Tissue-dependent subcellular localization of Drosophila arginine methyl-transferase 4 (DART4), a coactivator whose overexpression affects neither viability nor differentiation

Drosophila arginine methyl-transferase 4 (DART4) belongs to the type I class of arginine methyltransferases. It catalyzes the methylation of arginine residues to monomethylarginines and asymmetrical dimethylarginines. The DART4 sequence is highly similar to mammalian PRMT4/CARM1, and DART4 substrate specificity has been conserved, too. Recently it was suggested that DART4/Carmer functions in ecdysone receptor mediated apoptosis of the polytene larval salivary glands and an apparent up-regulation of DART4/Carmer mRNA levels before tissue histolysis was reported. Here we show that in Drosophila larvae, DART4 is mainly expressed in the imaginal disks and in larval brains, and to a much lesser degree in the polytene larval tissue such as salivary glands. In glands, DART4 protein is present in the cytoplasm and the nucleus. The nuclear signal emanates from the extrachromosomal domain and gets progressively restricted to the region of the nuclear lamina upon pupariation. Surprisingly, DART4 levels do not increase in salivary glands during pupariation, and overexpression of DART4 does not cause precautious cell death in the glands. Furthermore, over- and misexpression of DART4 under the control of the alpha tubulin promoter do not lead to any major problem in the life of a fly. This suggests that DART4 activity is regulated at the posttranslational level and/or that it acts as a true cofactor in vivo. We present evidence that nuclear localization of DART4 may contribute to its function because DART4 accumulation changes from a distribution with a strong cytoplasmic component during the transcriptional quiescence of the young embryo to a predominantly nuclear one at the onset of zygotic transcription.

Major surface glycoproteins of insect forms of Trypanosoma brucei are not essential for cyclical transmission by tsetse

Procyclic forms of Trypanosoma brucei reside in the midgut of tsetse flies where they are covered by several million copies of glycosylphosphatidylinositol-anchored proteins known as procyclins. It has been proposed that procyclins protect parasites against proteases and/or participate in tropism, directing them from the midgut to the salivary glands. There are four different procyclin genes, each subject to elaborate levels of regulation. To determine if procyclins are essential for survival and transmission of T. brucei, all four genes were deleted and parasite fitness was compared in vitro and in vivo. When co-cultured in vitro, the null mutant and wild type trypanosomes (tagged with cyan fluorescent protein) maintained a near-constant equilibrium. In contrast, when flies were infected with the same mixture, the null mutant was rapidly overgrown in the midgut, reflecting a reduction in fitness in vivo. Although the null mutant is patently defective in competition with procyclin-positive parasites, on its own it can complete the life cycle and generate infectious metacyclic forms. The procyclic form of T. brucei thus differs strikingly from the bloodstream form, which does not tolerate any perturbation of its variant surface glycoprotein coat, and from other parasites such as Plasmodium berghei, which requires the circumsporozoite protein for successful transmission to a new host.

Characterizing New Fluorescent Tools for Studying 5-HT3 Receptor Pharmacology

The pharmacological characterization of ligands depends upon the ability to accurately measure their binding properties. Fluorescence provides an alternative to more traditional approaches such as radioligand binding. Here we describe the binding and spectroscopic properties of eight fluorescent 5-HT3 receptor ligands. These were tested on purified receptors, expressed receptors on live cells, or in vivo. All compounds had nanomolar affinities with fluorescent properties extending from blue to near infra-red emission. A fluorescein-derivative had the highest affinity as measured by fluorescence polarization (FP; 1.14 nM), flow cytometry (FC; 3.23 nM) and radioligand binding (RB; 1.90 nM). Competition binding with unlabeled 5-HT3 receptor agonists (5-HT, mCPBG, quipazine) and antagonists (granisetron, palonosetron, tropisetron) yielded similar affinities in all three assays. When cysteine substitutions were introduced into the 5-HT3 receptor binding site the same changes in binding affinity were seen for both granisetron and the fluorescein-derivative, suggesting that they both adopt orientations that are consistent with co-crystal structures of granisetron with a homologous protein (5HTBP). As expected, in vivo live imaging in anaesthetized mice revealed staining in the abdominal cavity in intestines, but also in salivary glands. The unexpected presence of 5-HT3 receptors in mouse salivary glands was confirmed by Western blots. Overall, these results demonstrate the wide utility of our new high-affinity fluorescently-labeled 5-HT3 receptor probes, ranging from in vitro receptor pharmacology, including FC and FP ligand competition, to live imaging of 5-HT3 expressing tissues.

Social motility of African trypanosomes is a property of a distinct life-cycle stage that occurs early in tsetse fly transmission

The protozoan pathogen Trypanosoma brucei is transmitted between mammals by tsetse flies. The first compartment colonised by trypanosomes after a blood meal is the fly midgut lumen. Trypanosomes present in the lumen-designated as early procyclic forms-express the stage-specific surface glycoproteins EP and GPEET procyclin. When the trypanosomes establish a mature infection and colonise the ectoperitrophic space, GPEET is down-regulated, and EP becomes the major surface protein of late procyclic forms. A few years ago, it was discovered that procyclic form trypanosomes exhibit social motility (SoMo) when inoculated on a semi-solid surface. We demonstrate that SoMo is a feature of early procyclic forms, and that late procyclic forms are invariably SoMo-negative. In addition, we show that, apart from GPEET, other markers are differentially expressed in these two life-cycle stages, both in culture and in tsetse flies, indicating that they have different biological properties and should be considered distinct stages of the life cycle. Differentially expressed genes include two closely related adenylate cyclases, both hexokinases and calflagins. These findings link the phenomenon of SoMo in vitro to the parasite forms found during the first 4-7 days of a midgut infection. We postulate that ordered group movement on plates reflects the migration of parasites from the midgut lumen into the ectoperitrophic space within the tsetse fly. Moreover, the process can be uncoupled from colonisation of the salivary glands. Although they are the major surface proteins of procyclic forms, EP and GPEET are not essential for SoMo, nor, as shown previously, are they required for near normal colonisation of the fly midgut.

A cysteine protease inhibitor of plasmodium berghei is essential for exo-erythrocytic development

Plasmodium parasites express a potent inhibitor of cysteine proteases (ICP) throughout their life cycle. To analyze the role of ICP in different life cycle stages, we generated a stage-specific knockout of the Plasmodium berghei ICP (PbICP). Excision of the pbicb gene occurred in infective sporozoites and resulted in impaired sporozoite invasion of hepatocytes, despite residual PbICP protein being detectable in sporozoites. The vast majority of these parasites invading a cultured hepatocyte cell line did not develop to mature liver stages, but the few that successfully developed hepatic merozoites were able to initiate a blood stage infection in mice. These blood stage parasites, now completely lacking PbICP, exhibited an attenuated phenotype but were able to infect mosquitoes and develop to the oocyst stage. However, PbICP-negative sporozoites liberated from oocysts exhibited defective motility and invaded mosquito salivary glands in low numbers. They were also unable to invade hepatocytes, confirming that control of cysteine protease activity is of critical importance for sporozoites. Importantly, transfection of PbICP-knockout parasites with a pbicp-gfp construct fully reversed these defects. Taken together, in P. berghei this inhibitor of the ICP family is essential for sporozoite motility but also appears to play a role during parasite development in hepatocytes and erythrocytes.

The Social Life of African Trypanosomes

The unicellular parasite Trypanosoma brucei shuttles between its definitive host, the tsetse fly, and various mammals including humans. In the fly digestive tract, T. brucei must first migrate to the ectoperitrophic space, establish a persistent infection of the midgut and then migrate to the salivary glands before being transmitted to a new mammalian host. In 2010, it was shown that insect stages of the parasite (procyclic forms) exhibit social motility (SoMo) when cultured on a semi-solid surface, and it was postulated that this behaviour might reflect a migration step in the tsetse fly. Now, almost 5 years after the initial report, several new publications shed some light on the biological function of SoMo and provide insights into the underlying signalling pathways.

Apocrine papillary cystadenoma of a minor salivary gland on the lower lip: case presentation

Cystadenomas are a rare, painless, and slow-growing benign epithelial tumor of the salivary gland. This article describes the case of a papillary cystadenoma in the lower lip of a 46-year-old man. The lesion was removed using a carbon dioxide laser, and there were no signs of recurrence 1 year postoperatively.

What Is a Minor Stroke?

Background and Purpose— The term “minor stroke” is often used; however a consensus definition is lacking. We explored the relationship of 6 “minor stroke” definitions and outcome and tested their validity in subgroups of patients. Methods— A total of 760 consecutive patients with acute ischemic strokes were classified according to the following definitions: A, score ≤1 on every National Institutes of Health Stroke Scale (NIHSS) item and normal consciousness; B, lacunar-like syndrome; C, motor deficits with or without sensory deficits; D, NIHSS ≤9 excluding those with aphasia, neglect, or decreased consciousness; E, NIHSS ≤9; and F, NIHSS ≤3. Short-term outcome was considered favorable when patients were discharged home, and favorable medium-term outcome was defined as a modified Rankin Scale score of ≤2 at 3 months. The following subgroup analyses were performed by definition: sex, age, anterior versus posterior and right versus left hemispheric stroke, and early (0 to 6 hours) versus late admission (6 to 24 hours) to the hospital. Results— Short-term and medium-term outcomes were most favorable in patients with definition A (74% and 90%, respectively) and F (71% and 90%, respectively). Patients with definition C and anterior circulation strokes were more likely to be discharged home than patients with posterior circulation strokes (P=0.021). The medium-term outcome of older patients with definition E was less favorable compared with the outcome of younger ones (P=0.001), whereas patients with definition A, D, and F did not show different outcomes in any subgroup. Conclusions— Patients fulfilling definition A and F had best short-term and medium-term outcomes. They would be best suited to the definition of “minor stroke.”

Primary tumors of the submandibular glands: a retrospective study based on 41 cases

We report our experience on the diagnostic approach, treatment, and follow-up of primary submandibular gland tumors. Retrospective review. Tertiary referral center. Forty-one adult patients, 22 male and 19 female, with primary submandibular gland tumors, 20 benign and 21 malignant. Age, gender, clinical findings, cyto- and histopathology, treatment and outcome were analyzed. Most tumors presented as a painless submandibular mass. Thirty three patients underwent a fine needle aspiration, the sensitivity, specificity and accuracy of which--in detecting malignant tumors--were 79%, 100% and 88%, respectively. Preoperative radiological imaging was performed in 30 cases. Patients with benign tumors were treated with surgery. Most malignant tumors were treated with a combined modality, including neck dissection and radiation therapy. Five patients developed a postoperative complication. Recurrent disease was encountered in 5 malignant tumors. The 2, 5 and 10 year disease-specific survival of patients with malignancy were 84%, 75% and 41%, respectively. The preoperative assessment of the nature of submandibular gland tumors remains challenging. Aggressive treatment of patients with malignant disease may help to avoid poor prognosis.

Effects of enamel abrasion, salivary pellicle, and measurement angle on the optical assessment of dental erosion

The present study assessed the effects of abrasion, salivary proteins, and measurement angle on the quantification of early dental erosion by the analysis of reflection intensities from enamel. Enamel from 184 caries-free human molars was used for in vitro erosion in citric acid (pH 3.6). Abrasion of the eroded enamel resulted in a 6% to 14% increase in the specular reflection intensity compared to only eroded enamel, and the reflection increase depended on the erosion degree. Nevertheless, monitoring of early erosion by reflection analysis was possible even in the abraded eroded teeth. The presence of the salivary pellicle induced up to 22% higher reflection intensities due to the smoothing of the eroded enamel by the adhered proteins. However, this measurement artifact could be significantly minimized (p<0.05) by removing the pellicle layer with 3% NaOCl solution. Change of the measurement angles from 45 to 60 deg did not improve the sensitivity of the analysis at late erosion stages. The applicability of the method for monitoring the remineralization of eroded enamel remained unclear in a demineralization/remineralization cycling model of early dental erosion in vitro.

Dental erosion and salivary flow rate in cerebral palsy individuals with gastroesophageal reflux

A high prevalence of gastroesophageal reflux (GERD) has been observed in individuals with cerebral palsy (CP). One of the main risks for dental erosion is GERD. This study aimed to evaluate the presence of GERD, variables related to dental erosion and associated with GERD (diet consumption, gastrointestinal symptoms, bruxism), and salivary flow rate, in a group of 46 non-institutionalized CP individuals aged from 3 to 13 years.

Comparison of three strip-type tests and two laboratory methods for salivary buffering analysis

This study evaluated the correlation between three strip-type, colorimetric tests and two laboratory methods with respect to the analysis of salivary buffering. The strip-type tests were saliva-check buffer, Dentobuff strip and CRT(®) Buffer test. The laboratory methods included Ericsson's laboratory method and a monotone acid/base titration to create a reference scale for the salivary titratable acidity. Additionally, defined buffer solutions were prepared and tested to simulate the carbonate, phosphate and protein buffer systems of saliva. The correlation between the methods was analysed by the Spearman's rank test. Disagreement was detected between buffering capacity values obtained with three strip-type tests that was more pronounced in case of saliva samples with medium and low buffering capacities. All strip-type tests were able to assign the hydrogencarbonate, di-hydrogenphosphate and 0.1% protein buffer solutions to the correct buffer categories. However, at 0.6% total protein concentrations, none of the test systems worked accurately. Improvements are necessary for strip-type tests because of certain disagreement with the Ericsson's laboratory method and dependence on the protein content of saliva.

Tritrichomonas foetus isolates from cats and cattle show minor genetic differences in unrelated loci ITS-2 and EF-1alpha

The protozoan parasite Tritrichomonas foetus is well known as an important causative agent of infertility and abortion in cattle (bovine trichomonosis). This World Organisation for Animal Health (O.I.E.) notifiable disease is thought to be under control in many countries including Switzerland. In recent studies, however, T. foetus has also been identified as an intestinal parasite that causes chronic large-bowel diarrhoea in cats. Since the feline isolates were considered indistinguishable from bovine isolates, the possibility and risk of parasite transmission from cats to cattle and vice versa has been intensively discussed in current literature. Therefore, we investigated if cat and cattle isolates are genetically distinct from each other or in fact represent identical genotypes. For this purpose, two independent genetic loci were selected that turned out to be well-suited for a PCR sequencing-based genotyping of trichomonad isolates: (i) previously published internal transcribed spacer region 2 (ITS-2) and (ii) a semi-conserved sequence stretch of the elongation factor-1 alpha (EF-1alpha) gene used for the first time in the present study. Respective comparative analyses revealed that both loci were sufficiently variable to allow unambiguous genetic discrimination between different trichomonad species. Comparison of both genetic loci confirmed that T. suis and T. mobilensis are phylogenetically very close to T. foetus. Moreover, these two genetic markers were suited to define host-specific genotypes of T. foetus. Both loci showed single base differences between cat and cattle isolates but showed full sequence identity within strains from either cat or cattle isolates. Furthermore, an additional PCR with a forward primer designed to specifically amplify the bovine sequence of EF-1alpha was able to discriminate bovine isolates of T. foetus from feline isolates and also from other trichomonads. The implications these minor genetic differences may have on the biological properties of the distinct isolates remain to be investigated.

Reasons for ordering computed tomography scans of the head in patients with minor brain injury

Minor brain injury is a frequent condition. Validated clinical decision rules can help in deciding whether a computed tomogram (CT) of the head is required. We hypothesized that institutional guidelines are not frequently used, and that psychological factors are a common reason for ordering an unnecessary CT.