Identifying Features of Cardiac Disease Phenotypes Based on Mechanical Function in a Catecholaminergic Polymorphic Ventricular Tachycardia Model.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is characterized by an arrhythmogenic mechanism involving disruption of calcium handling. This genetic disease can lead to sudden death in children and young adults during physical or emotional stress. Prior CPVT studies have focused on calcium handling, but mechanical functionality has rarely been investigated in vitro. In this research we combine stem cell-derived cardiomyocytes from a CPVT patient (RyR2-H2464D mutation) and a healthy familial control with an engineered culture platform to evaluate mechanical function of cardiomyocytes. Substrates with Young's modulus ranging from 10 to 50 kPa were used in conjunction with microcontact printing of ECM proteins into defined patterns for subsequent attachment. Digital Image Correlation (DIC) was used to evaluate collections of contracting cells. The amplitude of contractile strain was utilized as a quantitative indicator of functionality and disease severity. We found statistically significant differences: the maximum contractile strain was consistently higher in patient samples compared to control samples on all substrate stiffnesses. Additionally, the patient cell line had a statistically significantly slower intrinsic contraction rate than the control, which agrees with prior literature. Differences in mechanical strain have not been previously reported, and hypercontractility is not a known characteristic of CPVT. However, functional changes can occur as the disease progresses, thus this observation may not represent behavior observed in adolescent and adult patients. These results add to the limited studies of mechanical function of CPVT CMs reported in literature and identify functional differences that should be further explored.

Micropattern platform promotes extracellular matrix remodeling by human PSC-derived cardiac fibroblasts and enhances contractility of co-cultured cardiomyocytes.

In native heart tissue, cardiac fibroblasts provide the structural framework of extracellular matrix (ECM) while also influencing the electrical and mechanical properties of cardiomyocytes. Recent advances in the field of stem cell differentiation have led to the availability of human pluripotent stem cell-derived cardiac fibroblasts (iPSC-CFs) in addition to cardiomyocytes (iPSC-CMs). Here we use a novel 2D in vitro micropatterned platform that provides control over ECM geometry and substrate stiffness. When cultured alone on soft micropatterned substrates, iPSC-CFs are confined to the micropatterned features and remodel the ECM into anisotropic fibers. Similar remodeling and ECM production occurs when cultured with iPSC-CMs in a co-culture model. In addition to modifications in the ECM, our results show that iPSC-CFs influence iPSC-CM function with accelerated Ca2+ transient rise-up time and greater contractile strains in the co-culture conditions compared to when iPSC-CMs are cultured alone. These combined observations highlight the important role cardiac fibroblasts play in vivo and the need for co-culture models like the one presented here to provide more representative in vitro cardiac constructs.

Aligned human cardiac syncytium for in vitro analysis of electrical, structural, and mechanical readouts.

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have emerged as an exciting new tool for cardiac research and can serve as a preclinical platform for drug development and disease modeling studies. However, these aspirations are limited by current culture methods in which hPSC-CMs resemble fetal human cardiomyocytes in terms of structure and function. Herein we provide a novel in vitro platform that includes patterned extracellular matrix with physiological substrate stiffness and is amenable to both mechanical and electrical analysis. Micropatterned lanes promote the cellular and myofibril alignment of hPSC-CMs while the addition of micropatterned bridges enable formation of a functional cardiac syncytium that beats synchronously over a large two-dimensional area. We investigated the electrophysiological properties of the patterned cardiac constructs and showed they have anisotropic electrical impulse propagation, as occurs in the native myocardium, with speeds 2x faster in the primary direction of the pattern as compared to the transverse direction. Lastly, we interrogated the mechanical function of the pattern constructs and demonstrated the utility of this platform in recording the strength of cardiomyocyte contractions. This biomimetic platform with electrical and mechanical readout capabilities will enable the study of cardiac disease and the influence of pharmaceuticals and toxins on cardiomyocyte function. The platform also holds potential for high throughput evaluation of drug safety and efficacy, thus furthering our understanding of cardiovascular disease and increasing the translational use of hPSC-CMs.

Channelopathy as a SUDEP Biomarker in Dravet Syndrome Patient-Derived Cardiac Myocytes.

Dravet syndrome (DS) is a severe developmental and epileptic encephalopathy with a high incidence of sudden unexpected death in epilepsy (SUDEP). Most DS patients carry de novo variants in SCN1A, resulting in Nav1.1 haploinsufficiency. Because SCN1A is expressed in heart and in brain, we proposed that cardiac arrhythmia contributes to SUDEP in DS. We generated DS patient and control induced pluripotent stem cell-derived cardiac myocytes (iPSC-CMs). We observed increased sodium current (INa) and spontaneous contraction rates in DS patient iPSC-CMs versus controls. For the subject with the largest increase in INa, cardiac abnormalities were revealed upon clinical evaluation. Generation of a CRISPR gene-edited heterozygous SCN1A deletion in control iPSCs increased INa density in iPSC-CMs similar to that seen in patient cells. Thus, the high risk of SUDEP in DS may result from a predisposition to cardiac arrhythmias in addition to seizures, reflecting expression of SCN1A in heart and brain.

Vulvar cancer: Two pathways with different localization and prognosis.

BACKGROUND: Two etiologic pathways for vulvar squamous cell carcinoma (SCC) are described: in a background of lichen sclerosus and/or differentiated vulvar intraepithelial neoplasia and related to high-risk human papillomavirus (HPV) infection with high grade squamous intraepithelial lesion (HSIL) as precursor. The aim was to compare the predilection site and survival of HPV-related to non HPV-related vulvar SCCs. METHODS: Data of patients treated for primary vulvar SCC at the Radboudumc between March 1988 and January 2015 were analyzed. All histological specimens were tested for HPV with the SPF10/DEIA/LiPA25 system assay and p16INK4a staining was performed using CINtec® histology kit. Vulvar SCCs were considered HPV-related in case of either >25% p16INK4a expression and HPV positivity or >25% p16INK4a expression and HSIL next to the tumor without HPV positivity. Tumor localization, disease specific survival (DSS), disease free survival (DFS) and overall survival (OS) of patients with HPV-related and non HPV-related vulvar SCC were compared. RESULTS: In total 318 patients were included: 55 (17%) had HPV-related (Group 1) and 263 (83%) had non HPV-related vulvar SCC (Group 2). Tumors in Group 1 were significantly more often located at the perineum compared to Group 2, 30% and 14%, respectively (p=0.001). The DSS, DFS and OS were significantly better in HPV-related than in non HPV-related vulvar SCC patients. CONCLUSION: HPV-related vulvar SCCs are more frequently located at the perineum and have a favorable prognosis compared to non HPV-related vulvar SCCs. Both localization and HPV-relation could explain this favorable prognosis. HPV-related vulvar SCC seems to be a separate entity.

hERG 1b is critical for human cardiac repolarization.

The human ether-à-go-go-related gene (hERG; or KCNH2) encodes the voltage-gated potassium channel underlying IKr, a repolarizing current in the heart. Mutations in KCNH2 or pharmacological agents that reduce IKr slow action potential (AP) repolarization and can trigger cardiac arrhythmias associated with long QT syndrome. Two channel-forming subunits encoded by KCNH2 (hERG 1a and 1b) are expressed in cardiac tissue. In heterologous expression systems, these subunits avidly coassemble and exhibit biophysical and pharmacological properties distinct from those of homomeric hERG 1a channels. Despite these findings, adoption of hERG 1a/1b heteromeric channels as a model for cardiac IKr has been hampered by the lack of evidence for a direct functional role for the 1b subunit in native tissue. In this study, we measured IKr and APs at physiological temperature in cardiomyocytes derived from human induced pluripotent stem cells (iPSC-CMs). We found that specific knockdown of the 1b subunit using shRNA caused reductions in 1b mRNA, 1b protein levels, and IKr magnitude by roughly one-half. AP duration was increased and AP variability was enhanced relative to controls. Early afterdepolarizations, considered cellular substrates for arrhythmia, were also observed in cells with reduced 1b expression. Similar behavior was elicited when channels were effectively converted from heteromers to 1a homomers by expressing a fragment corresponding to the 1a-specific N-terminal Per-Arnt-Sim domain, which is omitted from hERG 1b by alternate transcription. These findings establish that hERG 1b is critical for normal repolarization and that loss of 1b is proarrhythmic in human cardiac cells.

Virulence of Leptographium serpens on Longleaf Pine Seedlings Under Varying Soil Moisture Regimes.

Recently, Leptographium serpens has been recovered from the roots of declining and dead longleaf pine (Pinus palustris) in stands associated with various abiotic stresses. Although most data suggest that L. serpens is pathogenic to various Pinus spp., there is little known of its virulence on longleaf pine or its relationship with abiotic stress in causing disease. These trials examined the effects of L. serpens infection coupled with drought stress. Trials began with wound inoculations of bareroot longleaf pine seedlings in spring 2006 and 2007 at the seedling stress facility at Auburn University. Soon after inoculation, seedlings were also subjected to adequate moisture, moderate drought, or severe drought. Sixteen weeks after inoculation, longleaf pine survival, L. serpens virulence, and seedling growth characteristics were measured. Longleaf pine seedlings inoculated with L. serpens had 33% mortality (138/420) which was significantly greater than nonwounded control seedlings (22%, 47/211). Survival and lesion size on longleaf pine suggests that L. serpens is moderately pathogenic to longleaf pine seedlings. Separately, moisture stress associated with low soil moisture also contributed to seedling mortality. Results suggest that L. serpens infection and moisture stress commonly experienced by southern pines act independently to stress longleaf pine.

Drug-induced long QT syndrome: hERG K+ channel block and disruption of protein trafficking by fluoxetine and norfluoxetine.

BACKGROUND AND PURPOSE: Fluoxetine (Prozac) is a widely prescribed drug in adults and children, and it has an active metabolite, norfluoxetine, with a prolonged elimination time. Although uncommon, Prozac causes QT interval prolongation and arrhythmias; a patient who took an overdose of Prozac exhibited a prolonged QT interval (QTc 625 msec). We looked for possible mechanisms underlying this clinical finding by analysing the effects of fluoxetine and norfluoxetine on ion channels in vitro. EXPERIMENTAL APPROACH: We studied the effects of fluoxetine and norfluoxetine on the electrophysiology and cellular trafficking of hERG K+ and SCN5A Na+ channels heterologously expressed in HEK293 cells. KEY RESULTS: Voltage clamp analyses employing square pulse or ventricular action potential waveform protocols showed that fluoxetine and norfluoxetine caused direct, concentration-dependent, block of hERG current (IhERG). Biochemical studies showed that both compounds also caused concentration-dependent reductions in the trafficking of hERG channel protein into the cell surface membrane. Fluoxetine had no effect on SCN5A channel or HEK293 cell endogenous current. Mutations in the hERG channel drug binding domain reduced fluoxetine block of IhERG but did not alter fluoxetine's effect on hERG channel protein trafficking. CONCLUSIONS AND IMPLICATIONS: Our findings show that both fluoxetine and norfluoxetine at similar concentrations selectively reduce IhERG by two mechanisms, (1) direct channel block, and (2) indirectly by disrupting channel protein trafficking. These two effects are not mediated by a single drug binding site. Our findings add complexity to understanding the mechanisms that cause drug-induced long QT syndrome.

Pathogenicity of Leptographium Species Associated with Loblolly Pine Decline.

Freshly lifted seedlings and 21-year-old trees of loblolly pine were wound-inoculated with Leptographium species recovered from the soil and/or roots of trees with loblolly decline symptoms in central Alabama. Seedlings inoculated with L. procerum in the greenhouse produced significantly fewer root initials and a smaller root mass than control seedlings. Vertical lesions produced in seedlings by L. serpens and L. terebrantis were significantly longer than in controls. Lesions produced in mature trees by L. serpens and L. lundbergii were significantly longer than in controls. Of the fungi tested, L. serpens, L. terebrantis, and L. lundbergii were the most aggressive and may pose the greatest threat to loblolly pines.

Unique function for carboxyl-terminal domain of Oct-2 in Ig-secreting cells.

The activity of Ig gene promoters and enhancers is regulated by two related transcription factors, Oct-1 (ubiquitous) and Oct-2 (B lineage specific), which bind the octamer motif (ATTTGCAT) present in these elements. As Ig promoter-binding factors, Oct-1 and Oct-2 each work together with a B lymphocyte-specific cofactor OCA-B/OBF-1/Bob-1 that interacts with them through their POU (DNA-binding) domains. Because both can mediate Ig promoter activity in B cells, there has been some question as to whether these two octamer-binding factors serve distinct functions in lymphocytes. We have shown previously that the silencing of B lymphocyte-specific genes in plasmacytoma x T lymphoma hybrids can be prevented by preserving Oct-2 expression. The pronounced effect of this transcription factor on the phenotype of plasmacytoma x T lymphoma hybrids established a critical role for Oct-2 not only in maintaining Ig gene expression, but in maintaining the overall genetic program of Ig-secreting cells. In the present study, we have explored the functional differences between Oct-1 and Oct-2 using chimeric Oct-1/Oct-2 proteins in cell fusion assays. Our results provide further evidence for an essential role for Oct-2 in Ig-secreting cells and identify the C-terminal domain of Oct-2 as responsible for its unique function in these cells.

Promoting sun safety among zoo visitors.

BACKGROUND: Each year, millions of children visit zoological parks, where they are exposed to long bouts of ultraviolet radiation (UVR). We conducted a study in the winter and replicated it in the summer to evaluate an intervention for reducing UVR exposure during the zoo visit. METHODS: Each study used a nonequivalent control group design: one zoological site received the intervention and a second received evaluation only. Key outcome measures consisted of observed prevalence of hat use by exiting children (N = 8,721 and 8,524, respectively, in winter and summer studies) and purchase rates of sunscreen and hats in zoo gift shops. Intervention consisted of tip sheets for parents, children's activities, prompts, and discounts off the price of sunscreen and sun-protective hats. RESULTS: In the summer study, sales of both sunscreen and target hats increased significantly at the intervention site relative to the control site, whereas in the winter study, only sunscreen sales at the intervention site had a significant (relative) increase. Children's hat use increased significantly at the intervention site, but only in the winter study. CONCLUSIONS: The multicomponent program was effective in promoting purchases of sun-safe items, but its impact on children's hat use was inconclusive.

Deletional analyses reveal an essential role for the hs3b/hs4 IgH 3' enhancer pair in an Ig-secreting but not an earlier-stage B cell line.

The Ig heavy chain (IgH) locus is controlled by multiple regulatory sequences mapping both within the IgH transcription unit (E mu) and downstream (3') of IgH coding sequences (hs3a, hs1,2, hs3b and hs4). Enhancer knockout studies in mice have implicated E mu in the control of IgH variable region gene assembly, but single-enhancer knockouts involving the 3' IgH enhancers have yet to shed light on their function. Transfection studies in mice and cell lines have suggested that the 3' enhancers behave similarly to a locus control region as first identified in the beta-globin locus. We have exploited this property to form mini-loci in a surface Ig(+) and an Ig-secreting cell line as a means for studying the functions of the 3' IgH enhancers. Importantly, this experimental system allows for the analysis of enhancer function within the context of chromatin. The mini-loci consisted of an Ig gamma 2b transcription unit linked to the four murine 3' IgH enhancers. Using targeted deletions of enhancer pairs within these mini-loci, we have discovered a critical and apparently developmentally regulated role for the hs3b/hs4 enhancer pair in IgH transgene expression.

How does endotoxin trigger inflammation in otitis media with effusion?

OBJECTIVES: The relationship among microorganisms, endotoxin, and inflammatory mediators in otitis media with effusion (OME) was examined. STUDY DESIGN: Analysis of 152 middle ear effusions aspirated at the time of ventilation tube insertion from children with OME. METHODS: Effusion samples were cultured for pathogenic bacteria The two primary cytokines, interleukin-1beta (IL1beta) and tumor necrosis factor (TNFalpha), and the adhesion molecules, intercellular and vascular adhesion molecule (ICAM-1 and VCAM-1), were quantified using the enzyme-linked immunosorbent assay (ELISA) technique. Endotoxin concentration was measured with a limulus amebocyte lysate assay, and total protein concentration was quantified using the Biorad microassay. RESULTS: The cultures of pathogenic bacteria were positive in 33 of the 152 effusions (22%), which contained more endotoxin and more of the primary cytokines than the 119 culture-negative effusions. Endotoxin and the primary cytokines were positively correlated, both in the whole material and in the sterile effusions alone. The adhesion molecules were positively correlated with each other, but not with endotoxin or the primary cytokines. CONCLUSIONS: We found a positive correlation between endotoxin and the primary cytokines TNFalpha and IL1beta in culture-positive OME effusions as well as in culture-negative ones, suggesting endotoxin-induced local production of TNFalpha and IL1beta in the middle ear. ICAM-1 and VCAM-1 were also present in the middle ear, but their concentrations were not directly correlated to endotoxin or the primary cytokines.

Compliance with federal and state legislation by indoor tanning facilities in San Diego.

BACKGROUND: The prevalence rates of both skin cancers and indoor tanning among the US population are high and have increased substantially in recent years. Low compliance by indoor tanning facilities with safety regulations may place consumers at greater risk of skin and ocular damage. OBJECTIVE: This study quantified the level of compliance by indoor tanning facilities with selected federal and state regulations and recommendations. METHODS: Tanning facilities (N = 54) in San Diego County, California, were visited by a confederate posing as a prospective customer. Compliance with 13 regulations/recommendations were assessed by either direct query or observation of the presence/absence of signs and warning labels. Operators' responses to 5 risk-based questions also were noted. RESULTS: No facility complied with all 13 regulations/recommendations. Compliance with 3 protective eyewear regulations was high (89%-100%). In contrast, compliance with maximum tanning frequency recommendations (approximately 6%) and parental consent regulations (approximately 43%) was disturbingly low. CONCLUSION: The investigators recommend instituting mandatory, comprehensive training for operators, as well as systematic compliance monitoring with enforcement of penalties for noncompliance.

Confidence intervals for the risk ratio under cluster sampling based on the beta-binomial model.

In cohort studies, the risk ratio (RR) is one of the most commonly used epidemiologic indices to quantify the effect of a suspected risk factor on the probability of developing a disease. When we employ cluster sampling to collect data, an interval estimator that does not account for the intraclass correlation between subjects within clusters is likely inappropriate. In application of the beta-binomial model to account for the intraclass correlation, we develop four asymptotic interval estimators of the RR, which are direct extensions of some recently developed estimators for independent binomial sampling. We then use Monte Carlo simulation to evaluate the finite-sample performance of these four interval estimators in a variety of situations. We find that the estimator using the logarithmic transformation generally performs well and is preferable to the other three estimators in most of the situations considered here. Finally, we include an example from a study of an educational intervention with emphasis on behaviour change to illustrate the use of the estimators developed in this paper.

Tobacco mosaic virus replicase-mediated cross-protection: contributions of RNA and protein-derived mechanisms.

Specific sequences of the tobacco mosaic virus (TMV) RNA-dependent RNA-polymerase (RdRp) gene were investigated for their ability to confer cross-protection. Nine overlapping segments ranging from 713 to 1070 nucleotides in length and covering the methyltransferase, helicase, and polymerase (POL) domains of the TMV RdRp open reading frame were systemically expressed in Nicotiana benthamiana using a potato X virus (PVX) vector [Chapman, S., Kavanagh, T., and Baulcombe, D. C. (1992). Plant J., 1, 549-557]. PVX-infected plants were subsequently challenge inoculated with 10 microg of wild-type TMV and monitored for TMV accumulation. Mock inoculated plants and plants preinfected with the unmodified PVX vector rapidly accumulated high levels of challenge virus. In contrast, plants preinfected with PVX vectors expressing segments of the TMV RdRp open reading frame displayed either high or low levels of protection. High protection levels were observed for PVX constructs expressing segments of the TMV POL domain, whereas low protection levels were observed for PVX constructs expressing segments covering the methyltransferase and helicase domains. Frameshift mutations that blocked protein expression from RdRp segments disrupted only the high levels of protection derived from POL segments and not the low levels derived from the other segments. However, all RdRp segments conferred similarly high levels of protection against a TMV construct with restricted local movement. Thus both RNA and protein sequences in conjunction with the speed of the infecting challenge virus can affect the protection derived from the TMV RdRp gene.

A national survey of sun safety activities at U.S. zoos.

Skin cancer incidence in the United States has increased. Exposure to ultraviolet radiation (UVR) is a major risk factor for skin cancer. Every year, millions of children with their families visit outdoor leisure facilities such as zoos where overexposure to UVR may occur. This study was conducted to assess the proportion of U.S. zoos that provided sun safety activities during the summer of 1998 and their willingness to incorporate skin cancer prevention strategies at their facility in the future. A 56 item self-administered questionnaire was mailed to 140 zoos accredited by the American Zoo and Aquarium Association. A follow-up telephone survey was also conducted with 33 mail non-respondents. The response rate was 97% (N = 136). Results revealed that in the summer of 1998, only 5% of zoos provided sun safety recommendations to visitors and 25% of zoos provided recommendations to their employees. The recommendations made most often to visitors and employees were to use sunscreen and wear protective clothing. Eighty-five percent of zoos indicated interest in providing sun safety activities at their facilities in the future. The next step will be to design environmentally appropriate sun safety programs for zoos and to encourage zoos to implement these programs.

Role of OCA-B in 3'-IgH enhancer function.

OCA-B (alternately called Bob1 and OBF-1) is a B cell-specific coactivator that interacts with the ubiquitously expressed Oct-1 and the B cell-restricted Oct-2 to activate transcription via the octamer site (5'-ATGCAAAT-3'). OCA-B-/- mice appear to undergo normal Ag-independent B cell maturation. However, Ag-dependent B cell differentiation, including germinal center formation, production of secondary Ig isotypes, and proliferation in response to surface Ig cross-linking, is greatly affected. We demonstrate that the observed reductions in expression of class-switched isotypes in OCA-B-/- mice may be due in part to deficiencies in the function of the 3'-IgH enhancer elements. Furthermore, we find that surface Ig cross-linking represses all the Ig enhancers and that this repression is absent in OCA-B-/- B cells. These results suggest an important role for OCA-B in Ig enhancer function in vivo.

Sonomorphologic evaluation of goiter in an iodine deficiency area in the Ivory Coast.

OBJECTIVES: This study evaluated the extent of thyroid abnormalities in a remote iodine-deficient area of the Ivory Coast. METHODS: Ultrasonography was used in detecting the presence of goiter. RESULTS: The overall prevalence rates of goiter were 64.7% among females and 53.3% among males. In children aged 6 to 15 years (n = 314), the prevalence of goiter was 62% regardless of sex. Thyroid volume increased steadily with age, with significantly larger goiters in women 25 years and older. Frequency of cysts and calcifications did not correlate with sex. CONCLUSIONS: Especially in developing countries, prophylaxis of iodine deficiency disorders must be improved in iodine-deficient areas to prevent substantial morbidity, which is more severe in women and elderly persons.

Promoting skin cancer prevention counseling by pharmacists.

OBJECTIVES: This study evaluated the effects of an intervention on rates of skin cancer prevention counseling by pharmacists. METHODS: Fifty-four pharmacies were randomly assigned to intervention or control conditions. Intervention consisted of training, feedback, and prompts. Counseling rates before and after the intervention were obtained from study confederates. RESULTS: At pretest, the proportions of control and intervention sites providing counseling at least once were 7.4% and 0%, respectively (NS). At posttest, these proportions were 3.7% and 66.7%, respectively (P < .001). CONCLUSIONS: The results indicated that the intervention was successful and that pharmacists can play an important role in educating the public about skin cancer prevention strategies.