The impact of the Fundamental Critical Course on knowledge acquisition in Rwanda.

Background: Emerging critical care systems have gained little attention in low- and middle-income countries. In sub-Saharan Africa, only 4% of the healthcare workforce is trained in critical care, and mortality rates are unacceptably high in this patient population. Objectives: We sought to retrospectively describe the knowledge acquisition and confidence improvement of practitioners who attend the Fundamental Critical Care Support (FCCS) course in Rwanda. Methods: We conducted a retrospective study in which we assessed survey data and multiple-choice question data that were collected before and after course delivery. The purpose of these assessments at the time of delivery was to evaluate participants' perception and acquisition of critical care knowledge. Results: Thirty-six interprofessional clinicians completed the training. Performance on the multiple-choice questions improved overall after the course (mean score pre-course of 56.5% to mean score post-course of 65.8%, p-value <0.001) and improved in all content areas with the exception of diagnosis and management of acute coronary syndrome and acute respiratory failure/mechanical ventilation. Both physicians and nurses improved their scores significantly (68.9% to 75.6%, p-value = 0.031 and 52.0% to 63.5%, p-value <0.001, respectively). Self-reported confidence in level of knowledge also increased in all areas. Survey respondents indicated on open-answer questions that they would like the course offerings at least annually, and that further dissemination of the course in Rwanda was warranted. Conclusion: Deploying the established FCCS course improved Rwandan healthcare provider knowledge and confidence across most critical care content areas. Therefore, this course represents a good first step in bridging the gaps noted in emerging critical care systems. Contributions of the study: Critical care education in sub-Saharan Africa is limited and few staff have formal training. The aim of the study was to determine whether a focused course delivered in Rwanda on critical care management improved knowledge in key areas. Our retrospective study on results from a multiple choice question test and survey indicate that short courses may improve knowledge of critical care management.

Expression of corticosteroid-binding globulin CBG in the human heart.

Glucocorticoids are known to be involved in myocardial regeneration and destruction. Cardiomyocytes are mostly devoid of nuclear glucocorticoid receptors (GRs) and it is generally assumed that effects of adrenal steroids in heart are mediated through the mineralocorticoid receptor (MR). Here we used immunocytochemistry to study localization of corticosteroid binding globulin (CBG) in semithin sections of human cardiac tissue samples. With staining of consecutive sections we examined colocalization with GR and MR immunoreactivities. While GR staining was almost undetectable, a portion of myocytes with MR immunostained nuclei was found. Almost all cardiomyocytes exhibited CBG immunostaining in cytoplasm and on the cell membrane. Most pronounced CBG immunoreactivities were found in Purkinje fibers and in smooth muscle cells of arterial walls. With RT-PCR, we found in homogenates of cardiac tissue detectable levels of CBG encoding mRNA. Our findings indicate that CBG is expressed in human heart. Known cardiac effects of adrenal steroids may in part be mediated through the binding globulin and its putative membrane receptor in addition to nuclear steroid receptors and direct genomic action. Highlights of our study: Human cardiomyocytes express mineralocorticoid receptors, but are mostly free of nuclear glucocorticoid receptors. CBG is expressed in myocardium and in Purkinje fibers. CBG in heart is colocalized with mineralocorticoid receptor. Endothelia and smooth muscle cells of arterial walls show colocalization of CBG and MR.

Waist circumference to assess reversal of insulin resistance following weight reduction after bariatric surgery: cohort and cross-sectional studies.

OBJECTIVE: To validate the use of waist circumference to assess reversal of insulin resistance after weight loss induced by bariatric surgery. DESIGN: In cross-sectional studies, threshold values for insulin resistance were determined with homeostasis model assessment of insulin resistance (HOMA-IR) (algorithm based on fasting plasma glucose and insulin) in 1018 lean subjects and by hyperinsulinemic euglycemic clamp (clamp) in 26 lean women. In a cohort study on 211 patients scheduled for bariatric surgery, HOMA-IR and waist circumference were measured before and 1.5-3 years after weight reduction. In a subgroup of 53 women, insulin sensitivity was also measured using clamp. RESULTS: The threshold for insulin resistance (90th percentile) was 2.21 (mg dl(-1) fasting glucose × mU l(-1) fasting insulin divided by 405) for HOMA-IR and 6.118 (mg glucose per kg body weight per minute) for clamp. Two methods to assess reversal of insulin resistance by measuring waist circumference were used. A single cutoff value to <100 cm for waist circumference was associated with reversal of insulin resistance with an odds ratio (OR) of 49; 95% confidence interval (CI)=7-373 and P=0.0002. Also, a diagram based on initial and weight loss-induced changes in waist circumference in patients turning insulin sensitive predicted reversal of insulin resistance following bariatric surgery with a very high OR (32; 95% CI=4-245; P=0.0008). Results with the clamp cohort were similar as with HOMA-IR analyses. CONCLUSIONS: Reversal of insulin resistance could either be assessed by a diagram based on initial waist circumference and reduction of waist circumference, or by using 100 cm as a single cutoff for waist circumference after weight reduction induced by bariatric surgery.

Lipolysis index: evaluation of a new tool for metabolic assessment in epidemiological studies on obesity.

Lipid mobilization through adipocyte lipolysis is central for energy metabolism and is decreased in obesity. However, the factors of importance for lipolytic activity in the general population are not known. To further examine this we performed a cross-sectional study on teenagers and adults. We constructed and evaluated a simple index of lipolytic activity (ratio of fasting p-glycerol and body fat %) in population based samples in 316 teenagers (BMI 16-51 kg/m (2)) and 3,039 adults (BMI 16-70 kg/m (2)). In the adults, multiple regression analysis showed that waist and BMI but not age, plasma insulin, plasma noradrenaline or waist-to-hip ratio contributed independently and inversely to lipolytic activity (partial r=-0.37 and -0.28, respectively, p<0.0001). Together waist and BMI explained about 45% of the variability of lipolysis. Waist was a stronger factor than BMI in stepwise regression. The same analysis in teenagers showed that only BMI contributed independently and inversely to lipolytic activity (partial r=-0.90, p<0.0001) and explained about 55% of lipolysis variation. BMI had the strongest effect on lipolysis in lean teenagers. The results were the same for men and women. At all levels of lipolytic activity plasma fatty acid levels were elevated in obese subjects (p<0.0001). We conclude that during adolescence BMI is the major factor negatively influencing lipolytic activity, in particular among lean young subjects. In adulthood central fat accumulation together with increasing BMI decreases lipolysis. In spite of low lipolytic activity circulating fatty acid levels are increased in obesity, probably due to an adipose mass effect.

[Current classification of precursor lesions of oral squamous cell carcinoma principles of the WHO classification 2005]. / Aktuelle Klassifikation der Präkursorläsionen des oralen Plattenepithelkarzinoms Prinzipien der WHO-Klassifikation 2005.

The WHO classification of oral tumours summarizes the precancerous squamous cell lesions under the term epithelial precursor lesions. For the first time three classification schemas that histologically categorize oral epithelial precursor lesions are used analogously. According to the WHO suggestion of 2005 the traditional schema of grading dysplasia as mild dysplasia, moderate dysplasia, severe dysplasia and carcinoma in situ continues to be used. In addition the concept of intraepithelial neoplasia is introduced as squamous intraepithelial neoplasia I-III. Squamous intraepithelial neoplasia III (SIN III) combines severe dysplasia and carcinoma in situ. The Ljubljana classification of squamous intraepithelial lesions was originally established to grade laryngeal epithelial precancerous lesions. The clear and succinct nomenclature and the simple clinical utility of the Ljubljana classification have also proven to be useful for oral epithelial precursor lesions: squamous cell (simple) hyperplasia; basal/parabasal cell hyperplasia (analogous to mild dysplasia and to SIN I); atypical hyperplasia (analogous to moderate-severe dysplasia and to SIN I-III and is also called risky epithelium); carcinoma in situ (analogous to WHO carcinoma in situ and to SIN III). Atypical hyperplasia (risky epithelium) and carcinoma in situ are defined as lesions requiring either total excision or close clinical monitoring.

[Central giant cell granuloma of the mandible. Definition and differential diagnosis]. / Zentrales Riesenzellgranulom des Unterkiefers. Begriffsbestimmung und Differenzialdiagnose.

The synopsis of radiographic examination (uni- or multilocular radiolucency), histologic findings (giant cells throughout a benign fibroblastic matrix), blood chemistry analysis (normal serum parathyroid hormone) and clinical features provides the definitive diagnosis of giant cell granuloma, allowing the clearly defined surgical management of this lesion. The case history of a 48-year-old female patient who presented with a giant cell granuloma in the right mandible is used to illustrate this controversially discussed intra-osseous lesion. The potential therapeutic change from radical operative treatment, including functional maintenance, to conservative procedures is emphasized.

[Paraneoplastic pityriasis rubra pilaris in metastatic adenocarcinoma without diagnosable primary]. / Paraneoplastische Pityriasis rubra pilaris bei metastasiertem Adenokarzinom ohne lokalisierbares Primum.

HISTORY AND ADMISSION FINDINGS: A 75-year-old woman, without a history of severe illness, developed an erythematosquamous skin disease on hands and forearms. After continued spreading of these cutaneous lesions, she was admitted to hospital, presenting with a generalised desquamating erythrodermia and marked pruritus. INVESTIGATIONS: Skin biopsy showed cornocutaneous signs with alternating ortho- and parakeratosis, typical for pityriasis rubra pilaris. Laboratory findings showed a chronic to acute inflammation with leukocytosis, granulocytosis in the differential blood count, raised C-reactive protein in a range from 54.7 to a maximum of 157.2 mg/l and a protein electrophoresis with elevated alpha1- and alpha2-fraction. TREATMENT AND COURSE: The erythrodermia only temporarily receded under systemic therapy with acitretin and prednisolone. The patient developed intermittent septic fever accompanied by reduction or loss of consciousness. The general condition of the patient worsened considerably. Out of a rapidly progressing pleural effusion malignant cells similar to adenocarcinoma were isolated. Because CA15-3 was elevated we conducted an extended search especially for a breast carcinoma, but found only pathologically enlarged axillary, mediastinal and abdominal lymph nodes in conventional X-ray, CT, ultrasound and endoscopic procedures. The patient died from paraneoplastic pulmonary embolism. At autopsy, the widespread metastatic dissemination from poorly differentiated adenocarcinoma was confirmed. A necrosis in the right breast containing tumour cell remnants could probably be regarded as the primary neoplasm. Immunohistochemically no definite proof of breast nor gastro-intestinal carcinoma could be found. CONCLUSIONS: This case presents a rare paraneoplastic cutaneous manifestation as pityriasis rubra pilaris triggered by a poorly differentiated adenocarcinoma. The primary neoplasm could not definitely be identified, neither pre nor post mortem.

Modulation of survival motor neuron pre-mRNA splicing by inhibition of alternative 3' splice site pairing.

Spinal muscular atrophy is caused by the loss of functional survival motor neuron (SMN1) alleles. A translationally silent nucleotide transition in the duplicated copy of the gene (SMN2) leads to exon 7 skipping and expression of a nonfunctional gene product. It has been suggested that differential SMN2 splicing is caused by the disruption of an exonic splicing enhancer. Here we show that the single nucleotide difference reduces the intrinsic strength of the 3' splice site of exon 7 2-fold, whereas the strength of the 5' splice site of the exon 7 is not affected. Thus, a decrease in splice site strength is magnified in the context of competing exons. These data suggest that lower levels of exon 7 definition not only reduce intron 6 removal but, more importantly, increase the efficiency of the competing exon 7 skipping pathway. Antisense oligonucleotides were tested to modulate exon 7 inclusion, which contains the authentic translation stop codon. Oligonucleotides directed toward the 3' splice site of exon 8 were shown to alter SMN2 splicing in favor of exon 7 inclusion. These results suggest that antisense oligonucleotides could be used as a therapeutic strategy to counteract the progression of SMA.

Exercise in the prevention and treatment of osteoporosis: the role of physical therapy and nursing.

Exercise plays an important role in the prevention and treatment of osteoporosis. This article cites several scientific studies that support exercise as a means of increasing bone mass and strength for the prevention of osteoporosis. The ultimate goal of intervention for individuals with osteoporosis is fracture prevention. A comprehensive approach, including exercise, is outlined in this article, and the roles of the physical therapist and nurse are discussed.

The role of U2AF35 and U2AF65 in enhancer-dependent splicing.

Splicing enhancers are RNA sequence elements that promote the splicing of nearby introns. The mechanism by which these elements act is still unclear. Some experiments support a model in which serine-arginine (SR)-rich proteins function as splicing activators by binding to enhancers and recruiting the splicing factor U2AF to an adjacent weak 3' splice site. In this model, recruitment requires interactions between the SR proteins and the 35-kDa subunit of U2AF (U2AF35). However, more recent experiments have not supported the U2AF recruitment model. Here we provide additional evidence for the recruitment model. First, we confirm that base substitutions that convert weak 3' splice sites to a consensus sequence, and therefore increase U2AF binding, relieve the requirement for a splicing activator. Second, we confirm that splicing activators are required for the formation of early spliceosomal complexes on substrates containing weak 3' splice sites. Most importantly, we find that splicing activators promote the binding of both U2AF65 and U2AF35 to weak 3' splice sites under splicing conditions. Finally, we show that U2AF35 is required for maximum levels of activator-dependent splicing. We conclude that a critical function of splicing activators is to recruit U2AF to the weak 3' splice sites of enhancer-dependent introns, and that efficient enhancer-dependent splicing requires U2AF35.

Impaired energy metabolism in hearts of septic baboons: diminished activities of Complex I and Complex II of the mitochondrial respiratory chain.

Recent findings support the view that the bioenergetic part of septic organ failure is not caused by insufficient supply of oxygen but by disturbances of the mitochondrial function. Therefore, the aim of the present study was to investigate key enzymes of energy metabolism in septic hearts to answer the question whether or not impairment of mitochondrial or glycolytic enzymes occur under these conditions. For this purpose the well established model of septic baboons was used. Baboons under general anesthesia were made septic by infusion of Escherichia coli. Single challenge with infusion of high amounts of bacteria was compared with a multiple challenge protocol (less bacteria infused). Some animals obtained no E. coli (sham). The hearts of the baboons were removed after 72 h (survival: yes) or after death (survival: no) of the animals, frozen in liquid nitrogen, and stored at -80 degrees C until spectrophotometrical measurement of nine mitochondrial and glycolytic enzymes. A reduction of the activity of NADH:cytochrome-c-reductase (Complex I + III) to 67% and succinate:cytochrome-c-reductase (Complex II + III) to 45% was found in the hearts of surviving animals after infusion of high amounts of bacteria. After multiple challenge with lesser amounts of bacteria, no significant changes in enzyme activity were detectable. After lethal septic shock, activities of Complex I + III (12%) and Complex II + III (13%) as well as of phosphofructokinase (16%) were found to be strongly diminished. Decylubiquinol:cytochrome-c-reductase (Complex III, 59%), cytochrome-c-oxidase (51%), succinate dehydrogenase (60%), glucosephosphate isomerase (61%), lactate dehydrogenase (61%), and citrate synthase (120%) were less or unaffected. Similar but less pronounced effects were found after infusion of lesser amounts of bacteria. By means of inhibitor titrations of succinate: cytochrome-c-reductase, it was shown that the loss of activity is not caused by Complex III but by disturbances in Complex II. It is concluded that E. coli-induced sepsis causes decreased activities of Complex I and Complex II in baboon heart mitochondria in a dose-dependent manner.

Serine-arginine (SR)-rich splicing factors have an exon-independent function in pre-mRNA splicing.

Two distinct functions have been proposed for the serine-arginine (SR)-rich family of splicing factors. First, SR proteins are essential splicing factors and are thought to function by mediating protein-protein interactions within the intron during spliceosome assembly. Second, SR proteins bind to exonic enhancer sequences and recruit spliceosome components to adjacent introns. The latter activity is required for splice-site recognition and alternative splicing. Until now it has not been possible to determine whether the requirement for SR proteins in the basic splicing reaction is a secondary consequence of their exon-dependent recruitment function. Here we show that RNA substrates containing only 1 nt of exon sequence can undergo the first step of the splicing reaction in vitro and that this activity requires SR proteins. Thus, we provide direct evidence that SR proteins have both exon-independent and exon-dependent functions in pre-mRNA splicing.

Thermodynamic dissection of the substrate-ribozyme interaction in the hammerhead ribozyme.

The free energy of substrate binding to the hammerhead ribozyme was compared for 10 different hammerheads that differed in the length and sequence of their substrate recognition helices. These hammerheads were selected because neither ribozyme nor substrate oligonucleotide formed detectable alternate secondary structures. The observed free energies of binding varied from -8 to -24 kcal/mol and agreed very well with binding energies calculated from the nearest-neighbor free energies if a constant energetic penalty of DeltaG degreescore = +3.3 +/- 1 kcal/mol is used for the catalytic core. A set of substrates that contained a competing hairpin secondary structure showed weaker binding to the ribozyme by an amount consistent with the predicted free energy for hairpin formation. These thermodynamic conclusions permit the prediction of substrate binding affinities for ribozyme-substrate pairs of any helix length and sequence, and thus, should be very valuable for the rational design of ribozymes directed toward gene inactivation.

A systematic analysis of the factors that determine the strength of pre-mRNA splicing enhancers.

We find that the strength of splicing enhancers is determined by the relative activities of the bound serine-arginine (SR)-rich splicing factors, the number of SR proteins within the enhancer complex and the distance between the enhancer and the intron. Remarkably, the splicing activity of the bound SR proteins is directly proportional to the number of RS tetrapeptide sequences within the RS domain. Quantitative analysis of the effects of varying the distance between the enhancer and the intron revealed that the splicing efficiency is directly proportional to the calculated probability of a direct interaction between the enhancer complex and the 3' splice site. These data are consistent with a model in which splicing enhancers function by increasing the local concentration of SR proteins in the vicinity of the nearby intron through RNA looping.

The function of multisite splicing enhancers.

Splicing enhancers are RNA sequences consisting of one or more binding sites (enhancer elements) for specific serine/arginine (SR)-rich proteins. When associated with these elements, SR proteins activate splicing by recruiting the splicing machinery to the adjacent intron through protein-protein interactions. Here, we show that the rate and efficiency of splicing increases linearly, rather than synergistically, as the number of identical or nonidentical enhancer elements present on pre-mRNA is increased. We conclude that only one splicing enhancer complex at a time is capable of interacting with the constitutive splicing machinery. Thus, the function of multisite enhancer elements to increase the probability of an interaction between the enhancer complex and the splicing machinery rather than to promote functional synergy.

Use of intrinsic binding energy for catalysis by an RNA enzyme.

The contribution of several individual ribozyme.substrate base pairs to binding and catalysis has been investigated using hammerhead ribozyme substrates that were truncated at their 3' or 5' ends. The base pairs at positions 1.1-2.1 and 15.2-16.2, which flank the conserved core, each contribute 10(4)-fold in the chemical step, without affecting substrate binding. In contrast, base pairs distal to the core contribute to substrate binding but have no effect on the chemical step. These results suggest a "fraying model" in which each ribozyme.substrate helix can exist in either an unpaired ("open") state or a helical ("closed") state, with the closed state required for catalysis. The base pairs directly adjacent to the conserved core contribute to catalysis by allowing the closed state to form. Once the number of base pairs is sufficient to ensure that the closed helical state predominates, additional residues provide stabilization of the helix, and therefore increase binding, but have no further effect on the chemical step. Remarkably, the >5 kcal/mol free energy contribution to catalysis from each of the internal base pairs is considerably greater than the free energy expected for formation of a base pair. It is suggested that this unusually large energetic contribution arises because free energy that is typically lost in constraining residues within a base pair is expressed in the transition state, where it is used for positioning. This extends the concept of "intrinsic binding energy" from protein to RNA enzymes, suggesting that intrinsic binding energy is a fundamental feature of biological catalysis.

Common themes in the function of transcription and splicing enhancers.

Regulation of both transcription and RNA splicing requires enhancer elements, that is, cis-acting DNA or RNA sequences that promote the activities of linked promoters or splice sites, respectively. Both types of enhancer associate with regulatory proteins to form multicomponent enhancer complexes that recruit the necessary enzymatic machinery to promoter or splice site recognition sequences. This recruitment occurs as a result of direct interactions between regulatory proteins in the enhancer complexes and components of the basic enzymatic machineries. Recent advances suggest that the high degree of regulatory specificity observed for both transcription and splicing is due, in large part, to the multicomponent nature of enhancer complexes and to their cooperative assembly.