Toward genome editing in X-linked RP-development of a mouse model with specific treatment relevant features.

Genome editing represents a powerful tool to treat inherited disorders. Highly specific endonucleases induce a DNA double strand break near the mutant site, which is subsequently repaired by cellular DNA repair mechanisms that involve the presence of a wild type template DNA. In vivo applications of this strategy are still rare, in part due to the absence of appropriate animal models carrying human disease mutations and knowledge of the efficient targeting of endonucleases. Here we report the generation and characterization of a new mouse model for X-linked retinitis pigmentosa (XLRP) carrying a point mutation in the mutational hotspot exon ORF15 of the RPGR gene as well as a recognition site for the homing endonuclease I-SceI. Presence of the genomic modifications was verified at the RNA and protein levels. The mutant protein was observed at low levels. Optical coherence tomography studies revealed a slowly progressive retinal degeneration with photoreceptor loss starting at 9 months of age, paralleling the onset of functional deficits as seen in the electroretinogram. Early changes to the outer retinal bands can be used as biomarker during treatment applications. We further show for the first time efficient targeting using the I-SceI enzyme at the genomic locus in a proof of concept in photoreceptors following adeno-associated virus mediated gene transfer in vivo. Taken together, our studies not only provide a human-XLRP disease model but also act as a platform to design genome editing technology for retinal degenerative diseases using the currently available endonucleases.

Integrative rehabilitation of residents chronic post-stroke in skilled nursing facilities: the design and evaluation of the BrightArm Duo.

PURPOSE: To describe the novel BrightArm Duo bimanual upper extremity (UE) rehabilitation system; to determine its technology acceptance and clinical benefit for older hemiplegic participants. METHODS: The system table tilted to adjust arm gravity loading. Participants wore arm supports that sensed grasp strength and wrist position on the table. Wrist weights further increased shoulder exertion. Games were designed to improve UE strength, motor function, cognition and emotive state and adapted automatically to each participant. The system underwent feasibility trials spanning 8 weeks in two skilled nursing facilities (SNFs). Participants were evaluated pre-therapy and post-therapy using standardized clinical measures. Computerized measures of supported arm reach, table tilt and number of arm repetitions were stored on a remote server. OUTCOMES: Seven participants had significant improvements in their active range of shoulder movement, supported arm reach, shoulder strength, grasp strength and their ability to focus. The group demonstrated higher arm function measured with FMA (p = 0.01) and CAHAI (p = 0.05), and had an improvement in depression (Becks Depression Inventory, II). BrightArm Duo technology was well accepted by participants with a rating of 4.4 out of 5 points. CONCLUSIONS: Given these findings, it will be beneficial to evaluate the BrightArm Duo application in SNF maintenance programs. Implications for Rehabilitation Integrative rehabilitation that addresses both physical and cognitive domains is promising for post-stroke maintenance in skilled nursing facilities. Simultaneous bilateral arm exercise may improve arm function in older hemiplegic patients several years after stroke. Virtual reality games that adapt to the patient can increase attention and working memory while decreasing depression in elderly.

Vaccination against pandemic H1N1 (2009) in patients after allogeneic hematopoietic stem cell transplantation: a retrospective analysis.

PURPOSE: Limited data are available on immunologic responses to primary pandemic H1N1 (2009) vaccination in recipients of allogeneic hematopoietic stem cell transplantation (HSCT) recipients. In 2009 serologic responses to either pandemic H1N1 (2009) vaccine (n = 36) or pandemic H1N1 (2009) infection (n = 2) were studied in 38 HSCT recipients. METHODS: Responses were measured with a standard hemagglutination-inhibition assay. Fourteen patients had active chronic graft-versus-host disease (cGvHD) at the time of vaccination/infection and seven patients had cGvHD in remission; 11 patients had no immunosuppressive therapy, and 27 patients were on immunosuppressive therapy. Nineteen patients (53%) responded to pandemic H1N1 (2009) vaccination. Two patients had pandemic H1N1 (2009) infection without prior vaccination, and one patient had severe pandemic H1N1 (2009) infection with acute respiratory distress syndrome despite prior single vaccination. RESULTS: Non-responders to pandemic H1N1 (2009) vaccination more often had cGvHD (65 vs. 53%) and received second- or third-line therapy (53 vs. 11%), while responders mostly had first-line therapy for cGvHD. While vaccine responders had no or single agent immunosuppressive therapy, non-responders frequently received moderate or intense immunosuppressive therapy. All vaccine recipients previously treated with rituximab were non-responders. CONCLUSIONS: In summary, the overall response to pandemic H1N1 (2009) vaccination in HSCT recipients was modest. Patients receiving combined immunosuppressive therapy for steroid-refractory cGvHD barely responded to pandemic H1N1 (2009) vaccination.

Hongoquercins A and B, new sesquiterpenoid antibiotics: isolation, structure elucidation, and antibacterial activity.

Two new sesquiterpenoid antibiotics, hongoquercins A and B, were isolated from the extracts of an unidentified fungus. The structures of both metabolites were determined by spectroscopic analysis. They are related to a class of compounds commonly found in brown algae and dictyoceratid sponges. Hongoquercin A exhibited moderate activity against gram-positive bacteria.

Imprinted H19 oncofetal RNA is a candidate tumour marker for hepatocellular carcinoma.

AIMS/BACKGROUND: To study the expression of the H19 gene in hepatocellular carcinoma. H19 is an imprinted, maternally expressed gene, which is tightly linked, both physically and functionally, to the paternally expressed insulin-like growth factor 2 (IGF II). IGF II is known to be involved in liver carcinogenesis. H19 was first discovered in the fetal mouse liver to be under the same regulatory genes as alpha fetoprotein (alpha FP), a widely used tumour marker for hepatocellular carcinoma. METHODS: Using both radioactive and non-radioactive in situ hybridisation, the expression of the H19 gene was compared with the presence of alpha FP, as demonstrated by immunohistochemistry, in 18 cases of hepatocellular carcinoma. RESULTS: H19 expression was present in 13 of 18 cases, whereas staining for alpha FP was positive in only nine of 18 cases. Concordance was found in 12 of 18 tumours (66.7%). In general, the staining pattern for H19 was more diffuse than the immunohistochemical staining for alpha FP. CONCLUSIONS: The addition of a non-radioactive in situ hybridisation assay for H19 RNA to the panel of tumour markers used for the histopathological and cytological diagnosis of hepatocellular carcinoma might be useful.

Polycystic ovary syndrome. An autoimmune disease?

OBJECTIVE: To determine whether there is an autoimmune cause of polycystic ovary disease (PCOD) implicating antiovarian antibodies in the pathophysiology of the syndrome. STUDY DESIGN: This study examined 31 women diagnosed as having PCOD according to internationally recognized criteria and who attended our infertility clinic during 1994-1995. RESULTS: We could not confirm the findings of previous studies. Only one patient in our group with type II diabetes mellitus had antiovarian antibodies present in her serum. CONCLUSION: It seems that an autoimmune mechanism probably is not of prime importance in this multifaceted syndrome.

Single-case experimental designs in medical education: an innovative research method.

This paper presents an argument for more extensive use of single-case experimental research designs in medical education research. Single-case experimental designs consist of a group of experimental techniques that are widely used in the social sciences but are just beginning to be utilized by medical researchers. The method emphasizes reliable observations of behavior, repeated measurements of outcome, and individualized tailoring of objectives for each subject; all of these occur within a system that allows an experimental analysis to be conducted. Single-case designs are particularly useful when only small numbers of participants are available for a relatively long period of time. Trends in medical education toward individualized instruction, adult-centered learning, and fine-grained analyses of medical skills and knowledge make this field especially amenable to single-case experimental designs. Issues of internal and external validity, generality, practicality, and ethics are discussed, and several typical designs are illustrated. While the emergence of qualitative research methods in medical education may prove useful, single-case designs can maintain experimental science's emphasis on methodologic rigor, while allowing the flexibility often needed to conduct research in applied settings.

Expression and translocation of the chlamydial major outer membrane protein in Escherichia coli.

The entire gene encoding the major outer membrane protein (MOMP) from Chlamydia psittaci strain GPIC has been cloned and expressed in Escherichia coli. A tightly regulated T7 promoter is used to control expression of the protein in Escherichia coli. Upon induction of expression, the precursor (pre-MOMP) is synthesized in the cell. This is followed by the appearance of a lower molecular weight protein that comigrates with mature MOMP from chlamydial elementary bodies by both one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis. When E. coli cells expressing MOMP are converted to spheroplasts and subjected to protease treatment, MOMP is quantitatively degraded while cytoplasmic pre-MOMP is protected from degradation. Whole cells subjected to the same protease treatment show no degradation of MOMP. Furthermore, MOMP is not detected in surface-labeling experiments using several MOMP-specific antibodies. These data indicate that pre-MOMP is translocated to the periplasmic space and processed but is not surface exposed in E. coli. Expression of MOMP in this system causes a significant reduction in cell viability. In addition, coexpression in E. coli of MOMP or a MOMP-PhoA fusion with various chaperone proteins does not alter the level of MOMP translocation.

A study on the mechanism of action of sceptrin, an antimicrobial agent isolated from the South Pacific sponge Agelas mauritiana.

The mechanism of action of sceptrin, an antimicrobial agent isolated from the sponge Agelas mauritiana, was investigated. Sceptrin has been reported to exhibit antibacterial and antifungal activities. In our studies, sceptrin demonstrated a bacteriostatic rather than bactericidal effect on exponentially growing Escherichia coli cells at the MIC. Under these conditions, the culture produced chains of cells, and incorporation of radio-labelled precursors into DNA, protein, and cell wall was unaffected, whereas incorporation of 3H-uridine into RNA was slightly inhibited. At concentrations higher than the MIC, sceptrin was bactericidal, inhibited the incorporation of all radiolabelled precursors, and induced the formation of unusual spheroplasts. Peptidoglycan turnover in E. coli appeared to be stimulated by sceptrin as demonstrated by a release of diaminopimelic acid-containing high molecular weight material. Subsequent studies of the release of potassium ions from E. coli and the lysis of red blood cells suggested that sceptrin disrupts the cell membranes of both prokaryotic and eukaryotic cells. It is proposed that spheroplasts formation may reflect a cell wall effect that occurs subsequent to membrane damage.

Blood rheology in acute myocardial infarction: effects of high-dose i.v. streptokinase compared to placebo.

To compare the haemorheological effects of an i.v. infusion of 1.5 MU of streptokinase with placebo, we investigated the time course of plasma fibrinogen concentration and the haemorheologic parameters plasma viscosity, erythrocyte aggregation and whole blood viscosity at different shear rates during the early phase of acute myocardial infarction until week 3 in 38 unselected patients from the ISAM and ISIS-2 study. Within 3 h, streptokinase led to a near afibrinogenaemia lasting for more than 24 h. Concomitantly, with streptokinase we found a reduction of plasma viscosity, erythrocyte aggregation and whole blood viscosity, whereas with placebo, values showed a slight increase, resulting in significant differences between the groups within the first 2 days. Thereafter, both groups showed an increase in all parameters, values reaching a maximum after 1 week. The streptokinase-induced reduction in blood viscosity may lead to an improvement in microcirculation in the infarction area during the early phase, whereas the hyperviscosity observed independently of therapy after 1 week may lead to an impairment of microcirculation.

Oxygen regulated 80 kDa protein and glucose regulated 78kDa protein are identical.

Ischemic stress of cells within solid tumors arises from inadequate perfusion of regions of the tumor and results in microenvironments which are hypoxic and deficient in nutrient delivery and waste product removal. Stressed cells within these microenvironments show growth inhibition and synthesize unique sets of proteins referred to as glucose and oxygen regulated proteins (GRPs and ORPs respectively). The commonality of proteins induced by glucose-starvation and hypoxia has not been proven. To this end, ORPs were induced in Chinese hamster ovary cells in the presence of high glucose concentration in the media and ORP 80 isolated from two dimension gels. Eleven tryptic peptides of the 80 kDa ORP were sequenced and found to be identical to GRP 78 sequences. The data demonstrate that GRP 78 and ORP 80 have the same primary amino acid sequence and suggest that glucose-starvation and hypoxia can induce the same cellular responses.

[A comparison of different methods of calculating clearances using 99mTc-mercaptoacetyltriglycine and 123I- or 131I-ortho-iodohippuric acid]. / Vergleich verschiedener Clearance-Berechnungs-methoden bei Verwendung von 99mTc-Mercaptoacetyltriglycin sowie 123J- oder 131J-Orthojod-hippursäure.

Using labelled MAG3 (n = 172) or OIH (n = 102) the results of three different techniques of calculating renal clearance were compared. The following techniques were used: Tauxe (single plasma concentration determination), Oberhausen (modified, combined partial body and plasma disappearance clearance) and Winkler (partial body clearance). For OIH, linear regression analysis revealed a positive correlation of Tauxe and Oberhausen clearances with a correlation coefficient (r) of 0.90 and a mean ratio (mean) Oberhausen/Tauxe clearances of 1.03. For MAG3, the Oberhausen values were found to be significantly lower than the Tauxe values with an equally good correlation. For this radiopharmaceutical the Winkler values were significantly higher than the Tauxe and Oberhausen values. For OIH, no such difference was noted. However, the correlation coefficient was significantly lower. Whereas with the Tauxe method, a mean ratio MAG3-/OIH clearances of 0.62 was observed in an earlier investigation, from the present data a ratio of 0.53 is calculated for the Oberhausen clearance. The difference between the Tauxe and Oberhausen clearances with MAG3 is of importance when comparing clearance values obtained with clearance calculation techniques and for establishing normal values for the MAG3 clearance.

Identification of consensus epitope structures expressed in recombinant DNA libraries.

In the course of screening cDNA expression libraries with a monospecific polyclonal antibody to topoisomerase I, we isolated three different immunopositive cDNA clones. By comparing their derived amino acid sequences, a consensus region of similarity in otherwise completely dissimilar sequences was identified as an epitope. The approach described here should identify both continuous and discontinuous topological epitopes. The probability of occurrence of "spurious" immunopositive clones is shown to depend on the number of codons of each critical amino acid within the antigenic determinant.

Subacute and chronic cerebral infarctions: SPECT and gadolinium-DTPA enhanced MR imaging.

Single photon emission computed tomography (SPECT) examinations of regional cerebral blood flow (rCBF) and regional cerebral blood volume (rCBV) were carried out in 23 patients with 19 subacute and 7 chronic cerebral infarctions. Corresponding magnetic resonance (MR) examinations (unenhanced and contrast enhanced scans) were done within a time interval of 2 days. Although all subacute lesions showed an increased rCBV, the rCBF could be either increased or decreased. Contrast enhancement in MR was associated with increased rCBV but not with increased rCBF. In all chronic lesions rCBV and rCBF were decreased and there was no contrast enhancement detectable in MR. The application of Gd-diethylenetriamine pentaacetic acid as contrast agent in MR demonstrates blood-brain barrier disruption as focal or gyral contrast enhancement in cerebral infarction. The SPECT examinations revealed an increased rCBV indicating a vasodilation or vasoparalysis. The rCBF was either increased or decreased depending on the collateral formation or recanalization of occluded vessels.

Grand multiparity: an obstetric or neonatal risk factor?

Grand multiparity has been considered to be a factor in maternal and neonatal morbidity. In addition, families with seven or more children have been associated with low socioeconomic status. To minimize the confounding effect of the socioeconomic status, the outcome of grand multiparity has been investigated in a mostly homogeneous, ultraorthodox Jewish community in Jerusalem, Israel. A total of 5916 deliveries in one community hospital (Bikur Cholim) were studied, of which 893 (13%) occurred in mothers who had given birth to seven or more infants. There was a significant decrease in the incidence of small for gestational age infants among the grand multiparous women (3.6% as opposed to 5.8% in the control population). This difference was independent of maternal age. Moreover, grand multiparous women gave birth to significantly more large for gestational age infants. No increase in obstetric complications or neonatal morbidity and mortality was found among the offspring of the grand multiparous mothers. Having taken socioeconomic status into account, we conclude that grand multiparity does not carry an increased risk of perinatal morbidity or mortality.

PIP: Grand multiparity has been considered to be a factor in maternal and neonatal morbidity. In addition, families with 7 or more children have been associated with low socioeconomic status. To minimize the confounding effect of the socioeconomic status, the outcome of grand multiparity has been investigated in a mostly homogeneous, ultraorthodox Jewish community in Jerusalem, Israel. A total of 5916 deliveries in 1 community hospital (Bikur Cholim) were studied, of which 893 (13%) occurred in mothers who had given birth to 7 or more infants. There was a significant decrease in the incidence of small for gestational age infants among the grand multiparous women (3.6% as opposed to 5.8% in the control population). This difference was independent of maternal age. Moreover, grand multiparous women gave birth to significantly more large for gestational age infants. No increase in obstetric complications or neonatal morbidity was found among the offspring of the grand multiparous mothers. Taking socioeconomic status into account, it is concluded that grand multiparity does not carry an increased risk of perinatal morbidity or mortality.