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Background: Accidental dural puncture (ADP) is the most frequent major complication when performing an epidural procedure in obstetrics. Consequently, loss of pressure in the cerebrospinal fluid (CSF) leads to the development of post-dural puncture headache (PDPH), which occurs in 16%-86% of cases. To date, the efficacy of epidural fibrin patches (EFP) has not been evaluated in a controlled clinical trial, nor in comparative studies with epidural blood patches (EBP)., Methods: The objective of the present study was to compare the efficacy of EFP with respect to EBP for the treatment of refractory accidental PDPH. This prospective, randomized, open-label, parallel, comparative study included 70 puerperal women who received an EBP or EFP (35 in each group) after failure of the conventional analgesic treatment for accidental PDPH in a hospital., Results: A higher percentage of women with EFP than EBP achieved complete PDPH relief after 2 (97.1% vs. 54.3%) and 12 h (100.0% vs. 65.7%) of the patch injection. The percentage of patients who needed rescue analgesia was significantly lower with EFP after 2 (2.9% vs. 48.6%) and 12 h (0.0% vs. 37.1%). After 24 h, PDPH was resolved in all women who received EFP. The recurrence of PDPH was reported in one woman from the EBP group (2.9%), who subsequently required a second patch. The mean length of hospital stay was significantly lower with EFP (3.9 days) than EBP (5.9 days). Regarding satisfaction, the mean value (Likert scale) was significantly higher with EFP (4.7 vs. 3.0)., Conclusions: EFP provided better outcomes than EBP for the treatment of obstetric PDPH in terms of efficacy, safety, and patient satisfaction., (© 2023 The Authors. Pain Practice published by Wiley Periodicals LLC on behalf of World Institute of Pain.)

Four new nitrogen-containing heterocyclic derivatives (acridine, quinoline, indole, pyridine) were synthesized and their biological properties were evaluated. The compounds showed affinity for DNA and HSA, with CAIC and CAAC displaying higher binding constants (Kb) of 9.54 × 10 4 and 1.06 × 10 6 , respectively. The fluorescence quenching assay (Ksv) revealed suppression values ranging from 0.34 to 0.64 × 10 3  M -1 for ethidium bromide (EB) and 0.1 to 0.34 × 10 3  M -1 for acridine orange (AO). Molecular docking confirmed the competition of the derivatives with intercalation probes at the same binding site. At 10 μM concentrations, the derivatives inhibited topoisomerase IIα activity. In the antiproliferative assays, the compounds demonstrated activity against MCF-7 and T47-D tumor cells and nonhemolytic profile. Regarding toxicity, no acute effects were observed in the embryos. However, some compounds caused enzymatic and cardiac changes, particularly the CAIC, which increased SOD activity and altered heart rate compared to the control. These findings suggest potential antitumor action of the derivatives and indicate that substituting the acridine core with different cores does not interfere with their interaction and topoisomerase inhibition. Further investigations are required to assess possible toxicological effects, including reactive oxygen species generation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

We previously described the participation of canonical phospholipase D isoforms (PLD1 and PLD2) in the inflammatory response of retinal pigment epithelium (RPE) cells exposed to high glucose concentrations (HG). Here, we studied the role of the PLD pathway in RPE phagocytic function. For this purpose, ARPE-19 cells were exposed to HG (33 mM) or to normal glucose concentration (NG, 5.5 mM) and phagocytosis was measured using pHrodo™ green bioparticles ® or photoreceptor outer segments (POS). HG exposure for 48 and 72 h reduced phagocytic function of ARPE-19 cells, and this loss of function was prevented when cells were treated with 5 μM of PLD1 (VU0359595 or PLD1i) or PLD2 (VU0285655-1 or PLD2i) selective inhibitors. Furthermore, PLD1i and PLD2i did not affect RPE phagocytosis under physiological conditions and prevented oxidative stress induced by HG. In addition, we demonstrated PLD1 and PLD2 expression in ABC cells, a novel human RPE cell line. Under physiological conditions, PLD1i and PLD2i did not affect ABC cell viability, and partial silencing of both PLDs did not affect ABC cell POS phagocytosis. In conclusion, PLD1i and PLD2i prevent the loss of phagocytic function of RPE cells exposed to HG without affecting RPE function or viability under non-inflammatory conditions., Competing Interests: The authors declare no conflict of interest.

Background/aim: Studies with acridine compounds have reported anticancer effects. Herein, we evaluated the toxicity and antitumor effect of the (E)-1'-((4-chlorobenzylidene)amino)-5'-oxo-1',5'-dihydro-10H-spiro[acridine-9,2'-pyrrole]-4'-carbonitrile (AMTAC-06), a promising anticancer spiro-acridine compound., Materials and Methods: The toxicity of AMTAC-06 was evaluated on zebrafish and mice. Antitumor activity was assessed in Ehrlich ascites carcinoma model. Effects on angiogenesis, cytokine levels and cell cycle were also investigated., Results: AMTAC-06 did not induce toxicity on zebrafish and mice (LD 50 approximately 5000 mg/kg, intraperitoneally). No genotoxicity was observed on micronucleus assay. AMTAC-06 significantly reduced the total viable Ehrlich tumor cells and increased sub-G 1 peak, suggesting apoptosis was triggered. Moreover, the compound significantly decreased the density of peritumoral microvessels, indicating an anti-angiogenic action, possibly dependent on the cytokine modulation (TNF-α, IL-1β and IFN-γ). No significant toxicological effects were recorded for AMTAC-06 on tumor transplanted animals., Conclusion: AMTAC-06 has low toxicity and a significant antitumor activity., (Copyright© 2020, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Intermediate filaments (IFs) contribute to force transmission, cellular integrity, and signaling in skeletal muscle. We previously identified keratin 19 (Krt19) as a muscle IF protein. We now report the presence of a second type I muscle keratin, Krt18. Krt18 mRNA levels are about half those for Krt19 and only 1:1,000th those for desmin; the protein was nevertheless detectable in immunoblots. Muscle function, measured by maximal isometric force in vivo, was moderately compromised in Krt18 -knockout ( Krt18 -KO) or dominant-negative mutant mice ( Krt18 DN), but structure was unaltered. Exogenous Krt18, introduced by electroporation, was localized in a reticulum around the contractile apparatus in wild-type muscle and to a lesser extent in muscle lacking Krt19 or desmin or both proteins. Exogenous Krt19, which was either reticular or aggregated in controls, became reticular more frequently in Krt19-null than in Krt18-null, desmin-null, or double-null muscles. Desmin was assembled into the reticulum normally in all genotypes. Notably, all three IF proteins appeared in overlapping reticular structures. We assessed the effect of Krt18 on susceptibility to injury in vivo by electroporating siRNA into tibialis anterior (TA) muscles of control and Krt19-KO mice and testing 2 wk later. Results showed a 33% strength deficit (reduction in maximal torque after injury) compared with siRNA-treated controls. Conversely, electroporation of siRNA to Krt19 into Krt18 -null TA yielded a strength deficit of 18% after injury compared with controls. Our results suggest that Krt18 plays a complementary role to Krt19 in skeletal muscle in both assembling keratin-based filaments and transducing contractile force.

Purpose: The aims of our study were to determine first circadian influences on central concentrations of the neuropeptides oxytocin and arginine-vasopressin and second to investigate if these central concentrations are associated with those in the peripheral compartments blood and saliva in neurocritical care patients. We therefore included patients with external ventricular drain who attended a neurosurgical intensive care unit and were not exposed to painful or stressful stimuli during the sampling period. For this purpose, blood, cerebrospinal fluid and saliva were collected in a 24-hour-interval at the timepoints 06:00, 12:00, 18:00 and 24:00., Results: In none of the three body fluids examined, significant time-dependent fluctuations of oxytocin and arginine-vasopressin concentrations could be detected during the 24-hour sampling period. The only exception was the subgroup of postmenopausal women whose oxytocin concentrations in cerebrospinal fluid at 12:00 were significantly higher than at 18:00. Correlations of blood and cerebrospinal fluid and blood and saliva neuropeptide levels were very weak to weak at each timepoint. Cerebrospinal fluid and saliva oxytocin levels showed a moderate correlation at 06:00 but did correlate very weak at the other timepoints., Conclusions: Central as well as peripheral oxytocin and arginine-vasopressin concentrations in neurocritical care patients did not show significant diurnal fluctuations. No strong correlations between central and peripheral neuropeptide concentrations could be detected under basal conditions. If investigators even though decide to use saliva concentrations as surrogate parameter for central neuropeptide activity, they have to consider that correlations of cerebrospinal fluid and saliva oxytocin seem to be highest in the early morning., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Key Points: Dysferlin, the protein missing in limb girdle muscular dystrophy 2B and Miyoshi myopathy, concentrates in transverse tubules of skeletal muscle, where it stabilizes voltage-induced Ca 2+ transients against loss after osmotic shock injury (OSI). Local expression of dysferlin in dysferlin-null myofibres increases transient amplitude to control levels and protects them from loss after OSI. Inhibitors of ryanodine receptors (RyR1) and L-type Ca 2+ channels protect voltage-induced Ca 2+ transients from loss; thus both proteins play a role in injury in dysferlin's absence. Effects of Ca 2+ -free medium and S107, which inhibits SR Ca 2+ leak, suggest the SR as the primary source of Ca 2+ responsible for the loss of the Ca 2+ transient upon injury. Ca 2+ waves were induced by OSI and suppressed by exogenous dysferlin. We conclude that dysferlin prevents injury-induced SR Ca 2+ leak., Abstract: Dysferlin concentrates in the transverse tubules of skeletal muscle and stabilizes Ca 2+ transients when muscle fibres are subjected to osmotic shock injury (OSI). We show here that voltage-induced Ca 2+ transients elicited in dysferlin-null A/J myofibres were smaller than control A/WySnJ fibres. Regional expression of Venus-dysferlin chimeras in A/J fibres restored the full amplitude of the Ca 2+ transients and protected against OSI. We also show that drugs that target ryanodine receptors (RyR1: dantrolene, tetracaine, S107) and L-type Ca 2+ channels (LTCCs: nifedipine, verapamil, diltiazem) prevented the decrease in Ca 2+ transients in A/J fibres following OSI. Diltiazem specifically increased transients by ∼20% in uninjured A/J fibres, restoring them to control values. The fact that both RyR1s and LTCCs were involved in OSI-induced damage suggests that damage is mediated by increased Ca 2+ leak from the sarcoplasmic reticulum (SR) through the RyR1. Congruent with this, injured A/J fibres produced Ca 2+ sparks and Ca 2+ waves. S107 (a stabilizer of RyR1-FK506 binding protein coupling that reduces Ca 2+ leak) or local expression of Venus-dysferlin prevented OSI-induced Ca 2+ waves. Our data suggest that dysferlin modulates SR Ca 2+ release in skeletal muscle, and that in its absence OSI causes increased RyR1-mediated Ca 2+ leak from the SR into the cytoplasm., (© 2017 The Authors. The Journal of Physiology © 2017 The Physiological Society.)

The increasing relevance of Information and Communication Technologies (ICTs) in medical care is indisputable. This evidence makes it necessary to start studies that analyse the scope these new forms of access to information and understanding of medicine have on the professional activity of the physician, on the attitude and on the knowledge of patients or, on the doctor-patient relationship. The purpose of this study is to explore some of these aspects in a group of physicians whose clinical activity is related to one of the greatest social impact health problems which is the treatment of chronic pain. Starting with the completion of a questionnaire, in the study group it is observed that the interaction between social structure, increase of information flows and ICTs generate transformations in social practices and behaviour of the actors of the health system. Internet is confirmed as an information space on the subject, but is shown as an underutilized space of interaction between the doctor and his patient.

Dysferlinopathies, most commonly limb girdle muscular dystrophy 2B and Miyoshi myopathy, are degenerative myopathies caused by mutations in the DYSF gene encoding the protein dysferlin. Studies of dysferlin have focused on its role in the repair of the sarcolemma of skeletal muscle, but dysferlin's association with calcium (Ca(2+)) signaling proteins in the transverse (t-) tubules suggests additional roles. Here, we reveal that dysferlin is enriched in the t-tubule membrane of mature skeletal muscle fibers. Following experimental membrane stress in vitro, dysferlin-deficient muscle fibers undergo extensive functional and structural disruption of the t-tubules that is ameliorated by reducing external [Ca(2+)] or blocking L-type Ca(2+) channels with diltiazem. Furthermore, we demonstrate that diltiazem treatment of dysferlin-deficient mice significantly reduces eccentric contraction-induced t-tubule damage, inflammation, and necrosis, which resulted in a concomitant increase in postinjury functional recovery. Our discovery of dysferlin as a t-tubule protein that stabilizes stress-induced Ca(2+) signaling offers a therapeutic avenue for limb girdle muscular dystrophy 2B and Miyoshi myopathy patients.

The aim of this pioneer study is to begin to create and validate a scale to measure Quality of Life in cancer patients in Puerto Rico (ECVCA-PR) in order to provide local health professionals with a reliable instrument that help to measure attitudes that could affect different patient's quality of life aspects and allows knowing the needs of those cancer patients. Sample consisted of 32 patients (9 men, 23 women), between ages of 30 to 83 years that were receiving services (i.e., hospitalization, treatment, and follow up) at Dr. Isaac González Martínez Oncological Hospital in San Juan, Puerto Rico. The psychometric properties of the instrument indicate a reliability index (Cronbach's Alpha) of 0.927 with 164 items, an excellent index according to the literature.

Fibulins are evolutionarily conserved extracellular matrix (ECM) proteins that assemble in elastic fibers and basement membranes. Caenorhabditis elegans has a single fibulin gene that produces orthologs of vertebrate fibulin-1 C and D splice forms. In a structure-function analysis of fibulin-1 domains, a series of deletion constructs show that EGF repeats 4 and 5 are required for the hemicentin-dependent assembly and function of fibulin-1D in native locations. In contrast, constructs missing the second EGF repeat of fibulin-1D (EGF2D) assemble in ectopic locations in a hemicentin dependent manner. Constructs that contain EGF2D are cleaved into two fragments, but constructs with EGF2D missing are not, suggesting that a protease binds and/or cleaves fibulin-1D at a site that is likely within EGF2D. Together, the data suggests that EGF repeats 4 and 5 promote interaction with hemicentin while a region within EGF2D suppresses ectopic interactions with hemicentin and this suppression may be protease dependent., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Purpose: To elucidate whether a dose of 2 g cefoxitin as a prophylactic agent in patients undergoing elective colorectal surgery is able to maintain free drug concentrations above the minimum inhibitory concentration of the microorganisms involved in surgical site infection., Methods: This was a prospective study involving 56 patients electively undergoing rectal or colon surgery. All plasma concentration-time data were analyzed simultaneously using the population approach to estimate population pharmacokinetic parameters and study the influence of the subjects' demographic characteristics, disease status, surgical procedure, and clinical laboratory values on the pharmacokinetic properties of cefoxitin., Results: A one-compartment open model was chosen to describe plasma concentrations of cefoxitin. Since cefoxitin is eliminated almost entirely via the kidney, creatinine clearance was identified as a covariate of cefoxitin clearance. The relationship between total cefoxitin clearance (CL) and creatinine clearance (CL(CR)) was best described using a nonlinear model [CL = 11.5 × (CL(CR)/77)(0.52)]. The population apparent volume of distribution was 12 L. Computer simulations carried out to determine the probability to maintain free plasma concentrations above 8 mg/L (the concentration threshold for susceptible bacteria) 2 h after drug administration revealed that this probability decreased from 84% in patients with a CL(CR) of 40 mL/min to 28% in patients with a CL(CR) of 100 mL/min., Conclusions: To ensure cefoxitin target concentrations during surgery, we recommend that cefoxitin be administered every 1.5 h in patients with a CL(CR) ≥ 60 mL/min and every hour if the CL(CR) is ≥ 100 mL/min. Administration by continuous infusion preceded by a bolus injection should also be considered.

Intermediate filaments, composed of desmin and of keratins, play important roles in linking contractile elements to each other and to the sarcolemma in striated muscle. Our previous results show that the tibialis anterior (TA) muscles of mice lacking keratin 19 (K19) lose costameres, accumulate mitochondria under the sarcolemma, and generate lower specific tension than controls. Here we compare the physiology and morphology of TA muscles of mice lacking K19 with muscles lacking desmin or both proteins [double knockout (DKO)]. K19-/- mice and DKO mice showed a threefold increase in the levels of creatine kinase (CK) in the serum. The absence of desmin caused a larger change in specific tension (-40%) than the absence of K19 (-19%) and played the predominant role in contractile function (-40%) and decreased tolerance to exercise in the DKO muscle. By contrast, the absence of both proteins was required to obtain a significantly greater loss of contractile torque after injury (-48%) compared with wild type (-39%), as well as near-complete disruption of costameres. The DKO muscle also showed a significantly greater misalignment of myofibrils than either mutant alone. In contrast, large subsarcolemmal gaps and extensive accumulation of mitochondria were only seen in K19-null TA muscles, and the absence of both K19 and desmin yielded milder phenotypes. Our results suggest that keratin filaments containing K19- and desmin-based intermediate filaments can play independent, complementary, or antagonistic roles in the physiology and morphology of fast-twitch skeletal muscle.

Background: Standard nonoperative therapy for acute muscle strains usually involves short-term rest, ice, and nonsteroidal anti-inflammatory medications, but there is no clear consensus on how to accelerate recovery., Hypothesis: Local delivery of platelet-rich plasma to injured muscles hastens recovery of function., Study Design: Controlled laboratory study., Methods: In vivo, the tibialis anterior muscles of anesthetized Sprague-Dawley rats were injured by a single (large strain) lengthening contraction or multiple (small strain) lengthening contractions, both of which resulted in a significant injury. The tibialis anterior either was injected with platelet-rich plasma, was injected with platelet-poor plasma as a sham treatment, or received no treatment., Results: Both injury protocols yielded a similar loss of force. The platelet-rich plasma only had a beneficial effect at 1 time point after the single contraction injury protocol. However, platelet-rich plasma had a beneficial effect at 2 time points after the multiple contraction injury protocol and resulted in a faster recovery time to full contractile function. The sham injections had no effect compared with no treatment., Conclusion: Local delivery of platelet-rich plasma can shorten recovery time after a muscle strain injury in a small-animal model. Recovery of muscle from the high-repetition protocol has already been shown to require myogenesis, whereas recovery from a single strain does not. This difference in mechanism of recovery may explain why platelet-rich plasma was more effective in the high-repetition protocol, because platelet-rich plasma is rich in growth factors that can stimulate myogenesis., Clinical Relevance: Because autologous blood products are safe, platelet-rich plasma may be a useful product in clinical treatment of muscle injuries.

Objectives: To determine the utility of the nursing discharge summary and the degree of satisfaction with the information received by the relatives of infants' after surgery., Method: We conducted an observational, descriptive, cross-sectional study in the infants unit at the Juan Canalejo University Hospital Complex in La Coruña (Spain). A total of 110 patients were included, all aged between 1 month and 2 years, who were hospitalized between January and December 2006. A telephone survey was performed among the relatives of selected infants, using an adapted questionnaire consisting of eleven questions related to the aim of this study., Results: Eighty-five families were surveyed. The nursing discharge summary was given to 98.8% of the families. During hospitalization, 91.8% of the parents received information about the care to be continued at home. The relatives surveyed gave nursing explanations a mean score of 4.6 points (SD = 0.61) (1 "bad", 5 "excellent"); 96.5% considered the contents of the nursing discharge summary to be adequate, and 97.6% found the information easy to understand. A total of 17.6% resorted to the primary health care center to continue with care and 7.1% to clarify doubts. The mean score for overall satisfaction with the summary was 4.67 points (SD = 0.54) out of a possible maximum of 5 points., Conclusions: The nursing discharge summary used in the infants' unit provides the relatives and guardians of infants with sufficient information that is easy to understand and matches their needs. We highlight the high degree of satisfaction with the summary.

Hemicentins are conserved extracellular matrix proteins discovered in Caenorhabditis elegans, with orthologs in all vertebrate species including human and mouse. Hemicentins share a single, highly conserved amino-terminal von Willebrand A domain, followed by a long (>40) stretch of immunoglobulin repeats, multiple tandem epidermal growth factors and a fibulin-like carboxy-terminal module. C. elegans has a single hemicentin gene that has pleiotropic functions in transient cell contacts that are required for cell migration and basement membrane invasion and in stable contacts at hemidesmosome-mediated cell junctions and elastic fiber-like structures. Here, we summarize what is known about the function of hemicentin in C. elegans and discuss implications for hemicentin function in other species.

Fibulin-1C and fibulin-1D splice variants have been conserved throughout metazoan evolution and have distinct functions in Caenorhabditis elegans development. Both splice variants are required for the assembly of hemidesmosome-mediated mechanosensory neuron and uterine attachments, although the molecular associations that underlie their distinct functions at these locations are not known. Here, we show that the assembly of fibulin-1C and fibulin-1D splice variants at these anchorages is dependent upon distinct components of the extracellular matrix (ECM): Fibulin-1D assembly at uterine and mechanosensory neurons attachments is dependent upon a perlecan/ UNC-52 splice variant that includes alternately spliced IG8-IG10, whereas the assembly of fibulin-1C at mechanosensory neuron attachments is dependent upon laminin/ EPI-1. These data not only indicate that fibulin-1C and fibulin-1D are components of distinct networks of ECM but also demonstrates a novel function for a major class of perlecan splice variants found in C. elegans and mouse. In addition, we demonstrate that overexpression of another ECM protein, collagen XVIII, can suppress gonad morphogenesis defects associated with loss of fibulin-1C, suggesting that some genetic defects that result in a weakened basement membrane can be compensated by overexpression of genes for ECM components that stabilize basement membranes., ((c) 2006 Wiley-Liss, Inc.)

Hemicentins are conserved extracellular matrix proteins characterized by a single von Willebrand A (VWA) domain at the amino terminus, a long stretch (>40) of tandem immunoglobulin domains, multiple tandem epidermal growth factors (EGFs), and a single fibulin-like carboxyl-terminal module. In Caenorhabditis elegans, hemicentin is secreted from muscle and gonadal leader cells and assembles at multiple locations into discrete tracks that constrict broad regions of cell contact into adhesive and flexible line-shaped junctions. To determine hemicentin domains critical for function and assembly, we have expressed fragments of hemicentin as GFP tagged fusion proteins in C. elegans. We find that a hemicentin fragment containing the VWA domain can target to multiple assembly sites when expressed under the control of either endogenous hemicentin regulatory sequences or the muscle-specific unc-54 promoter. A hemicentin fragment containing the EGF and fibulin-like carboxyl-terminal modules can co-assemble with existing hemicentin polymers in wild-type animals but has no detectable function in the absence of endogenous hemicentin. The data suggest that the VWA domain is a cell binding domain whose function is to target hemicentin to sites of assembly and the EGF/fibulin-like carboxyl-terminal modules constitute an assembly domain that mediates direct interactions between hemicentin monomers during the hemicentin assembly process.

Fibulins are a family of extracellular glycoproteins associated with basement membranes and elastic fibers in vertebrates. Conservation of the fibulin-1 gene throughout metazoan evolution includes fibulin-1C and fibulin-1D alternate splice variants, although little is known about variant specific functions that would justify this striking structural conservation. We have therefore investigated the structure, localization and loss-of-function phenotype specific to both fibulin-1 variants in C. elegans. We find that fibulin-1C has specific roles during pharynx, intestine, gonad and muscle morphogenesis, being required to regulate cell shape and adhesion, whereas fibulin-1D assembles in flexible polymers that connect the pharynx and body-wall-muscle basement membranes. The assembly of fibulin-1C and fibulin-1D in multiple locations is dependent upon the presence of hemicentin, a recently described extracellular member of the immunoglobulin superfamily. We suggest that the distinct developmental roles and hemicentin-dependent assembly for fibulin-1 splice variants demonstrated here may be relevant to fibulin-1 and possibly other fibulin family members in non-nematode species.

Several recent outbreaks of toxoplasmosis were related to drinking water. We propose a strategy for Toxoplasma oocyst detection as part of an approach to detecting multiple waterborne parasites, including Giardia and Cryptosporidium spp., by the U.S. Environmental Protection Agency method with the same sample. Water samples are filtered to recover Toxoplasma oocysts and purified on a sucrose density gradient. Detection is based on PCR and mouse inoculation (bioassay) to determine the presence and infectivity of recovered oocysts. In an experimental seeding assay with 100 liters of deionized water, a parasite density of 1 oocyst/liter was successfully detected by PCR in 60% of cases and a density of 10 oocysts/liter was detected in 100% of cases. The sensitivity of the PCR assay varied from less than 10 to more than 1000 oocysts/liter, depending on the sample source. PCR was always more sensitive than mouse inoculation. This detection strategy was then applied to 139 environmental water samples collected over a 20-month period. Fifty-three samples contained PCR inhibitors, which were overcome in 39 cases by bovine serum albumin addition. Among 125 interpretable samples, we detected Toxoplasma DNA in 10 cases (8%). None of the samples were positive by mouse inoculation. This strategy efficiently detects Toxoplasma oocysts in water and may be suitable as a public health sentinel method.

The cuticle of the nematode Caenorhabditis elegans is a collagenous extracellular matrix which forms the exoskeleton and defines the shape of the worm. We have characterized the C. elegans gene M142.2, and we show that this is a developmentally regulated gene important for cuticle structure. Transgenic worms expressing M142.2 promoter fused to green fluorescent protein showed that M142.2 is expressed in late embryos and L2d predauers, in the hypodermal cells which synthesize the cuticle. The same temporal pattern was seen by RT-PCR using RNA purified from specific developmental stages. A recombinant fragment of M142.2 was expressed in Escherichia coli and used to raise an antiserum. Immunohistochemistry using the antiserum localized M142.2 to the periphery of the alae of L1 and dauers, forming two longitudinal ribbons over the hypodermal cells. Loss-of-function of M142.2 by RNAi resulted in a novel phenotype: dumpy dauers which lacked alae. M142.2 therefore plays a major role in the assembly of the alae and the morphology of the dauer cuticle; because of its similarity to the other cut genes of the cuticle, we have named the gene cut-6.

A ubiquitous feature of collagens is protein interaction, the trimerization of monomers to form a triple helix followed by higher order interactions during the formation of the mature extracellular matrix. The Caenorhabditis elegans cuticle is a complex extracellular matrix consisting predominantly of cuticle collagens, which are encoded by a family of approximately 154 genes. We identify two discrete interacting sets of collagens and show that they form functionally distinct matrix substructures. We show that mutation in or RNA-mediated interference of a gene encoding a collagen belonging to one interacting set affects the assembly of other members of that set, but not those belonging to the other set. During cuticle synthesis, the collagen genes are expressed in a distinct temporal series, which we hypothesize exists to facilitate partner finding and the formation of appropriate interactions between encoded collagens. Consistent with this hypothesis, we find for the two identified interacting sets that the individual members of each set are temporally coexpressed, whereas the two sets are expressed approximately 2 h apart during matrix synthesis.