Highly efficient subcloning of rodent malaria parasites by injection of single merosomes or detached cells

This protocol describes a method for obtaining rodent Plasmodium parasite clones with high efficiency, which takes advantage of the normal course of Plasmodium in vitro exoerythrocytic development. At the completion of development, detached cells/merosomes form, which contain hundreds to thousands of merozoites. As all parasites within a single detached cell/merosome derive from the same sporozoite, we predicted them to be genetically identical. To prove this, hepatoma cells were infected simultaneously with a mixture of Plasmodium berghei sporozoites expressing either GFP or mCherry. Subsequently, individual detached cells/merosomes from this mixed population were selected and injected into mice, resulting in clonal blood stage parasite infections. Importantly, as a large majority of mice become successfully infected using this protocol, significantly less mice are necessary than for the widely used technique of limiting dilution cloning. To produce a clonal P. berghei blood stage infection from a non-clonal infection using this procedure requires between 4 and 5 weeks.

Redistribution of cobalt and nickel in detached wheat shoots: effects of steam-girdling and of cobalt and nickel supply

Detached wheat shoots (ear with peduncle and flag leaf) were incubated for 4 d in a solution containing 1 mM RbCl and 1 mM SrCl2 as well as 10, 40 or 160 µM NiCl2 and CoCl2. The phloem of some plants was interrupted by steam-girdling the stem below the ear to distinguish between xylem and phloem transport. The phloem-immobile Sr flowed mainly to the leaf lamina and to the glumes via the xylem. The Sr transport was not sensitive to steam-girdling. In contrast, the phloem-mobile Rb accumulated during the incubation time mainly in the stem and the leaf sheath. The Rb transport to the grains was impaired by steam-girdling as well as by elevated Ni and Co concentrations in the incubation solution indicating that Rb was transported via the phloem to the maturing grains and that this transport was affected by the heavy metals. Ni was removed more efficiently from the xylem in the peduncle than Co (but far less efficiently than Rb). It became evident that the two heavy metals can also be transferred from the xylem to the phloem in the stem of wheat and reach the maturing grains via the phloem.

Role of intra- and peritumoral budding in the interdisciplinary management of rectal cancer patients

The presence of tumor budding (TuB) at the invasive front of rectal cancers is a valuable indicator of tumor aggressiveness. Tumor buds, typically identified as single cells or small tumor cell clusters detached from the main tumor body, are characterized by loss of cell adhesion, increased migratory, and invasion potential and have been referred to as malignant stem cells. The adverse clinical outcome of patients with a high-grade TuB phenotype has consistently been demonstrated. TuB is a category IIB prognostic factor; it has yet to be investigated in the prospective setting. The value of TuB in oncological and pathological practice goes beyond its use as a simple histomorphological marker of tumor aggressiveness. In this paper, we outline three situations in which the assessment of TuB may have direct implications on treatment within the multidisciplinary management of patients with rectal cancer: (a) patients with TNM stage II (i.e., T3/T4, N0) disease potentially benefitting from adjuvant therapy, (b) patients with early submucosally invasive (T1, sm1-sm3) carcinomas at a high risk of nodal positivity and (c) the role of intratumoral budding assessed in preoperative biopsies as a marker for lymph node and distant metastasis thus potentially aiding the identification of patients suitable for neoadjuvant therapy.

Bioabsorbable pins for treatment of osteochondral fractures of the knee after acute patella dislocation in children and young adolescents

A retrospective study was performed on the use of bioabsorbable pins in the fixation of osteochondral fractures (OCFs) after traumatic patellar dislocation in children. Eighteen children (13 females, 5 males) aged 11 to 15 years (mean age 13.1 years) with osteochondral fracture (OCF) of the knee joint were treated at the authors' institution. Followup ranged from 22 months to 5 years. Diagnosis was verified by X-ray and magnetic resonance imaging (MRI) of the knee and patella. In seven patients the osteochondral fragment was detached from the patella and in 11 it was detached from the lateral femoral condyle. All patients were subjected to open reduction and fixation of the lesion with bioabsorbable pins. Postoperatively, the knee was immobilized in a cast and all patients were mobilized applying a standardized protocol. Bone consolidation was successful in 17 of the 18 patients. Bioabsorbable pins reliably fix OCF in children and adolescents, demonstrating a high incidence of consolidation of the detached osteochondral fragment in short- and middle-term followup without requiring further operative procedures.

[Medium-term results after m. vastus medialis obliquus-plasty for lateral patellar dislocation]

Progressive retropatellar arthrosis is often seen in dated rigid distal realignment (i.e. osteotomy of tuberositas) at long-term follow-ups. Therefore, operations for lateral dislocation of the patella are still discussed controversially. Dynamic, proximal realignments seem to have lower rates of arthrosis but higher rates of redislocation. Recently, in anatomic and biomechanic studies, the m. vastus medialis obliquus (vmo) was found to be one of the most important proximal restraints to lateral dislocation of the patella.A total of 28 patients (mean age 21.5 years) were treated between 1994 and 2003 with a plasty of the vmo for lateral patellar dislocation. The technique was performed for most etiologies of femoropatellar instability.For this proximal soft tissue technique, the muscle tendon is detached from its patellar insertion. Subsequently, the tendon is reinserted at the patella 10-15 mm more distally and fixed with Mitek anchors. Full weight bearing in extension is possible immediately after surgery. An active vastus medialis training is started after 6 weeks.Of the patients, 27 were evaluated clinically and radiologically in 2004 (a mean of 5 years postoperatively). A total of 83% of the patients estimated the result to be good or excellent, 10% were satisfied and 7% were discontent. The mean Lysholm-Knee-Score was 83.1 points. Two patients suffered a patella redislocation (7%). A statistically significant improvement of the congruence angle was noted in the radiographs, even in medium-term controls. In 89% of the cases no or only little retropatellar arthrosis was observed. These 5 year results are comparable to those of other techniques for distal or proximal realignments. The rate of redislocation was below average. Compared to the rate of retropatellar arthrosis in long-term results of rigid distal realignment, our patients demonstrated a relative low rate after 5 years. We attribute this to the minimal interference in physiological joint mechanics and to the restored anatomy. In terms of future long-term results, our findings are promising. The idea of a proximal dynamic stabilization and the causal operative approach at the origin of pathology using vmo-plasty was confirmed in recent anatomic and biomechanic studies. Over or under correction of soft tissues could be adapted. More rigid techniques of distal realignment do not allow an adaptation to this extent and can lead to prearthrotic hyperpression in the medial femoropatellar and femorotibial joints.

Unattended emotional intonations modulate linguistic prosody processing

Prosody or speech melody subserves linguistic (e.g., question intonation) and emotional functions in speech communication. Findings from lesion studies and imaging experiments suggest that, depending on function or acoustic stimulus structure, prosodic speech components are differentially processed in the right and left hemispheres. This direct current (DC) potential study investigated the linguistic processing of digitally manipulated pitch contours of sentences that carried an emotional or neutral intonation. Discrimination of linguistic prosody was better for neutral stimuli as compared to happily as well as fearfully spoken sentences. Brain activation was increased during the processing of happy sentences as compared to neutral utterances. Neither neutral nor emotional stimuli evoked lateralized processing in the left or right hemisphere, indicating bilateral mechanisms of linguistic processing for pitch direction. Acoustic stimulus analysis suggested that prosodic components related to emotional intonation, such as pitch variability, interfered with linguistic processing of pitch course direction.

Scleral buckling versus primary vitrectomy in rhegmatogenous retinal detachment study (SPR Study): recruitment list evaluation. Study report no. 2

BACKGROUND: Accompanying the patient recruitment within the "Scleral buckling versus primary vitrectomy in rhegmatogenous retinal detachment multicentre trial (SPR)", all patients with primary rhegmatogenous retinal detachment (RRD) had to be documented in a detailed recruitment list. The main goal of this analysis was to estimate the prevalence of "medium-severe" RRD (SPR Study eligible) as defined by the SPR Study inclusion criteria. In addition, the detailed anatomical situation of medium-severe RRD is investigated. METHODS: SPR Study recruitment was evaluated via a standardised questionnaire, which contained a coloured fundus drawing and information regarding possible reasons for exclusion from the SPR Study in each case. A team of three experienced vitreoretinal surgeons evaluated all fundus drawings from a 1-year period. The review led to a decision on SPR Study eligibility on the pure basis of anatomical assessment. The main outcome measures were assessment of feasible inclusion into the SPR Study by the evaluation team based on the fundus drawing and anatomical details. RESULTS: A total of 1,115 patients with RRD from 13 European centres were prospectively enrolled in the year 2000. The quality of the drawings sufficed for assessment in 1,107 cases (99.3%). Three hundred and twelve fundus drawings (28.2%) met the anatomic inclusion criteria of the SPR Study. RRD of medium severity is characterised by an average number of 2.6 (SD 2.4) retinal breaks, 5.8 (SD 2.8) clock hours of detached retina, unclear hole situation in 15.1% of cases (n=47), attached macula in 42.9% (n=134), bullous detachment in 15.1% (n=47) and vitreous haemorrhage/opacity in 7.7% (n=24). CONCLUSIONS: In the recruitment lists of the SPR Study of the year 2000, RRD of medium severity was present in nearly one third of the patients with primary RRD. These findings emphasise the clinical relevance of the SPR Study.

Factor XIII activation peptide is released into plasma upon cleavage by thrombin and shows a different structure compared to its bound form

The first step of coagulation factor XIII (FXIII) activation involves cleavage of the FXIII activation peptide (FXIII-AP) by thrombin. However, it is not known whether the FXIII-AP is released into plasma upon cleavage or remains attached to activated FXIII. The aim of the present work was to study the structure of free FXIII-AP, develop an assay for FXIII-AP determination in human plasma, and to answer the question whether FXIII-AP is released into plasma. We used ab-initio modeling and molecular dynamics simulations to study the structure of free FXIII-AP. We raised monoclonal and polyclonal antibodies against FXIII-AP and developed a highly sensitive and specific ELISA method for direct detection of FXIII-AP in human plasma. Structural analysis showed a putative different conformation of the free FXIII-AP compared to FXIII-AP bound to the FXIII protein. We concluded that it might be feasible to develop specific antibodies against the free FXIII-AP. Using our new FXIII-AP ELISA, we found high levels of FXIII-AP in in-vitro activated plasma samples and serum. We showed for the first time that FXIIIAP is detached from activated FXIII and is released into plasma, where it can be directly measured. Our findings may be of major clinical interest in regard to a possible new marker in thrombotic disease.

Angioscopy for experimental evaluation of optional IVC filters

PURPOSE: To demonstrate the feasibility of direct angioscopic visualization of an optional inferior vena cava (IVC) filter in situ and during retrieval. MATERIALS AND METHODS: Angioscopy was used for direct visualization of optional IVC filters in six sheep. Cavograms were obtained before the filters were retrieved. After successful filter retrieval, segmental IVC perfusion was performed to evaluate filter retrieval-related damage to the IVC wall. Therefore, all branch vessels were ligated before the IVC segment was flushed with normal saline solution until it was fully distended. Then, the inflow was terminated and the IVC segment observed for deflation. Subsequently, the IVC was harvested en bloc, dissected, and inspected macroscopically. RESULTS: The visibility of IVC filters at angioscopy was excellent. During the retrieval procedure, filter collapse and retraction into the sheath were clearly demonstrated. Angioscopy provided additional information to that obtained with cavography, demonstrating adherent material in three filters. Three filters in place for more than 2 months could not be retrieved because the filter legs were incorporated into the IVC wall. After filter retrieval, there was no perforation at segmental IVC perfusion. At macroscopic inspection of the IVC lumen, a small piece of detached endothelium was found in one animal. CONCLUSION: Angioscopy enabled the direct evaluation of optional IVC filters in situ and during retrieval. Compared with cavography, angioscopy provided additional information about the filter in situ and the retrieval procedure. Future applications of this technique could include studies of filter migration, compression, and clot-trapping efficacy.

Probing the function of ionotropic and G protein-coupled receptors in surface-confined membranes

This article reports on recent electrical and optical techniques for investigating cellular signaling reactions in artificial and native membranes immobilized on solid supports. The first part describes the formation of planar artificial lipid bilayers on gold electrodes, which reveal giga-ohm electrical resistance and the insertion and characterization of ionotropic receptors therein. These membranes are suited to record a few or even single ion channels by impedance spectroscopy. Such tethered membranes on planar arrays of microelectrodes offer mechanically robust, long-lasting measuring devices to probe the influence of different chemistries on biologically important ionotropic receptors and therefore will have a future impact to probe the function of channel proteins in basic science and in biosensor applications. In a second part, we present complementary approaches to form inside-out native membrane sheets that are immobilized on micrometer-sized beads or across submicrometer-sized holes machined in a planar support. Because the native membrane sheets are plasma membranes detached from live cells, these approaches offer a unique possibility to investigate cellular signaling processes, such as those mediated by ionotropic or G protein-coupled receptors, with original composition of lipids and proteins.

Linkage in narratives: A comparison between monolingual speakers of French and Italian, and early and late French-Italian bilinguals

In the course of language acquisition learners have to deal with the task of producing narrative texts that are coherent across a range of conceptual domains (space, time, entities) -- both within as well as across utterances. The organization of information is analyzed in this study, on the basis of retellings of a silent film, in terms of devices used in the coordination and subordination of events within the narrative sequence. The focus on subordination reflects a core grammatical difference between Italian and French, as Italian is a null-subject language while French is not. The implications of this contrast for information structure include differences in topic management within the sequence of events. The present study investigates in how far Italian-French bilingual speakers acquire the patterns of monolingual speakers of Italian. It compares how early and late bilinguals of these two languages proceed when linking information in narratives in Italian.

The Spanish variety of Malaga: From real to mental to virtual speech

Internet has affected our lives and society in manifold ways, and partly, in fundamental ways. Therefore, it is no surprise that one of the affected areas is language and communication itself. Over the last few years, online social networks have become a widespread and continuously expanding medium of communication. Being a new medium of social interaction, online social networks produce their own communication style, which in many cases differs considerably from real speech and is also perceived differently. The focus of analysis of my PhD thesis is how social network users from the city of Malaga create this virtual style by means of phonic features typical of the Andalusian variety of Spanish and how the users’ language attitude has an influence on the use of these phonic features. The data collection was fourfold: 1) a main corpus was compiled from 240 informants’ utterances on Facebook and Tuenti; 2) a corpus constituted of broad transcriptions of recordings with 120 people from Malaga served as a comparison; 3) a survey in which 240 participants rated the use of said phonetic variants on the following axes: “good–bad”, “correct–incorrect” and “beautiful–ugly” was carried out; 4) a survey with 240 participants who estimated with which frequency the analysed features are used in Malaga was conducted. For the analysis, which is quantitative and qualitative, ten variables were chosen. Results show that the studied variants are employed differently in virtual and real speech depending on how people perceive these variants. In addition, the use of the features is constrained by social factors. In general, people from Malaga have a more positive attitude towards non-­‐standard features if they are used in virtual speech than in real speech. Thus, virtual communication is seen as a style serving to create social meaning and to express linguistic identity. These stylistic practices reflect an amalgam of social presuppositions about usage conventions and individual strategies for handling a new medium. In sum, the virtual style is an initiative deliberately taken by the users, to create their, real and virtual, identities, and to define their language attitudes towards the features of their variety of speech.

Counting scars on tree stems to assess rockfall hazards: A low effort approach, but how reliable?

Rockfall is a widespread and hazardous process in mountain environments, but data on past events are only rarely available. Growth-ring series from trees impacted by rockfall were successfully used in the past to overcome the lack of archival records. Dendrogeomorphic techniques have been demonstrated to allow very accurate dating and reconstruction of spatial and temporal rockfall activity, but the approach has been cited to be labor intensive and time consuming. In this study, we present a simplified method to quantify rockfall processes on forested slopes requiring less time and efforts. The approach is based on a counting of visible scars on the stem surface of Common beech (Fagus sylvatica L.). Data are presented from a site in the Inn valley (Austria), where rocks are frequently detached from an ~ 200-m-high, south-facing limestone cliff. We compare results obtained from (i) the “classical” analysis of growth disturbances in the tree-ring series of 33 Norway spruces (Picea abies (L.) Karst.) and (ii) data obtained with a scar count on the stem surface of 50 F. sylvatica trees. A total of 277 rockfall events since A.D. 1819 could be reconstructed from tree-ring records of P. abies, whereas 1140 scars were observed on the stem surface of F. sylvatica. Absolute numbers of rockfalls (and hence return intervals) vary significantly between the approaches, and the mean number of rockfalls observed on the stem surface of F. sylvatica exceeds that of P. abies by a factor of 2.7. On the other hand, both methods yield comparable data on the spatial distribution of relative rockfall activity. Differences may be explained by a great portion of masked scars in P. abies and the conservation of signs of impacts on the stem of F. sylvatica. Besides, data indicate that several scars on the bark of F. sylvatica may stem from the same impact and thus lead to an overestimation of rockfall activity.

Adaptation to Changes in the Coffee Value Chain and the Price of Coiffee among Coffee Producers in Two Villages in Kilimanjaro, Tanzania

Market liberalization in Tanzania has eroded the monopoly of the cooperative unions by allowing private coffee buyers (PCBs) to compete with them on equal footing. Similarly, farmers groups and primary societies are now allowed to sell coffee at auction. Thus, farmers have various options for selling their coffee. Similarly, the coffee industry has experienced large fluctuations in prices and stagnation in production. How do farmers react to these changes? Can and do farmers profit from different market conditions and sell to different traders at the lower end of the value chain, or do they remain with cooperatives or farmers groups? This study was conducted in Mruwia and Mshiri villages in Moshi Rural district. Whereas Mshiri village remains attached to the Kilimanjaro Native Cooperative Union (KNCU), Mruwia has detached from this organization and sells coffee independently. The sample (103) was randomly selected from the coffee farmers in the two villages. Data were collected through surveys, focus group discussions (FGDs), and socio-anthropological methods (participant-observation, biographies, and thematic interviews). Results indicate that the selection of whom to sell coffee depends largely on farmers’ dependence on coffee and prices, other benefits accrued, and whether the initial costs are covered by buyers. Additionally, most respondents did not sell coffee to PCBs. Thus, prices, the institutional infrastructure, and the structure of local communities were important when making decisions about how and with whom to trade.