysbiosis in the cervical canal, but there was a decrease in the content of lactobacilli, in the absence of clinical manifestations. They were additionally prescribed the use of Acylact DUO to stimulate the growth of their own lactobacilli. The inclusion of ozone therapy in the complex of therapeutic measures for inflammatory diseases of the genitals is highly effective, since it eliminates hypoxia underlying inflammation, has an anti-inflammatory, anti-stress, desensitizing and immunomodulatory effect. This allows timely treatment, prevention of chronization of the process.

Primary Submandibular Gland Tubercular Sialadenitis: A Case Report

Tuberculosis of the submandibular gland is a rare pathology, the diagnosis of which is difficult, given the non- pathognomic clinical presentation and the absence of diagnostic guidelines, the portal of entry can be by hematogenous route or through the excretory canal of the gland. We present the case of a 33-year-old patient who presented with recurrent submandibular sialadenitis without explanation. In order to make a diagnosis a submandibulectomy was performed. The histological study showed a tubercular epitheliogigantocellular granuloma; the patient was put on antitubercular drugs for 6 months with a good evolution.

An examination of nervous system revealed unexpected immunoreactivity of both secretory apparatus and excretory canals in plerocercoids of two broad tapeworms (Cestoda: Diphyllobothriidea).

Dibothriocephalus ditremus and Dibothriocephalus latus are diphyllobothriidean tapeworms autochthonous to Europe. Their larval stages (plerocercoids) may seriously alter health of their intermediate fish hosts (D. ditremus) or cause intestinal diphyllobothriosis of the final human host (D. latus). Despite numerous data on the internal structure of broad tapeworms, many aspects of the morphology and physiology related to host–parasite co-existence remain unclear for these 2 species. The main objective of this work was to elucidate functional morphology of the frontal part (scolex) of plerocercoids, which is crucial for their establishment in fish tissues and for an early attachment in final hosts. The whole-mount specimens were labelled with different antibodies and examined by confocal microscope to capture their complex 3-dimensional microanatomy. Both species exhibited similar general pattern of immunofluorescent signal, although some differences were observed. In the nervous system, FMRF amide-like immunoreactivity (IR) occurred in the bi-lobed brain, 2 main nerve cords and surrounding nerve plexuses. Differences between the species were found in the structure of the brain commissures and the size of the sensilla. Synapsin IR examined in D. ditremus occurred mainly around FMRF amide-like IR brain lobes and main cords. The unexpected finding was an occurrence of FMRF amide-like IR in terminal reservoirs of secretory gland ducts and excretory canals, which has not been observed previously in any tapeworm species. This may indicate that secretory/excretory products, which play a key role in host–parasite relationships, are likely to contain FMRF amide-related peptide/s.

Study of a piscian cestode, Probothriocephalus pathakpuraensis, n.sp. from Mastacembelus armatus from Pathakpura Orai, district Jalaun (U.P.) India

Twenty five fishes, Mastacembelus armatus were examined at Pathakpura, Orai, district Jalaun (U.P.) India. Seven alike cestodes were reported in it’s intestine. After morphological study of the worm we reached on the conclusion that present species differs from all known species of genus Probothriocephalus in the presence of internal and external seminal vesicles, testes in four bands located in partly cortical and partly medullary regions which cross the ventral longitudinal excretory canals and prominent mehlis gland.

A review of Stree sharira from classical Ayurvedic literature

Finding the proper etio-pathogenesis and treating gynaecological problems requires knowledge of stree-sharira (anatomy and physiology of the female reproductive system). The classics of Ayurveda have focussed on the practical application of knowledge and hence they discussed sharira rather than micro structural details. The name Yoni denotes the entire genital canal. Similar descriptions of the vagina are explained by modern science as a fibro-musculo-membranous sheath connecting the uterine chamber to the outside at the vulva. It contains the uterine secretory and menstrual blood excretory canal. Garbhashaya is located in the third avarta of the yoni, behind the bladder, between pittashaya and pakvashaya. Sushruta used the term plalamntargata, which is comparable to the ovaries or beeja granthi. Three doshas are in charge of the rutuchakra (menstrual cycle). The Rajasrava kala (menstrual cycle duration) can last anywhere between three and seven days. The mechanism of artava pravrutti has been clearly defined by Ayurveda. There is menstrual blood loss throughout the three to five or seven-day Rajahkala (menstrual phase), and vata dosha predominates. During this phase, the drava and sara gunas of the pitta dosha and the chala guna and kshepana karma of the vata dosha both function. Ritu-kala, or the proliferative phase, lasts for 12 or 16 days or the entire cycle. Even without menstruation, navina rajah (new cycles) are established at this time, and fertility chances are at their highest. These functions share similarities with the kapha dosha, indicating that kapha is the dominant dosha in this period.

A metazoan-specific C-terminal motif in EXC-4 and Gα-Rho/Rac signaling regulate cell outgrowth during tubulogenesis in C. elegans.

Chloride intracellular channels (CLICs) are conserved proteins for which the cellular and molecular functions remain mysterious. An important insight into CLIC function came from the discovery that Caenorhabditis elegans EXC-4/CLIC regulates morphogenesis of the excretory canal (ExCa) cell, a single-cell tube. Subsequent work showed that mammalian CLICs regulate vascular development and angiogenesis, and human CLIC1 can rescue exc-4 mutants, suggesting conserved function in biological tube formation (tubulogenesis) and maintenance. However, the cell behaviors and signaling pathways regulated by EXC-4/CLICs during tubulogenesis in vivo remain largely unknown. We report a new exc-4 mutation, affecting a C-terminal residue conserved in virtually all metazoan CLICs, that reveals a specific role for EXC-4 in ExCa outgrowth. Cell culture studies suggest a function for CLICs in heterotrimeric G protein (Gα/β/γ)-Rho/Rac signaling, and Rho-family GTPases are common regulators of cell outgrowth. Using our new exc-4 mutant, we describe a previously unknown function for Gα-encoding genes (gpa-12/Gα12/13, gpa-7/Gαi, egl-30/Gαq and gsa-1/Gαs), ced-10/Rac and mig-2/RhoG in EXC-4-mediated ExCa outgrowth. Our results demonstrate that EXC-4/CLICs are primordial players in Gα-Rho/Rac-signaling, a pathway that is crucial for tubulogenesis in C. elegans and in vascular development.

Cestode Parasite (Platyhelminthes) of Rodent Rattus rattus (Linnaeus, 1758) from Aurangabad, Maharashtra, India with a Redescription of Hymenolepis diminuta (Rudolphi, 1819)

In this study Cestodes collected from a species of rodents of the genera Rattus rattus (Linnaeus, 1758) from Aurangabad district Maharashtra, India are reported. Sixty three specimens of the cestode parasites were collected from 23 infected intestines after examining 110 host house rat (Rattus rattus) at Aurangabad, (MS), India during the period of April, 2005-March, 2007. The present form of cestode parasite is redescribed on the basis of some similar characters with the previously described species Hymenolepis diminuta (Rudolphi, 1819). The characters of present form are similar with Hymenolepis diminuta having the scolex well-marked rostellum, hooks in single crown on rostellum, oval cirrus pouch, three testes, one poral and two aporal, two pairs longitudinal excretory canal and pre-ovarian ootype but differs due to globular scolex, squarish mature proglottids and differs in size of ovary (0.526 - 0.552 x 0.114 - 0.184 mm).

Inositol polyphosphate multikinase IPMK-1 regulates development through IP3/calcium signaling in Caenorhabditis elegans

Inositol polyphosphate multikinase (IPMK) is a conserved protein that initiates the production of inositol phosphate intracellular messengers and is critical for regulating a variety of cellular processes. Here, we report that the C. elegans IPMK-1, which is homologous to the mammalian inositol polyphosphate multikinase, plays a crucial role in regulating rhythmic behavior and development. The deletion mutant ipmk-1(tm2687) displays a long defecation cycle period and retarded postembryonic growth. The expression of functional ipmk-1::GFP was detected in the pharyngeal muscles, amphid sheath cells, the intestine, excretory (canal) cells, proximal gonad, and spermatheca. The expression of IPMK-1 in the intestine was sufficient for the wild-type phenotype. The IP3-kinase activity of IPMK-1 is required for defecation rhythms and postembryonic development. The defective phenotypes of ipmk-1(tm2687) could be rescued by a loss-of-function mutation in type I inositol 5-phosphatase homolog (IPP-5) and improved by a supplemental Ca2+ in the medium. Our work demonstrates that IPMK-1 and the signaling molecule inositol triphosphate (IP3) pathway modulate rhythmic behaviors and development by dynamically regulating the concentration of intracellular Ca2+ in C. elegans. Advances in understanding the molecular regulation of Ca2+ homeostasis and regulation of organism development may lead to therapeutic strategies that modulate Ca2+ signaling to enhance function and counteract disease processes. Unraveling the physiological role of IPMK and the underlying functional mechanism in C. elegans would contribute to understanding the role of IPMK in other species, especially in mammals, and benefit further research on the involvement of IPMK in disease.

A Series of Tubes: The C. elegans Excretory Canal Cell as a Model for Tubule Development.

Formation and regulation of properly sized epithelial tubes is essential for multicellular life. The excretory canal cell of C. elegans provides a powerful model for investigating the integration of the cytoskeleton, intracellular transport, and organismal physiology to regulate the developmental processes of tube extension, lumen formation, and lumen diameter regulation in a narrow single cell. Multiple studies have provided new understanding of actin and intermediate filament cytoskeletal elements, vesicle transport, and the role of vacuolar ATPase in determining tube size. Most of the genes discovered have clear homologues in humans, with implications for understanding these processes in mammalian tissues such as Schwann cells, renal tubules, and brain vasculature. The results of several new genetic screens are described that provide a host of new targets for future studies in this informative structure.

Terminal web and vesicle trafficking proteins mediate nematode single-cell tubulogenesis.

Single-celled tubules represent a complicated structure that forms during development, requiring extension of a narrow cytoplasm surrounding a lumen exerting osmotic pressure that can burst the luminal membrane. Genetic studies on the excretory canal cell of Caenorhabditis elegans have revealed many proteins that regulate the cytoskeleton, vesicular transport, and physiology of the narrow canals. Here, we show that βH-spectrin regulates the placement of intermediate filament proteins forming a terminal web around the lumen, and that the terminal web in turn retains a highly conserved protein (EXC-9/CRIP1) that regulates apical endosomal trafficking. EXC-1/IRG, the binding partner of EXC-9, is also localized to the apical membrane and affects apical actin placement and RAB-8-mediated vesicular transport. The results suggest that an intermediate filament protein acts in a novel pathway to direct the traffic of vesicles to locations of lengthening apical surface during single-celled tubule development.

Ultrastructure of the intestinal system in unfed larvae of Limnesia maculata (O.F. Müller, 1776) (Acariformes, Limnesiidae)

The intestinal system of unfed fresh-water mite larvae Limnesia maculata (O.F. Müller, 1776) (Acariformes, Limnesiidae) has been studied with transmission electron microscopy. The intestinal system is composed of the foregut, including the pharynx and the esophagus, the sac-like blind midgut and the excretory organ. The pharynx begins with the mouth covered by the labrum. The pharynx runs along the bottom of the gnathosoma and shows ventral dilators. No valves are expressed between the pharynx and the esophagus. The esophagus possesses strongly plicate walls and, before entering the midgut, passes through the brain. The sac-like midgut does not reveal a well-pronounced lumen, developed epithelium and separate lobes. It consists of the two cell types of endoderm origin mixed in the midgut volume. The first type – the vacuolated cells – does not possess Golgi bodies and lysosomal apparatus and shows electron-lucent vacuoles with a granular inclusion inside. These cells apparently do not take part in digestion of the embryonic yolk. The second type – the non-vacuolated cells – shows both, a well developed Golgi complex and large heterolysosomes, and obviously digests the embryonic yolk. Consequently, they may be attributed as specialized vitellophages. Nevertheless, both cell types may take part in formation of the definitive midgut epithelium. The sac-like thin-walled excretory organ is strongly dilated and contains the embryonic wastes in the form of electron-dense globules and birefringent particles. No muscle envelope surrounds the excretory organ. The embryonic wastes together with wastes accumulated during feeding may be evacuated from the organ only after completion of feeding. The excretory canal on this developmental stage is not connected with the excretory organ. It opens to the outside with a simple slit-like excretory pore. Before feeding, larvae have to pass the process of the post-molt development before their midgut would be ready to receive nutrients.

Morphological Characterization of Monezia expansa Rudolphi, 1810 (F: Anoplocephalidae) isolated from the intestine of the domestic sheep, Ovis aries (Bovidae) by light microscopy

In the present study, A cestode parasite was recovered from the intestine of the domestic sheep, Ovis aries (Bovidae) from the main slaughterhouse of Cairo, Egypt during the year of 2015. The parasite was observed attached to the wall of the host intestine by unarmed scolex with suckers only. Five out of 10 gastrointestinal tracts (50%) were found to be infected. Light microscopic examination showed that the adult worm of this species was whitish in color measured 421-492 (470±0.4) cm in length and 4.2-6.99 (5.6±0.7) mm in width. The scolex was with prominent four suckers measured 0.38-0.42mm in diameter. The scolex led into a long neck followed by segments. The mature segment was broader than longer, each proglottid measured 3.23-5.29 (4.6± 0.2) mm in width and 1.0-1.64 (1.4±0.02) mm in width and showed a two set of genital organs. The ovaries and the vitelline glands formed a ring on either side, median to the longitudinal excretory canals, while the testes were distributed throughout the central field or they may be concentrated toward the sides. Each ovary was of ovoid shape, measured about 0.16-0.19 (0.14±0.01) × 0.12-0.15 (0.13±0.02) mm and was located 0.8± 0.1 mm apart from the lateral side. The testes were concentrated on both sides. At the posterior border of each proglottid, a row of interproglotidal glands was arranged around small pits. Gravid segments measured 0.022-0.076 (0.048±0.2) mm in length and 2.10-5.02 (3.05±0.02) mm in width. The present parasite was compared with the previous species of the same host which showed that the parasite isolated is Monezia expansa.

Osmoregulatory performance and immunolocalization of Na+/K+-ATPase in the branchiopod Artemia salina from the Sebkha of Sidi El Hani (Tunisia)

Artemia salina is an extremophile species that tolerates a wide range of salinity, especially hypertonic media considered lethal for the majority of other aquatic species. In this study, A. salina cysts were hatched in the laboratory and nauplii were acclimated at three different salinities (60, 139 and 212 ppt). Once in the adult phase, their hemolymph osmolality was measured. The animals were strong hypo-osmoregulators in the entire range of tested salinities, with up to 10 fold lower hemolymph osmolalities than their surrounding environment. Immunostaining of Na+/K+-ATPase was done on sections and on whole body mounts of adults in order to localize the ionocytes in different organs. An intense Na+/K+-ATPase immunostaining throughout the cells was observed in the epithelium of the ten pairs of metepipodites. A positive immunoreactivity for Na+/K+-ATPase was also detected in the maxillary glands, in the epithelium of the efferent tubule and of the excretory canal, as well as in the anterior digestive tract. This study confirms the strong hypo-osmotic capacity of this species and affords an overview of the different organs involved in osmoregulation in A. salina adults.

Intrahepatic ectopic pancreas

Ectopic pancreas (or aberrant or pancreatic heterotopia) is defined as pancreatic tissue outside the boundaries of the pancreas, without anatomic connection to the main gland. Its incidence is between 0.5% and 14% in autopsy series, but it is probably underestimated because it is often asymptomatic. It most commonly affects the duodenum (30-35%), the stomach (30%), and the jejunum (15%). Hepatic involvement is exceptional. We here report a new case of unexpected detection. The study involved a 54-year-old, hypertense female patient who had undergone surgery for adenocarcinoma of the sigmoid classified pT3N1b (A) two years before. Disease progression was marked by the occurrence of multiple predominant secondary nodules in the right hepatic lobe (B). Neoadjuvant chemotherapy (4 cicles of FOLFOX-ERBITUX) was started. The patient received right lobectomy. The histological examination of the different surgical specimens confirmed the presence of secondary, moderately differentiated hepatic adenocarcinoma of colorectal origin with healthy surgical excision margins. The surrounding liver was the site of unsystematized macrovacuolar steatosis in 20% of cases. It was associated with a focus of pancreatic heterotopia, including some acini and excretory canals arranged in lobules (C, D). The postoperative course was simple; however, patient’s outcome was marked by recurrence of cancer in the left lobe of the liver.

Analysis of the novel excretory cell expressed ECP-1 protein and its proposed ECP-1/IFC-2 fusion protein EXC-2 in the nematode Caenorhabditis elegans

The multigene family of cytoplasmic intermediate filament (IF) proteins in C. elegans covers eleven members, of which four (IFA-1 to IFA-3, IFB-1), which form an obligate heteropolymeric IF system, are essential for development. The six other C. elegans IF proteins IFB-2, IFC-1, IFC-2, IFD-1, IFD-2 and IFP-1 are co-expressed in the intestinal terminal web during different developmental stages, reveal various differently penetrant RNAi phenotypes and form another heteropolymeric IFB-2/IFCDP-1 IF system in C. elegans. Interestingly, the alternatively spliced IFC-2 variant, called EXC-2, was recently found also to be needed for a normal excretory system maturation in C. elegans. In order to better understand the IFC-2 function in the nematode tissue, we retrieved from the WormBase its multiple predicted alternatively spliced transcripts and analysed them using the molecular, immunofluorescence and RNAi approaches. We found that the 21-exon long genomic fragment encodes, besides the two different intestinal IFC-2a and IFC-2b IF proteins, also the novel excretory cell/IF unrelated protein ECP-1 and probably also the large ECP-1/IFC-2 fusion protein EXC-2, which all seem to be tissue-specific regulated from different promoters. Our analyses provide a framework for investigating interactions between the novel ECP-1, EXC-2 and some other proteins, including IFs, which show a similar excretory canal phenotype and are essential for development of the C. elegans excretory cells.

Novel exc Genes Involved in Formation of the Tubular Excretory Canals of Caenorhabditis elegans.

Regulation of luminal diameter is critical to the function of small single-celled tubes, of which the seamless tubular excretory canals of Caenorhabditis elegans provide a tractable genetic model. Mutations in several sets of genes exhibit the Exc phenotype, in which canal luminal growth is visibly altered. Here, a focused reverse genomic screen of genes highly expressed in the canals found 18 genes that significantly affect luminal outgrowth or diameter. These genes encode novel proteins as well as highly conserved proteins involved in processes including gene expression, cytoskeletal regulation, and vesicular and transmembrane transport. In addition, two genes act as suppressors on a pathway of conserved genes whose products mediate vesicle movement from early to recycling endosomes. The results provide new tools for understanding the integration of cytoplasmic structure and physiology in forming and maintaining the narrow diameter of single-cell tubules.