Novel indices reveal that pollinator exposure to pesticides varies across biological compartments and crop surroundings.

Declines in insect pollinators have been linked to a range of causative factors such as disease, loss of habitats, the quality and availability of food, and exposure to pesticides. Here, we analysed an extensive dataset generated from pesticide screening of foraging insects, pollen-nectar stores/beebread, pollen and ingested nectar across three species of bees collected at 128 European sites set in two types of crop. In this paper, we aimed to (i) derive a new index to summarise key aspects of complex pesticide exposure data and (ii) understand the links between pesticide exposures depicted by the different matrices, bee species and apple orchards versus oilseed rape crops. We found that summary indices were highly correlated with the number of pesticides detected in the related matrix but not with which pesticides were present. Matrices collected from apple orchards generally contained a higher number of pesticides (7.6 pesticides per site) than matrices from sites collected from oilseed rape crops (3.5 pesticides), with fungicides being highly represented in apple crops. A greater number of pesticides were found in pollen-nectar stores/beebread and pollen matrices compared with nectar and bee body matrices. Our results show that for a complete assessment of pollinator pesticide exposure, it is necessary to consider several different exposure routes and multiple species of bees across different agricultural systems.

Endothelial Cell Dysfunction and Hypoxia as Potential Mediators of Pain in Fabry Disease: A Human-Murine Translational Approach.

Fabry disease (FD) is caused by α-galactosidase A (AGAL) enzyme deficiency, leading to globotriaosylceramide accumulation (Gb3) in several cell types. Pain is one of the pathophysiologically incompletely understood symptoms in FD patients. Previous data suggest an involvement of hypoxia and mitochondriopathy in FD pain development at dorsal root ganglion (DRG) level. Using immunofluorescence and quantitative real-time polymerase chain reaction (qRT PCR), we investigated patient-derived endothelial cells (EC) and DRG tissue of the GLA knockout (KO) mouse model of FD. We address the question of whether hypoxia and mitochondriopathy contribute to FD pain pathophysiology. In EC of FD patients (P1 with pain and, P2 without pain), we found dysregulated protein expression of hypoxia-inducible factors (HIF) 1a and HIF2 compared to the control EC (p < 0.01). The protein expression of the HIF downstream target vascular endothelial growth factor A (VEGFA, p < 0.01) was reduced and tube formation was hampered in the P1 EC compared to the healthy EC (p < 0.05). Tube formation ability was rescued by applying transforming growth factor beta (TGFß) inhibitor SB-431542. Additionally, we found dysregulated mitochondrial fusion/fission characteristics in the P1 and P2 EC (p < 0.01) and depolarized mitochondrial membrane potential in P2 compared to control EC (p < 0.05). Complementary to human data, we found upregulated hypoxia-associated genes in the DRG of old GLA KO mice compared to WT DRG (p < 0.01). At protein level, nuclear HIF1a was higher in the DRG neurons of old GLA KO mice compared to WT mice (p < 0.01). Further, the HIF1a downstream target CA9 was upregulated in the DRG of old GLA KO mice compared to WT DRG (p < 0.01). Similar to human EC, we found a reduction in the vascular characteristics in GLA KO DRG compared to WT (p < 0.05). We demonstrate increased hypoxia, impaired vascular properties, and mitochondrial dysfunction in human FD EC and complementarily at the GLA KO mouse DRG level. Our data support the hypothesis that hypoxia and mitochondriopathy in FD EC and GLA KO DRG may contribute to FD pain development.

A whole-brain connectivity map of mouse insular cortex.

The insular cortex (IC) plays key roles in emotional and regulatory brain functions and is affected across psychiatric diseases. However, the brain-wide connections of the mouse IC have not been comprehensively mapped. Here, we traced the whole-brain inputs and outputs of the mouse IC across its rostro-caudal extent. We employed cell-type-specific monosynaptic rabies virus tracings to characterize afferent connections onto either excitatory or inhibitory IC neurons, and adeno-associated viral tracings to label excitatory efferent axons. While the connectivity between the IC and other cortical regions was highly bidirectional, the IC connectivity with subcortical structures was often unidirectional, revealing prominent cortical-to-subcortical or subcortical-to-cortical pathways. The posterior and medial IC exhibited resembling connectivity patterns, while the anterior IC connectivity was distinct, suggesting two major functional compartments. Our results provide insights into the anatomical architecture of the mouse IC and thus a structural basis to guide investigations into its complex functions.

Treatment with mononuclear cell populations improves post-infarction cardiac function but does not reduce arrhythmia susceptibility.

BACKGROUND: Clinical and experimental data give evidence that transplantation of stem and progenitor cells in myocardial infarction could be beneficial, although the underlying mechanism has remained elusive. Ventricular tachyarrhythmia is the most frequent and potentially lethal complication of myocardial infarction, but the impact of mono nuclear cells on the incidence of ventricular arrhythmia is still not clear. OBJECTIVE: We aimed to characterize the influence of splenic mononuclear cell populations on ventricular arrhythmia after myocardial infarction. METHODS: We assessed electrical vulnerability in vivo in mice with left ventricular cryoinfarction 14 days after injury and intramyocardial injection of specific subpopulations of mononuclear cells (MNCs) (CD11b-positive cells, Sca-1-positive cells, early endothelial progenitor cells (eEPCs)). As positive control group we used embryonic cardiomyocytes (eCMs). Epicardial mapping was performed for analysing conduction velocities in the border zone. Left ventricular function was quantified by echocardiography and left heart catheterization. RESULTS: In vivo pacing protocols induced ventricular tachycardia (VT) in 30% of non-infarcted mice. In contrast, monomorphic or polymorphic VT could be evoked in 94% of infarcted and vehicle-injected mice (p<0.01). Only transplantation of eCMs prevented post-infarction VT and improved conduction velocities in the border zone in accordance to increased expression of connexin 43. Cryoinfarction resulted in a broad aggravation of left ventricular function. All transplanted cell types augmented left ventricular function to a similar extent. CONCLUSIONS: Transplantation of different MNC populations after myocardial infarction improves left ventricular function similar to effects of eCMs. Prevention of inducible ventricular arrhythmia is only seen after transplantation of eCMs.

Inflatable penile prosthesis as tissue expander: what is the evidence?

ABSTRACT Objective: Many patients who undergo inflatable penile prosthesis (IPP) replacement are often upsized to larger cylinders, suggesting the IPP may serve as a tissue expander and increase internal penile length. The objective of this study is to evaluate whether cylinder length increases with subsequent IPP insertion. Materials and Methods: We queried American Medical Systems and Coloplast Patient Information Form databases to identify patients who underwent IPP placement and replacement between 2004-2013. Patients were grouped by device type and time to replacement (<2 or ≥2 years). We selected the 2-year mark for subgroup analysis to allow time for tissue expansion to occur and to exclude patients who underwent early explantation (e.g. erosion or infection). Results: Two thousand, seven hundred and forty nine patients (1,532 AMS 700 LGX, 717 AMS 700 CX, and 500 Coloplast Titan) met the inclusion criteria. Mean time between implants was earlier for LGX (29 months) than CX (39 months) and Titan (48 months) patients (p<0.001). Patients who underwent device replacement at <2 years did not experience an increase in mean cylinder length. On the contrary, patients who underwent device replacement at ≥2 years did experience significant increases in mean cylinder length (LGX 1.2 cm, CX 1.1 cm, and Titan 0.9 cm, p<0.001). The mean increases in length at ≥2 years were similar between the 3 devices (p=0.20). Sixty percent of patients demonstrated increases of >0.5 cm and 40% demonstrated increases of ≥1 cm. Conclusions: As demonstrated, the IPP may provide tissue expansion over time. Further evaluation is needed to determine if increased cylinder length correlates to increased functional length and patient satisfaction.

Standing cough test for evaluation of post-prostatectomy incontinence: a pilot study.

INTRODUCTION: We implemented a standardized Standing Cough Test (SCT) for assessment of men with post-prostatectomy incontinence (PPI) and stratified results according to an objective clinical grading scale in an attempt to facilitate male anti-incontinence surgical procedure selection. MATERIALS AND METHODS: SCT was routinely performed during the initial outpatient consultation for PPI. Incontinence severity was recorded based on a novel Male Stress Incontinence Grading Scale (MSIGS) to stratify PPI. Each patient was assigned an incontinence grade score of 0 through 4 during the SCT. Men with mild stress urinary incontinence (SUI) (grades 0-2) were offered sling surgery while those with heavier SUI (grades 3-4) were offered artificial urinary sphincter (AUS). MSIGS grade was correlated to preoperative patient-reported pads per day (PPD), and patient-reported outcomes of anti-incontinence surgery were assessed. RESULTS: Among 62 consecutive PPI patients, 20 (32%) were graded as mild based on SCT, while the majority (42/62, 68%) were graded as moderate-severe. Average time from prostatectomy to treatment was 6 years. MSIGS grade demonstrated a strong correlation with preoperative PPD (r = 0.74). Among the 53 patients who underwent surgery for PPI, 14 with mild SUI received a sling, while 39 (74%) more severe cases received an AUS. Patient-reported improvement was high overall in both groups (median 95%). CONCLUSION: Most men with chronic PPI present for definitive treatment in a delayed manner after prostatectomy despite having severe incontinence. The SCT provides immediate, objective information about the severity of PPI which strongly correlates with patient-reported pads-per-day and may expedite anti-incontinence surgical procedure selection.

Low Serum Testosterone Level Predisposes to Artificial Urinary Sphincter Cuff Erosion.

OBJECTIVE: To examine the association between decreased serum testosterone levels and artificial urinary sphincter (AUS) cuff erosion. MATERIALS AND METHODS: We evaluated serum testosterone levels in 53 consecutive patients. Low testosterone was defined as <280 ng/dL and found in 30/53 patients (56.6%). Chi-square and Student t tests, Kaplan-Meier analysis, binary logistic regression, and Cox regression analysis were used to determine statistical significance. RESULTS: Nearly all men with AUS cuff erosions had low serum testosterone (18/20, 90.0%) compared to those without erosions (12/33, 36.4%, P < .001). Mean time to erosion was 1.70 years (0.83-6.86); mean follow-up was 2.76 years (0.34-7.92). Low testosterone had a hazard ratio of 7.15 for erosion in a Cox regression analysis (95% confidence interval 1.64-31.17, P = .009) and Kaplan-Meier analysis demonstrated decreased erosion-free follow-up (log-rank P = .002). Low testosterone was the sole independent risk factor for erosion in a multivariable model including coronary artery disease and radiation (odds ratio 15.78; 95% confidence interval 2.77-89.92, P = .002). Notably, history of prior AUS, radiation, androgen ablation therapy, or concomitant penile implant did not confound risk of cuff erosion in men with low testosterone levels. CONCLUSION: Men with low testosterone levels are at a significantly higher risk to experience AUS cuff erosion. Appropriate counseling before AUS implantation is warranted and it is unclear whether testosterone resupplementation will mitigate this risk.

Rapid excision of massive localized lymphedema of the male genitalia with vessel sealing device.

INTRODUCTION: To present a series of patients who underwent surgical treatment for massive localized lymphedema (MLL) of the male genitalia and explore the utility of the LigaSure hemostatic vessel sealing device (VSD) for resection of advanced cases. MATERIALS AND METHODS: Although conservative and microsurgical treatments have been reported, MLL of the male genitalia requires open surgical resection with primary reconstruction. We reviewed our prospectively maintained database of all lymphedema excisions performed between January 2007 and December 2014 comparing resection with Bovie electrocautery to resection with the LigaSure VSD. Our analysis focused on any significant differences in rate of resection, estimated blood loss (EBL), and recurrence. RESULTS: Nineteen patients with MLL of the male genitalia underwent excision with either LigaSure (8 patients) or conventional Bovie electrocautery (11 patients). Rate of resection was significantly faster with LigaSure compared to Bovie (33.74 g/min versus 5.32 g/min, p = .035). Additionally, estimated EBL per gram of tissue resected was decreased in the LigaSure group (0.41 mL/g versus 0.17 mL/g, p = .057). Two of the 11 Bovie patients (18%) had recurrence of lymphedema requiring repeat resection, while none of the LigaSure patients developed recurrence. CONCLUSIONS: Resection of genital lymphedema using the LigaSure device offers promising results in managing advanced MLL of the male genitalia with the potential for faster resections, less EBL per tissue resected, and a lower rate of recurrence.

Male Stress Incontinence Grading Scale (MSIGS) for Evaluation of Men with Post-Prostatectomy Incontinence: A Pilot Study.

INTRODUCTION: We developed an objective clinical grading scale to characterize post-prostatectomy incontinence (PPI) and evaluated its use as a tool to facilitate male anti-incontinence procedure selection. PROTOCOL: Between September 2014 and July 2015, we prospectively implemented a novel Male Stress Incontinence Grading Scale (MSIGS) to stratify PPI patients based on incontinence severity. Patients included were those referred for PPI who had no prior anti-incontinence surgery. During the initial outpatient consultation, each patient was prospectively assigned an incontinence grade score of 0 through 4 based on the consensus of 2 examiners performing a standing cough test (SCT). All patients refrained from voiding for at least 60 minutes prior to the SCT. Men with mild SUI (MSIGS grades 0-2) were offered AdVance sling surgery while those with heavier SUI (MSIGS grades 3-4) were offered artificial urinary sphincter (AUS). MSIGS grade was correlated to patient-reported pads-per-day (PPD), and patient-reported outcomes of anti-incontinence surgery were assessed. OUTCOME: Of 62 consecutive new PPI patients, 20 were graded as mild based on SCT [five (8%) grade 0, 10 (16%) grade 1, five (8%) grade 2] while 42 were graded as moderate-severe [10 (16%) grade 3, 32 (52%) grade 4]. MSIGS grade demonstrated a strong correlation with preoperative PPD (r=0.74). Among the 53 patients who underwent surgical intervention for PPI, 14 with mild SUI were treated with AdVance® male urethral sling (MSIGS grade 0, 1, or 2) while 39 more severe cases received AUS (MSIGS grade 3 or 4). Patient-reported improvement was high overall (median 95%) and similar for sling and AUS patients (95% and 96.5% respectively, p=0.596). The median time from radical prostatectomy to anti-incontinence surgery was 5.4 years (range 1-20). DISCUSSION: The Male Stress Incontinence Grading Scale provides a rapid, simple, non-invasive, objective assessment of PPI severity which strongly correlates with patient-reported pads-per-day and appears to facilitate anti-incontinence surgical procedure selection.

Smoking Cessation Intervention on Facebook: Which Content Generates the Best Engagement?

BACKGROUND: Social media offer a great opportunity to deliver smoking cessation treatment to young adults, but previous online and social media interventions targeting health behavior change have struggled with low participant engagement. We examined engagement generated by content based on the Transtheoretical Model of Behavior Change (TTM) in a motivationally tailored smoking cessation intervention on Facebook. OBJECTIVE: This study aimed to identify which intervention content based on the TTM (Decisional Balance and 10 processes of change) generated the highest engagement among participants in pre-action stages of change (Precontemplation, Contemplation, and Preparation). METHODS: Participants (N=79, 20% female, mean age 20.8) were assessed for readiness to quit smoking and assigned to one of 7 secret Facebook groups tailored to their stage of change. Daily postings to the groups based on TTM Decisional Balance and the 10 processes of change were made by research staff over 3 months. Engagement was operationalized as the number of participant comments to each post. TTM content-based predictors of number of comments were analyzed and stratified by baseline stage of change, using negative binomial regression analyses with and without zero inflation. RESULTS: A total of 512 TTM-based posts generated 630 individual comments. In Precontemplation and Contemplation groups, Decisional Balance posts generated above average engagement (P=.01 and P<.001). In Contemplation groups, posts based on the TTM processes Dramatic Relief and Self-Liberation resulted in below average engagement (P=.01 and P=.005). In Preparation groups, posts based on Consciousness Raising generated above average engagement (P=.009). Participant engagement decreased over time and differed between groups within Precontemplation and Contemplation stages, but was independent of day of the week and time of day the content was posted to the groups. No participant baseline characteristics significantly predicted engagement. CONCLUSIONS: Participants not ready to quit in the next 30 days (in Precontemplation or Contemplation) engaged most when prompted to think about the pros and cons of behavior change, while those in the Preparation stage engaged most when posts increased awareness about smoking and smoking cessation. Findings support tailoring intervention content to readiness to quit and suggest intervention components that may be most effective in generating high participant engagement on social media.

'7-flap' perineal urethrostomy: an effective option for obese men with devastated urethras.

INTRODUCTION: To present an updated experience using our previously reported lateral perineal '7-flap' technique for perineal urethrostomy (PU), highlighting its role in a variety of patients with advanced urethral stricture disease. MATERIALS AND METHODS: All patients who underwent 7-flap PU from 2009-2013 were reviewed. PU was constructed by advancing a "7"-shaped laterally based perineal skin flap into a spatulated, amputated bulbomembranous urethra. The contralateral side of the amputated proximal urethra was then matured to the advanced perineal skin. Patients were stratified by body mass index (BMI) and outcomes were compared. RESULTS: Among 748 patients undergoing urethroplasty during the study period, 22 men (2.9%; mean age 61, range 31-80) received a 7-flap PU for advanced stricture disease (mean follow up 32 months). A majority of patients (14/22, 64%) were obese (BMI = 30). Disease etiologies consisted primarily of lichen sclerosus (9/22, 41%) while 6/22 (27%) had failed prior urethral reconstructions elsewhere. Mean operative time was 108 min (range 54-214), mean estimated blood loss (EBL) was 76 cc (30-200), and all patients were discharged immediately after surgery. Urethrostomy creation was possible in all patients regardless of BMI (mean 33, range 22-43), and there were no differences with regards to EBL (p = 0.71), operative time (p = 0.38), or success rate (p = 0.76) in obese versus non-obese patients undergoing 7-flap PU. Nearly all patients (21/22, 95%) are voiding spontaneously on follow up without the need for any additional procedure. CONCLUSION: In our updated experience, performance of 7-flap urethrostomy has resulted in durable long term success with acceptable performance in technically challenging cases.

Intraoperative Decision-making for Precise Penile Straightening During Inflatable Penile Prosthesis Surgery.

OBJECTIVE: To present a novel algorithm for definitive reconstruction of penile curvature in men undergoing inflatable penile prosthesis (IPP) surgery as an alternative to manual penile modeling and grafting procedures. METHODS: Patients with erectile dysfunction and concomitant penile curvature undergoing IPP placement were divided into 2 treatment groups: (1) group 1, penile deformity known preoperatively, and (2) group 2, penile curvature recognized intraoperatively after IPP placement. Group 1 patients underwent penile plication after artificial erection and immediately before IPP insertion via the same penoscrotal incision, whereas group 2 patients were treated with a Yachia (Heineke-Mikulicz) corporoplasty over the intact cylinders. Patients completed postoperative Patient Global Impression of Improvement (PGI-I) questionnaires assessing overall satisfaction. RESULTS: Among 405 men receiving IPP at our institution from 2007 to 2014, 30 patients received synchronous correction of penile curvature (7%). Group 1 included 23 of 30 (77%) patients, and 7 of 30 (23%) were in group 2. Overall mean initial curvature was 36°, and all patients were corrected to < 10°. Average operative times were 18 minutes longer compared with patients who underwent IPP placement alone (82 vs 64 minutes, P <.05). At an average follow-up of 13 months (range 7-32), 19 of 20 (95%) group 1 and 6 of 7 (86%) group 2 patients who completed surveys reported an improved overall condition. No patient reported chronic pain, recurrent deformity, or device malfunction. CONCLUSION: Penile curvature can be safely and reliably corrected at the time of IPP placement, regardless of whether the deformity was identified preoperatively.

Repeat Excision and Primary Anastomotic Urethroplasty for Salvage of Recurrent Bulbar Urethral Stricture.

PURPOSE: We compared the results of initial excision and primary anastomosis urethroplasty to the excision and primary anastomosis outcomes of other challenging reoperative clinical settings, including secondary cases (prior urethroplasty of any technique other than excision and primary anastomosis) and repeat cases (prior excision and primary anastomosis). MATERIALS AND METHODS: We reviewed our database of patients who underwent excision and primary anastomosis urethroplasty for bulbar urethral stricture at our tertiary referral center from 2007 to 2014. Patients without available data and those with a history of lichen sclerosus, radiation, pelvic fracture urethral injuries, distal strictures and/or hypospadias were excluded from analysis. Patient characteristics and outcomes were compared between those undergoing initial, secondary, and repeat excision and primary anastomosis urethroplasty for bulbar urethral stricture. RESULTS: Among 898 urethroplasties performed during the study period we identified 305 men who underwent excision and primary anastomosis urethroplasty of the bulbar urethra, including an initial procedure in 268 of 305 (88%) and reoperation in 37 (12%). Of patients with reoperation 18 of 37 (49%) underwent secondary excision and primary anastomosis following a different type of prior urethroplasty and 19 (51%) underwent repeat excision and primary anastomosis. Repeat excision and primary anastomosis in the bulbar urethra was successful in 18 of 19 patients (95%), which was comparable to the success rate of initial bulbar excision and primary anastomosis (251 of 268 or 94%) as well as secondary bulbar excision and primary anastomosis (17 of 18 or 94%, p = 0.975) with a similar mean stricture length. Mean followup for all patients was 41.5 months (range 6 to 90) and mean followup in each group was greater than 30 months. CONCLUSIONS: Repeat excision and primary anastomosis urethroplasty has excellent results for short bulbar strictures, comparable to those achieved in the initial and secondary setting.

Ventral Slit Scrotal Flap: A New Outpatient Surgical Option for Reconstruction of Adult Buried Penis Syndrome.

INTRODUCTION: We present a novel technique using ventral slit with scrotal skin flaps (VSSF) for the reconstruction of adult buried penis without skin grafting. TECHNICAL CONSIDERATIONS: An initial ventral slit is made in the phimotic ring, and the penis is exposed. To cover the defect in the ventral shaft skin, local flaps are created by making a ventral midline scrotal incision with horizontal relaxing incisions. The scrotal flaps are rotated to resurface the ventral shaft. Clinical data analyzed included preoperative diagnoses, length of stay, blood loss, and operative outcomes. Complications were also recorded. Fifteen consecutive patients with a penis trapped due to lichen sclerosus (LS) or phimosis underwent repair with VSSF. Each was treated in the outpatient setting with no perioperative complications. Mean age was 51 years (range, 26-75 years), and mean body mass index was 42.6 kg/m(2) (range, 29.8-53.9 kg/m(2)). The majority of patients (13 of 15, 87%) had a pathologic diagnosis of LS. Mean estimated blood loss was 57 cc (range, 25-200 cc), mean operative time was 83 minutes (range, 35-145 minutes), and all patients were discharged on the day of surgery. The majority of patients (11 of 15, 73.3%) remain satisfied with their results and have required no further intervention. Recurrences in 3 of 15 (20.0%) were due to LS, panniculus migration, and concealment by edematous groin tissue; 2 of these patients underwent subsequent successful skin grafting. CONCLUSION: VSSF is a versatile, safe, and effective reconstructive option in appropriately selected patients with buried penis, which enables reconstruction of penile shaft skin defects without requiring complex skin grafting.

Does Pressure Regulating Balloon Location Make a Difference in Functional Outcomes of Artificial Urinary Sphincter?

PURPOSE: We compared functional outcomes in patients who received an artificial urinary sphincter in the space of Retzius vs the same device placed at a high submuscular location. MATERIALS AND METHODS: We reviewed a prospectively maintained database of patients who received an artificial urinary sphincter between July 2007 and December 2014. After cuff placement was completed via a perineal incision, a 61 to 70 cm H2O pressure regulating balloon was placed through a separate high scrotal incision in the space of Retzius or in a high submuscular tunnel. Demographics, perioperative comorbidities and functional outcomes were compared between the groups. RESULTS: A total of 294 consecutive patients underwent artificial urinary sphincter placement. Mean followup was 23 months. Space of Retzius and high submuscular placement was performed in 140 (48%) and 154 patients (52%), respectively. Functional outcomes were similar between the groups, including the continence rate (defined as 0 or 1 pad daily) in 81% vs 88% (p = 0.11), the erosion rate in 9% vs 8% (p = 0.66) and the explantation rate in 10% vs 11% (p = 0.62). Artificial urinary sphincter revision for persistent incontinence was required in a similar proportion of the 2 groups (13% vs 8%, p = 0.16) with a comparable mean followup (24 vs 23 months, p = 0.30). Kaplan-Meier analysis revealed no difference between the groups in the rate of explantation (p = 0.70) or revision (p = 0.06). CONCLUSIONS: High submuscular placement of a pressure regulating balloon at artificial urinary sphincter surgery is a safe, effective alternative with functional outcomes equivalent to those of traditional placement in the space of Retzius.

Embryonic cardiomyocyte, but not autologous stem cell transplantation, restricts infarct expansion, enhances ventricular function, and improves long-term survival.

AIMS: Controversy exists in regard to the beneficial effects of transplanting cardiac or somatic progenitor cells upon myocardial injury. We have therefore investigated the functional short- and long-term consequences after intramyocardial transplantation of these cell types in a murine lesion model. METHODS AND RESULTS: Myocardial infarction (MI) was induced in mice (n = 75), followed by the intramyocardial injection of 1-2×10(5) luciferase- and GFP-expressing embryonic cardiomyocytes (eCMs), skeletal myoblasts (SMs), mesenchymal stem cells (MSCs) or medium into the infarct. Non-treated healthy mice (n = 6) served as controls. Bioluminescence and fluorescence imaging confirmed the engraftment and survival of the cells up to seven weeks postoperatively. After two weeks MRI was performed, which showed that infarct volume was significantly decreased by eCMs only (14.8±2.2% MI+eCM vs. 26.7±1.6% MI). Left ventricular dilation was significantly decreased by transplantation of any cell type, but most efficiently by eCMs. Moreover, eCM treatment increased the ejection fraction and cardiac output significantly to 33.4±2.2% and 22.3±1.2 ml/min. In addition, this cell type exclusively and significantly increased the end-systolic wall thickness in the infarct center and borders and raised the wall thickening in the infarct borders. Repetitive echocardiography examinations at later time points confirmed that these beneficial effects were accompanied by better survival rates. CONCLUSION: Cellular cardiomyoplasty employing contractile and electrically coupling embryonic cardiomyocytes (eCMs) into ischemic myocardium provoked significantly smaller infarcts with less adverse remodeling and improved cardiac function and long-term survival compared to transplantation of somatic cells (SMs and MSCs), thereby proving that a cardiomyocyte phenotype is important to restore myocardial function.

Transcriptional regulation of serine/threonine kinase-15 (STK15) expression by hypoxia and HIF-1.

The serine/threonine kinase-15 (STK15) acts as a cell cycle regulator being overexpressed in various tumors. One mechanism that could contribute to overexpression of STK15 is tumor hypoxia where hypoxia-inducible factor-1 (HIF-1) is a major regulator of transcription. Therefore, we analyzed whether hypoxia and HIF-1 could contribute to overexpression of STK15. We found that hypoxia increased STK15 expression and STK15 promoter activity in HepG2 tumor cells. Overexpression of HIF-1 alpha induced STK15 gene transcription, whereas HIF-1 alpha siRNA and overexpression of prolyl hydroxylase 2 (PHD-2), a negative regulator of HIF-1 alpha, reversed this effect. In addition, site-directed mutagenesis experiments and chromatin immunoprecipitation revealed that from the three putative hypoxia responsive elements (HRE) within the STK15 promoter only HRE-2 was functional and bound HIF-1. Further, siRNA against STK15 inhibited proliferation of HepG2 cells induced by hypoxia. These results show that STK15 gene transcription can be regulated by hypoxia and HIF-1 via HRE-2 of the STK15 promoter. Thus, tumor hypoxia may trigger overexpression of STK15 observed in various tumors.

Midgut epithelial endocrine cells are a rich source of the neuropeptides APSGFLGMRamide (Cancer borealis tachykinin-related peptide Ia) and GYRKPPFNGSIFamide (Gly1-SIFamide) in the crabs Cancer borealis, Cancer magister and Cancer productus.

Over a quarter of a century ago, Mykles described the presence of putative endocrine cells in the midgut epithelium of the crab Cancer magister (Mykles, 1979). In the years that have followed, these cells have been largely ignored and nothing is known about their hormone content or the functions they play in this species. Here, we used a combination of immunohistochemistry and mass spectrometric techniques to investigate these questions. Using immunohistochemistry, we identified both SIFamide- and tachykinin-related peptide (TRP)-like immunopositive cells in the midgut epithelium of C. magister, as well as in that of Cancer borealis and Cancer productus. In each species, the SIFamide-like labeling was restricted to the anterior portion of the midgut, including the paired anterior midgut caeca, whereas the TRP-like immunoreactivity predominated in the posterior midgut and the posterior midgut caecum. Regardless of location, label or species, the morphology of the immunopositive cells matched that of the putative endocrine cells characterized ultrastructurally by Mykles (Mykles, 1979). Matrix-assisted laser desorption/ionization-Fourier transform mass spectrometry identified the peptides responsible for the immunoreactivities as GYRKPPFNGSIFamide (Gly1-SIFamide) and APSGFLGMRamide [Cancer borealis tachykinin-related peptide Ia (CabTRP Ia)], respectively, both of which are known neuropeptides of Cancer species. Although the function of these midgut-derived peptides remains unknown, we found that both Gly1-SIFamide and CabTRP Ia were released when the midgut was exposed to high-potassium saline. In addition, CabTRP Ia was detectable in the hemolymph of crabs that had been held without food for several days, but not in that of fed animals, paralleling results that were attributed to TRP release from midgut endocrine cells in insects. Thus, one function that midgut-derived CabTRP Ia may play in Cancer species is paracrine/hormonal control of feeding-related behavior, as has been postulated for TRPs released from homologous cells in insects.

Different mechanisms of mitotic instability in cancer cell lines.

Chromosomes of human malignant tumours display not only structural recombinations but also a wide variety of mostly non-random numerical aberrations. However, only little is known about the mechanisms leading to recurrent aneuploidies. We therefore investigated whether the malsegregation of specific chromosomes is due to a defect of the mitotic spindle apparatus. We analyzed mitoses of cell lines of six gliomas and of one breast carcinoma by combined immunohistochemistry and fluorescence in situ hybridization for non-disjunction of chromosomes 7, 8, 10, 12, 17, and 18 and observed three different phenomena. i) Five of six glioma cell lines showed a bipolar spindle but displayed a chromosome-specific malsegregation of all chromosomes studied with high but significantly different frequencies. Chromosomes 7 and 8 showed non-disjunction in about 75 and 50%, respectively. Although chromosomes 10, 12, 17, and 18 displayed equal separation during mitosis in 72, 86, 73, and 78%, respectively, a relevant percentage of an average of 24% of dividing cells showed even malsegregation of these chromosomes. ii) Only one of the glioma cell lines displayed multipolar spindles in one-third of the investigated cells resulting in non-specific aneuploidy. iii) The breast cancer cell line MCF7 displayed a bipolar spindle, but high frequencies of non-disjunction of all six investigated chromosomes but without preferential loss or gain of specific chromosomes indicating a different mechanism of chromosome malsegregation. In a small percentage of mitoses the chromatids of both homologous chromosomes were not separated mimicking the mechanism in the first meiotic division. This mechanism of double non-disjunction, not detectable by conventional cytogenetic analysis, procreates cell clones with genomic separation for particular chromosomes resulting in homozygosity for mutations which had been present heterozygously in the initial tumour cells.

Detailed chromosomal characterization of the breast cancer cell line MCF7 with special focus on the expression of the serine-threonine kinase 15.

Although the cell line MCF7 is widely used in breast cancer research, its cytogenetic properties have not been thoroughly investigated so far. As conventional G-banding analysis cannot resolve the complex chromosome aberrations, we investigated MCF7 cells using molecular-cytogenetic methods, with particular attention to the DNA amplification site on chromosome 20q. With spectral karyotyping we found numerous unbalanced chromosome translocations, and with comparative genomic hybridization we detected many quantitative genomic imbalances. Furthermore, we analyzed the amplified region at 20q with the candidate tumour susceptibility gene STK15 in detail by fluorescence in situ hybridization, whole chromosome painting, immunohistochemistry, Western blot and expression analysis. In MCF7 interphase cells we found increased copy number of the STK15 gene associated with overexpression of STK15 mRNA. Accordingly, STK15 protein is overexpressed as compared to normal human fibroblasts in Western blot analysis. Overexpression of STK15 mRNA and protein is disproportionally stronger than that expected from the single additional copy of the STK15 gene. These data indicate that the highly increased level of STK15 protein in MCF7 cannot be explained by gene amplification alone. Apparently, secondary mechanisms of gene up-regulation are involved. This observation may be of general interest with regard to the activation of oncogenes in tumour cells.