[Fracture of the posterior malleolus : A paradigm shift]. / Fraktur des posterioren Malleolus : Ein Paradigmenwechsel.

Ankle fractures with involvement of the posterior malleolus have a poor prognosis. The traditional concept of addressing the posterior fragment as the final step in the surgical reconstruction depending on the fragment size manifest in the lateral X­ray, does not do justice to the anatomic and biomechanical relevance of the posterior malleolus. Intra-articular step-offs and fragment displacement ≥ 2 mm, impacted intercalary fragments, involvement of the fibular notch and instability of the syndesmosis, represent parameters relevant for the surgical reconstruction and the functional and radiomorphological outcome independent of fragment size. A valid assessment of these parameters can only be achieved via computed tomography (CT) including multiplanar 2D and 3D reconstruction. This is the foundation for the classification of posterior malleolar fractures according to Bartonícek et al., it forms the basis of the preoperative analysis of the fracture components and represents a decision-making tool for the indications for surgery. The individual fracture pattern guides the selection of the suitable approach or a combination of approaches and the surgical strategy. Making use of the posterior approaches enables the surgeon to have a direct view of the fracture components of the posterior malleolus as a precondition for an anatomic reduction. This central surgical step is frequently done as the first measure of the surgical reconstruction of the ankle joint. Furthermore, direct reduction and posterior stabilization is more accurate and stable than indirect reduction from an anterior approach with indirect fixation using lag screws. An additional temporary transfixation of the tibiofibular syndesmosis is often dispensable.

Minimally invasive internal fixation of calcaneal fractures or subtalar joint arthrodesis using the Calcanail®.

OBJECTIVE: A minimally invasive technique to prevent soft tissue problems using a calcaneal nail (Calcanail®, FH Orthopedics, Heimsbrunn, France) for calcaneal fractures or in subtalar joint arthrodesis is described. INDICATIONS: Displaced extra-articular calcaneal fractures involving the tuberosity fragment and in displaced intra-articular calcaneal fractures with impression and/or displacement of the subtalar joint surface. Subtalar joint arthrodesis for posttraumatic subtalar osteoarthritis. CONTRAINDICATIONS: Severely displaced fractures type Sanders IV with the purpose of internal fixation; peripheral calcaneal fractures; general contraindications for operative treatment. SURGICAL TECHNIQUE: Minimally invasive reduction of calcaneal fractures using a special distractor and a graft pusher through a calcaneal working channel. Fixation performed via the insertion of the Calcanail® and the two locking screws, optional additional screws. Subtalar joint arthrodesis with a Calcanail® used with optional three locking screws to fix the talus and calcaneus creating an angular stable construct. POSTOPERATIVE MANAGEMENT: Mobilization and restricted weight-bearing for 6 weeks in the patient's own shoes after fracture fixation or in a walker after arthrodesis. RESULTS: Preliminary results of 69 cases from three surgical centers have already been published. From 2013-2017, the technique was used in 48 of our own patients (42 calcaneal fracture reduction and fixation; 6 for subtalar joint arthrodesis). Mean postoperative hospital stay was 7 days for fracture reduction without any need of additional operations. During follow-up, 6 implant removals and 2 secondary subtalar fusions were noticed. All 6 cases of subtalar joint arthrodesis were planned in posttraumatic subtalar osteoarthritis.

[Joint-preserving treatment of medial femoral neck fractures with an angular stable implant]. / Gelenkerhaltende Versorgung der medialen Schenkelhalsfraktur mit einem winkelstabilen Implantat.

OBJECTIVE: Operative stabilization is recommended even for non-displaced or only slightly displaced femoral neck fractures. In addition to the known osteosynthetic procedures, an angular stabile implant system (Targon® FN) has been established since 2006 for treatment of such fractures. INDICATIONS: Displaced femoral neck fractures (Garden III and IV) and non-displaced fractures (Garden I and II). CONTRAINDICATIONS: Fractures close to the hip joint, which are not classified as typical medial femoral neck fractures and patients with advanced osteoarthritis of the hip who would profit from an endoprosthetic procedure. SURGICAL TECHNIQUE: The operative procedure is shown after fracture reposition and central positioning of the guide wire as a standard course. POSTOPERATIVE MANAGEMENT: Early postoperative mobilization under guidance of a physiotherapist. Initially, partial weight bearing only in selected cases with severe displacement. RESULTS: In our patients collective the Targon® FN has been implanted in over 100 cases. Revision indications and secondary endoprosthesis were documented in only 9 % of the cases. This angular stable screw osteosynthesis system is a safe procedure to achieve patient mobility if the indications are adhered to and implantation is correctly carried out.

Autophagy is required for zebrafish caudal fin regeneration.

Regeneration is the ability of multicellular organisms to replace damaged tissues and regrow lost body parts. This process relies on cell fate transformation that involves changes in gene expression as well as in the composition of the cytoplasmic compartment, and exhibits a characteristic age-related decline. Here, we present evidence that genetic and pharmacological inhibition of autophagy - a lysosome-mediated self-degradation process of eukaryotic cells, which has been implicated in extensive cellular remodelling and aging - impairs the regeneration of amputated caudal fins in the zebrafish (Danio rerio). Thus, autophagy is required for injury-induced tissue renewal. We further show that upregulation of autophagy in the regeneration zone occurs downstream of mitogen-activated protein kinase/extracellular signal-regulated kinase signalling to protect cells from undergoing apoptosis and enable cytosolic restructuring underlying terminal cell fate determination. This novel cellular function of the autophagic process in regeneration implies that the role of cellular self-digestion in differentiation and tissue patterning is more fundamental than previously thought.

Pharmacokinetics and penetration of linezolid into inflamed soft tissue in diabetic foot infections.

OBJECTIVE: Physiological changes and local and systemic inflammation may affect plasma and tissue pharmacokinetics of antimicrobial agents in diabetics. The aim of the study was to investigate the penetration of linezolid into inflamed areas of infected diabetic foot wounds and the pharmacokinetics in the risk population of diabetics. METHODS: Pharmacokinetics and tissue penetration of linezolid into inflamed diabetic foot infection (DFI) tissue were determined at steady state in 15 patients with diabetes type 2 and DFI following administration of multiple oral doses of 600 mg given every 12 h. Second debridement was performed on days 4-6, 3 h after linezolid administration. Linezolid concentrations were determined in perinecrotic wound tissue of inflamed diabetic foot by high-performance liquid chromatography (HPLC). RESULTS: A mean maximum plasma concentration (C(max)) in plasma of 14.3 mg/L was attained at a median of 2.0 h [time to reach C(max) (T(max)) range 0.5-6.0 h). Area under the concentration time curve from zero to 12 h (AUC(0-12 h)) with a mean of 114.1 mgh/L and C(min) of 5.4 mg/L were achieved in patients with diabetes mellitus type 2. Penetration of linezolid into inflamed areas of DFI with tissue/plasma ratios of mean 101.7% [95% confidence interval (CI) 56; 148%] produced a mean concentration of 9.6 microg/g (95% CI 7.4; 11.8 microg/g) greater than those predicted to be effective against methicillin-resistant staphylococci [minimum concentration that inhibits 90% of organisms (MIC(90)) of 4 mg/L]. Tissue/plasma ratios correlated positive with systemic inflammation. CONCLUSION: Plasma pharmacokinetics of linezolid in diabetics and adequate levels in inflamed areas of diabetic foot wound suggest that an oral dose of 600 mg bd of linezolid provides effective concentrations for treating methicillin-resistant Staphylococcus aureus (MRSA) in DFI.

Therapeutic silencing of mutant huntingtin with siRNA attenuates striatal and cortical neuropathology and behavioral deficits.

Huntington's disease (HD) is a neurodegenerative disorder caused by expansion of a CAG repeat in the huntingtin (Htt) gene. HD is autosomal dominant and, in theory, amenable to therapeutic RNA silencing. We introduced cholesterol-conjugated small interfering RNA duplexes (cc-siRNA) targeting human Htt mRNA (siRNA-Htt) into mouse striata that also received adeno-associated virus containing either expanded (100 CAG) or wild-type (18 CAG) Htt cDNA encoding huntingtin (Htt) 1-400. Adeno-associated virus delivery to striatum and overlying cortex of the mutant Htt gene, but not the wild type, produced neuropathology and motor deficits. Treatment with cc-siRNA-Htt in mice with mutant Htt prolonged survival of striatal neurons, reduced neuropil aggregates, diminished inclusion size, and lowered the frequency of clasping and footslips on balance beam. cc-siRNA-Htt was designed to target human wild-type and mutant Htt and decreased levels of both in the striatum. Our findings indicate that a single administration into the adult striatum of an siRNA targeting Htt can silence mutant Htt, attenuate neuronal pathology, and delay the abnormal behavioral phenotype observed in a rapid-onset, viral transgenic mouse model of HD.

Deterioro cognitivo leve: estudio de prevalencia y factores sociodemográficos en la ciudad de Córdoba, Argentina / Mild cognitive impairment: a prevalence and sociodemographic factors study in the city of Córdoba, Argentina

Introducción. El aumento de la expectativa de vida y un crecimiento significativo de la población de adultos mayores ha generado un interés por la prevención secundaria de diversas enfermedades, especialmente a partir de los 50 años. En Argentina, sólo los mayores de 65 años representan el 10% de la población total, por lo que resulta importante investigar sobre su estado mental. En este sentido, se realizó un estudio descriptivo transeccional en cuatro servicios de neuropsicología de la ciudad de Córdoba, en el marco de una campaña de evaluación de la memoria destinada a personas mayores de 50 años. Sujetos y métodos. En un período de tres meses, participaron 418 voluntarios con un promedio de edad de 64,24 años y de instrucción de 12,76 años, pertenecientes en su mayoría al medio urbano (80%). Se les administró el test minimental de Folstein, pruebas neuropsicológicas y medidas conductuales en dos sesiones de trabajo. Resultados. Se encontró que la mayoría (75,6%, n = 316) tuvo un rendimiento normal tanto neuropsicológico como conductual. En el límite de lo normal se halló un porcentaje menor (8,6%, n = 36). Se detectó un 9,1% (n = 38) con deterioro cognitivo leve de tipo amnésico y un 4,5% (n = 19) con deterioro leve de tipo multidominio, es decir, un 13,6% de personas con una mayor probabilidad de desarrollar demencia en los próximos años. Conclusión. El género femenino, la edad superior a 65 años, el menor nivel de instrucción y el número de hijos parecen ser las variables sociodemográficas que más influyen en el estado cognitivo

Introduction. The increase in life expectancy and the significant growth in the population of the elderly have generated an interest in secondary prevention of different diseases, especially from the age of 50. In Argentina, those over 65 represent 10% of the total population, what makes important to research into their mental health. A trans-sectional descriptive study has been carried out in four neuropsychology services in the city of Cordoba, within the framework of a campaign assessing memory for people over 50. Subjects and methods. In three months, 418 volunteers took part, with an average age of 64.24 years and 12.76 years of instruction, mostly belonging to a urban environment (80%). They were administered Folstein’s Minimental State, neuropsychological tests and behavioral measures in two work sessions. Results. The majority was found to have normal performances (75.6%, n = 316) at both behavioral and neuropsychological levels. A lower percentage was found at the borders of normality (8.6%, n = 36). 9.1% (n = 38) of those studied were found to have an amnesiac type of mild cognitive impairment, and 4.5% (n = 19) a multidomain type of deterioration. In sum, about 13.6% of people have a greater chance of developing dementia in coming years. Conclusion. The socio-demographic variables that most influence cognitive state appear to be the female gender, age higher than 65 years, lower levels of instruction, fewer children and fewer siblings

Gene expression profiling identifies FKBP39 as an inhibitor of autophagy in larval Drosophila fat body.

In Drosophila, the fat body undergoes a massive burst of autophagy at the end of larval development in preparation for the pupal transition. To identify genes involved in this process, we carried out a microarray analysis. We found that mRNA levels of the homologs of Atg8, the coat protein of early autophagic structures, and lysosomal hydrolases were upregulated, consistent with previous results. Genes encoding mitochondrial proteins and many chaperones were downregulated, including the inhibitor of eIF2alpha kinases and the peptidyl-prolyl cis-trans isomerase FK506-binding protein of 39 kDa (FKBP39). Genetic manipulation of FKBP39 expression had a significant effect on autophagy, potentially through modulation of the transcription factor Foxo. Accordingly, we found that Foxo mutants cannot properly undergo autophagy in response to starvation, and that overexpression of Foxo induces autophagy.

[Management problems in the diagnostic work-up and treatment of pathological fracture of the proximal femur. Clinical case report]. / Managementprobleme in Diagnostik und Therapie der pathologischen Schenkelhalsfraktur. Ein klinischer Fallbericht.

Pathological fractures caused by primary bone tumours are rare compared with fractures evolving in response to general osteoporosis or bone metastases. Nevertheless in cases of fracture due to inadequate trauma the underlying causality has to be clarified including histological analysis prior to the operating procedure. This case report demonstrates diagnostic and therapeutic standards in cases of pathological fracture of the proximal femur caused by osteosarcoma.

Proteolytic activity of 26s proteasomes isolated from muscles of the tobacco hornworm, Manduca sexta: differences between surviving muscles and those undergoing developmentally programmed cell death.

The intersegmental muscles (ISMs) of tobacco hornworm,Manduca sexta are a well-characterised model system for examining the biochemical changes that accompany programmed cell death during development. When the ISMs become committed to die, there are dramatic increases in both the ubiquitin-expression, and ubiquitin-dependent proteolysis. Since the 26S proteasome is responsible for ATP/ubiquitin-dependent proteolysis in cells, we examined its enzymatic properties. Specific chymotrypsin-like proteolytic activity of 26S proteasomes isolated from ISM is four times higher than that of surviving flight muscle (FM). However, specific activity does not change between developmental stages within ISM or FM. The difference between proteolytic capacity of the two kinds of muscles is even higher when the ISM become committed to die because 26S proteasome content of ISM increases just before cell death. These observations underline the role of 26S proteasome in programmed cell death.

A potential dual apical pathway in polarized regenerative cells of the midgut of Manduca sexta.

Two novel proteins with apparent molecular weight of 38 (Manduca sexta midgut MsM38) and 46kDa (MsM46) were isolated from midgut homogenates in wandering stage Manduca sexta larvae and both of them were found to be present exclusively in this tissue on Western blots. Immunocytochemical studies revealed that both proteins are expressed in the regenerative cells however, their distribution pattern is clearly different. MsM38 is localized in the cytoplasm of resting regenerative cells during the feeding period, and is accumulated in the calcospherits at the beginning of the wandering period. Along with the delamination of the larval epithelium, this protein is released apically from these vesicles. The antiserum labels an additional 76 kDa protein in the wandering larval midgut homogenates. The appearance of this 76 kDa protein coincides with the accumulation of the immunopositive material in the calcospherits. MsM46 is similarly distributed during the feeding period in the cytoplasm of regenerative cells. At the beginning of the wandering period it accumulates around the newly forming large apical vacuoles, that are released at the time of complete delamination of the larval epithelium. In parallel with this process MsM46, and another 40 kDa protein, that becomes labeled from this period on Western blots appeares on the apical microvillar projections. Thus both isolated proteins are directed apically from different compartments, that raises the possibility of a dual apical routing pathway in regenerative cells.

Localization of a cuticular protein during the postembryonal development of Manduca sexta.

The pattern of cuticle protein synthesis by the epidermis of insects changes during the last larval, pupal and adult development, leading to an alteration in cuticular stucture and feature. We have isolated a protein that had an apparent molecular mass of 33.1 kD from larval cuticle of Manduca sexta. Synthesis, transport and accumulation of MsCP33.1 were followed during metamorphosis by immunoblots and immunocytochemical methods using the antibody developed against this protein. Our data prove that the presence of MsCP33.1 in the larval cuticle is general while its appearance in the pupal or adult integument is restricted only in the cuticle of wings and apodemes. We established that the synthesis of 33.1 kD protein is negatively regulated by moulting hormone (20-hydroxyecdysone). Possible roles for this cuticular protein are discussed.

The immunoprotein scolexin and its synthesizing sites--the midgut epithelium and the epidermis.

Scolexin is one of the bacterial induced hemolymph proteins of tobacco homworm (Manduca sexta) larvae, that has hemocyte coagulation-provoking activity. The 72 kDa scolexin complex is composed of two 36 kDa subunits. To examine the protein secretory pathways in insect epithelia a polyclonal antibody was raised against the 36 kDa hemolymph protein. This MsH36 antibody recognised a 36 and a 72 kDa protein in tissue homogenates. On the basis of the characteristic labelling pattern observed on immunoblots and immunocytochemical sections we concluded that the 36 kDa protein in the hemolymph, in the midgut and in the epidermis was identical with the scolexin subunit. In present paper we report a labelling shift in the midgut epithelium between goblet and columnar cells that may be controlled by the hormonal system. A 72 kDa protein showed similar epitops and molecular weight to the scolexin complex and was detected in epidermis and in cuticle under both reducing and non-reducing conditions. Tissue localization of 36 kDa and 72 kDa MsH36 antibody labelling proteins indicated the possibility that the epidermal cells produce two kinds of scolexin-like proteins. The complex composed of 36 kDa subunits are transported basolaterally into the circulation and display hemocyte coagulation inducing activity while the 72 kDa form contains two subunits linked covalently secreted apically into the cuticle.

Distribution of serpins in the tissues of the tobacco hornworm (Manduca sexta) larvae. Existence of new serpins possibly encoded by a gene distinct from the serpin-1 gene.

Polyclonal antibodies were raised against the isolated hemolymph serine proteinase inhibitors (serpins) of Manduca sexta larvae. Two of these antibodies, MsH49a and MsH49b, displayed characteristic differences in labelling patterns of hemocytes, fat body, integumental epidermis and cuticle on immunoblots, and in light- and electronmicroscopic sections. The serpin composition of the latter three tissue homogenates was determined by native immunoblots and inhibitor binding assays. The results were compared to the hemolymph samples containing all the known inhibitors encoded by the well-characterized serpin-1 gene. The enzyme specificity of the MsH49b-labelled cuticular serpin was similar to serpin-1J, although its electrophoretic mobility on native PAGE was not identical with any of the known proteinase inhibitors encoded by the serpin-1 gene. Based on these data, we suggest that the cuticle and hemolymph may contain novel serpin(s) encoded by a gene other than the serpin-1 gene. Since the serpin-1J proved to be involved in the activation pathway of the prophenoloxidase system in the hemolymph, the in vivo function of cuticular MsH49b serpin was investigated by prophenoloxidase tests in native cuticular homogenates. Our results demonstrated that the cuticular serpin(s) that are labelled by the MsH49b antibody may play a determinant role in the regulation of the prophenoloxidase system of the integumental cuticle.

Immunoanalytical method for quality control of orange juice products.

The main goal of the present study is to develop an immunoanalytical method for the quality control of orange juice products. Peptides from various parts (juice, albedo, and flavedo) of citrus fruits (orange, mandarin, grapefruit, and lemon) were analyzed and isolated by SDS-PAGE. Antisera were developed in mice against the protein pool of orange juice and peel and tested by Western blot analysis. Using these antisera, some juice- and peel-specific peptides were detected. One of the antibodies in the antiserum developed against peel proteins recognized a single peel-specific peptide with a molecular mass of 28 kDa in 10000-fold dilution. It did not give any positive reactions against the sample prepared from the juice. The 24 and 27 kDa juice-specific peptides were isolated in electrophoretically pure form, and polyclonal antibodies were developed against them in mice. The anti-27 kDa antibody reacted with a 29 kDa protein in the peel sample, and it gave a positive reaction against the 27 kDa peptide of the juice. The antibodies developed in the course of the present work seem to be useful for determining the juice content in commercial citrus beverages and for evaluating the peel contamination in them.

Localisation of 26S proteasomes with different subunit composition in insect muscles undergoing programmed cell death.

The 26S proteasome is a large multisubunit complex involved in degrading both cytoplasmic and nuclear proteins. We have investigated the subcellular distribution of four regulatory ATPase subunits (S6 (TBP7/MS73), S6' (TBP1), S7 (MSS1), and S10b (SUG2)) together with components of 20S proteasomes in the intersegmental muscles (ISM) of Manduca sexta during developmentally programmed cell death (PCD). Immunogold electron microscopy shows that S6 is located in the heterochromatic part of nuclei of ISM fibres. S6' is present in degraded material only outside intact fibres. S7 can be detected in nuclei, cytoplasm and also in degraded material. S10b, on the other hand, is initially found in nuclei and subsequently in degraded cytoplasmic locations during PCD. 20S proteasomes are present in all areas where ATPase subunits are detected, consistent with the presence of intact 26S proteasomes. These results are discussed in terms of heterogeneity of 26S proteasomes, 26S proteasome disassembly and the possible role of ATPases in non-proteasome complexes in the process of PCD. Cell Death and Differentiation (2000) 7, 1210 - 1217.

Isolation and characterization of an apically sorted 41-kDa protein from the midgut of tobacco hornworm (Manduca sexta).

Immunocytochemical localization and sorting properties of a newly purified 41-kDa protein (MsM41) were investigated in an insect, the tobacco hornworm Manduca sexta. The protein purified from midgut homogenates of feeding fifth-stadium larvae was found exclusively in this tissue on Western blots. Presence of MsM41 protein was indicated in both anterior and posterior regions of the midgut during the whole fifth stadium. However, in the posterior region an additional 39-kDa protein was also detected during the feeding period of the last larval stage. Upon light-microscopic examination immunoreactivity was localized in the columnar cells, while the goblet, endocrine and regenerative cells remained unlabeled. Distribution of the label during the feeding period was different in the anterior and posterior regions. In the anterior region immunoreactivity was localized only to the brush border membrane of columnar cells, while in the posterior region some cytoplasmic structures identified as large trans-Golgi vesicles, endoplasmic reticulum and small secretory vesicles were also labeled. Large, apical extrusions remained immunonegative. In vitro translation confirmed that our protein was expressed only in the posterior region of the midgut. The primary translation product was a 39-kDa protein. Putative post-translational modifications yielded the 41-kDa form, which was then secreted apically. Its presence in the region of the anterior part microvilli was probably due to the countercurrent flux of the ectoperitrophic fluid.

45T-1, an established cell line with characteristics of Sertoli cells, forms organized aggregates in vitro after exposure to tumor necrosis factor alpha.

In the testis TNF is produced by germinal cells. The putative role of tumor necrosis factor alpha (TNF) in development and differentiation was investigated in 45T-1 mouse cell cultures, a cell line with characteristic markers of Sertoli cells, established from transgenic mouse families expressing the polyoma large T antigen in their testes. Exposure to TNF elicited a gradual assembly of the cells of the monolayer into highly organized spheroids. The first morphological sign of the changes was detected one week after TNF treatment by anti-desmin immunostaining which showed the formation of foci in the culture consisting of several hundred cells connected by an increasing number of cell contacts. Between days 10-20 the cells formed large ovoid or vermiform aggregates covered by several layers of flat, elongated cells. These cells extended septae into the inner mass of the spheroids consisting of loosely arranged, large polygonal or palisadic cells. The spheroids were surrounded by radially arranged elongated cells covered by small blebs. TNF treatment upregulated laminin expression in 45T-1 cell cultures, which is known to induce formation of cord-like structures by Sertoli cells in vitro. Coculturing 45T-1 cells with immortalized germinal cells or TNF-producing HeLa cells also lead to the formation of spheroids. These observations suggest that TNF production of germinal cells might contribute to the organization/differentiation of Sertoli cells.

Insect cuticle, an in vivo model of protein trafficking.

In the course of this study more than 20 proteins have been isolated from the larval cuticle of Manduca sexta. Synthesis, secretion, transport and accumulation of four particular proteins, representative members of four characteristic groups, were followed during metamorphosis by immunoblot and immuncytochemical methods and are described in detail in this paper. We established that only some of the proteins of the soft cuticle of Lepidopteran larvae are synthesized in epidermal cells at the beginning of the larval stages and are digested during the moulting period (MsCP29). Other proteins (MsCP30/11) are secreted into the cuticle by the epidermal cells in different forms during various developmental stages. Some proteins are secreted apically during the feeding period, but before ecdysis they are then taken up by epidermal cells and transported in a basolateral direction back into the hemolymph and saved in an immunologically intact form by the fat body cells (MsCP12.3). Some cuticle proteins have a non-epidermal origin. They are transported from the hemolymph into the cuticle. Before and during ecdysis these molecules reappear in the hemolymph and are detectable again in the pupal cuticle (MsCP78). Our data prove that the cuticle is not a non-living part of the insect body: it is not only an inert, protective armor, but maintains a continuous and dynamic metabolic connection with the other organs of the organism.