Creating saponin-free yellow pea seeds by CRISPR/Cas9-enabled mutagenesis on ß-amyrin synthase.

Dry pea (Pisum sativum) seeds are valuable sources of plant protein, dietary fiber, and starch, but their uses in food products are restricted to some extent due to several off-flavor compounds. Saponins are glycosylated triterpenoids and are a major source of bitter, astringent, and metallic off-flavors in pea products. ß-amyrin synthase (BAS) is the entry point enzyme for saponin biosynthesis in pea and therefore is an ideal target for knock-out using CRISPR/Cas9 genome editing to produce saponin deficient pea varieties. Here, in an elite yellow pea cultivar (CDC Inca), LC/MS analysis identified embryo tissue, not seed coat, as the main location of saponin storage in pea seeds. Differential expression analysis determined that PsBAS1 was preferentially expressed in embryo tissue relative to seed coat and was selected for CRISPR/Cas9 genome editing. The efficiency of CRISPR/Cas9 genome editing of PsBAS1 was systematically optimized in pea hairy roots. From these optimization procedures, the AtU6-26 promoter was found to be superior to the CaMV35S promoter for gRNA expression, and the use of 37°C was determined to increase the efficiency of CRISPR/Cas9 genome editing. These promoter and culture conditions were then applied to stable transformations. As a result, a bi-allelic mutation (deletion and inversion mutations) was generated in the PsBAS1 coding sequence in a T1 plant, and the segregated psbas1 plants from the T2 population showed a 99.8% reduction of saponins in their seeds. Interestingly, a small but statistically significant increase (~12%) in protein content with a slight decrease (~5%) in starch content was observed in the psbas1 mutants under phytotron growth conditions. This work demonstrated that flavor-improved traits can be readily introduced in any pea cultivar of interest using CRISPR/Cas9. Further field trials and sensory tests for improved flavor are necessary to assess the practical implications of the saponin-free pea seeds in food applications.

Germacrene A Synthases for Sesquiterpene Lactone Biosynthesis Are Expressed in Vascular Parenchyma Cells Neighboring Laticifers in Lettuce.

Sesquiterpene lactone (STL) and natural rubber (NR) are characteristic isoprenoids in lettuce (Lactuca sativa). Both STL and NR co-accumulate in laticifers, pipe-like structures located along the vasculature. NR-biosynthetic genes are exclusively expressed in laticifers, but cell-type specific expression of STL-biosynthetic genes has not been studied. Here, we examined the expression pattern of germacrene A synthase (LsGAS), which catalyzes the first step in STL biosynthesis in lettuce. Quantitative PCR and Illumina read mapping revealed that the transcripts of two GAS isoforms (LsGAS1/LsGAS2) are expressed two orders of magnitude (~100-200) higher in stems than laticifers. This result implies that the cellular site for LsGAS1/2 expression is not in laticifers. To gain more insights, promoters of LsGAS1/2 were cloned and fused to ß-glucuronidase (GUS), followed by transformations of lettuce with these promoter-GUS constructs. In in situ GUS assays, the GUS expression driven by the LsGAS1/2 promoters was tightly associated with vascular bundles. High-resolution microsections showed that GUS signals are not present in laticifers but are detected in the vascular parenchyma cells neighboring the laticifers. These results suggest that expression of LsGAS1/2 occurs in the parenchyma cells neighboring laticifers, while the resulting STL metabolites accumulate in laticifers. It can be inferred that active metabolite-trafficking occurs from the parenchyma cells to laticifers in lettuce.

Single-item measures for depression and anxiety: Validation of the Screening Tool for Psychological Distress in an inpatient cardiology setting.

BACKGROUND: Depression and anxiety are common among patients with cardiovascular disease (CVD) and confer significant cardiac risk, contributing to CVD morbidity and mortality. Unfortunately, due to the lack of screening tools that address the specific needs of hospitalized patients, few cardiac inpatient programs offer routine screening for these forms of psychological distress, despite recommendations to do so. AIMS: The purpose of this study was to validate single-item measures for depression and anxiety among cardiac inpatients. METHODS: Consecutive inpatients were recruited from the cardiology and cardiac surgery step-down units at a university-affiliated, quaternary-care hospital. Subjects completed a questionnaire that included: (a) demographics, (b) single-item-measures for depression and anxiety (from the Screening Tool for Psychological Distress (STOP-D)), and (c) Hospital Anxiety and Depression Scale (HADS). RESULTS: One hundred and five participants were recruited with a wide variety of cardiac diagnoses, having a mean age of 66 years, and 28% were women. Both STOP-D items were highly correlated with their corresponding validated measures and demonstrated robust receiver-operator characteristic curves. Severity scores on both items correlated well with established severity cut-off scores on the corresponding subscales of the HADS. CONCLUSIONS: The STOP-D is a self-administered, self-report measure using two independent items that provide severity scores for depression and anxiety. The tool performs very well compared with other previously validated measures. Requiring no additional scoring and being free, STOP-D offers a simple and valid method for identifying hospitalized cardiac patients who are experiencing psychological distress. This crucial first step triggers initiation of appropriate monitoring and intervention, thus reducing the likelihood of the adverse cardiac outcomes associated with psychological distress.

Digital breast tomosynthesis: lessons learned from early clinical implementation.

The limitations of mammography are well known and are partly related to the fact that with conventional imaging, the three-dimensional volume of the breast is imaged and presented in a two-dimensional format. Because normal breast tissue is similar in x-ray attenuation to some breast cancers, clinically relevant malignancies may be obscured by normal overlapping tissue. In addition, complex areas of normal tissue may be perceived as suspicious. The limitations of two-dimensional breast imaging lead to low sensitivity in detecting some cancers and high false-positive recall rates. Although mammographic screening has been shown to reduce breast cancer deaths by approximately 30%, controversy exists over when and how often screening mammography should occur. Digital breast tomosynthesis (DBT) is rapidly being implemented in breast imaging clinics around the world as early clinical data demonstrate that it may address some of the limitations of conventional mammography. With DBT, multiple low-dose x-ray images are acquired in an arc and reconstructed to create a three-dimensional image, thus minimizing the impact of overlapping breast tissue and improving lesion conspicuity. Early studies of screening DBT have shown decreased false-positive callback rates and increased rates of cancer detection (particularly for invasive cancers), resulting in increased sensitivity and specificity. In our clinical practice, we have completed more than 2 years of using two-view digital mammography combined with two-view DBT for all screening and select diagnostic imaging examinations (over 25,000 patients). Our experience, combined with previously published data, demonstrates that the combined use of DBT and digital mammography is associated with improved outcomes for screening and diagnostic imaging. Online supplemental material is available for this article.

Results of MR imaging screening for breast cancer in high-risk patients with lobular carcinoma in situ.

PURPOSE: To determine the outcome of screening breast magnetic resonance (MR) imaging examinations performed in patients with lobular carcinoma in situ (LCIS) at the authors' institution. MATERIALS AND METHODS: This study was approved by the institutional review board and was compliant with HIPAA. Retrospective review of screening breast MR imaging examinations at the institution from 1996 through September 2009 was performed in patients with prior biopsies demonstrating LCIS. Patients with prior breast cancer diagnosis were excluded. American College of Radiology Breast Imaging Reporting and Data System (BI-RADS) recommendations were recorded. Pathologic results of all consequent biopsies were obtained from the electronic medical records. RESULTS: A total of 445 breast MR examinations in 198 patients with LCIS were identified. Of these, 308 were screening examinations in 134 patients. One patient was a BRCA mutation carrier and was excluded. Of the remaining 307 screening examinations, 254 (82.7%) had BI-RADS category 1 or 2 findings; 27 (8.8%) had BI-RADS category 3 findings; and 27 (8.8%) had B-IRADS category 4 or 5 findings. Of the 27 studies that led to a biopsy recommendation, 10 (37%) yielded benign pathologic findings, five (18.5%) yielded malignant pathologic findings, and seven (25.9%) yielded high-risk lesions. Of the 27 studies with BI-RADS 3 findings, two (7.4%) resulted in biopsy, findings of both were benign. Overall, malignancy was detected in five of 307 screening studies (1.6%) and in five of 133 screened patients (3.8%). The positive predictive value (PPV) of these screening studies for which biopsy was recommended was 18.5%. The PPV 3 (studies for which biopsy was recommended and actually performed, as described in the BI-RADS guidelines) was 23.8%. CONCLUSION: Screening breast MR imaging helped identify breast cancer in LCIS patients at a rate similar to that shown in high-risk populations for whom screening breast MR imaging is currently consistently recommended.

Contexts of tobacco use and perspectives on smoking cessation among a sample of urban American Indians.

American Indians have the highest prevalence of cigarette use in the United States, but there is a shortage of knowledge about American Indians' own perspectives on smoking and cessation. The purpose of this exploratory qualitative study was to obtain information on American Indians' views that would be useful for subsequent intervention planning and development. Four focus groups were conducted with urban American Indians living in Maryland to explore the sociocultural contexts of tobacco use and their perspectives on various mainstream and culturally-specific smoking cessation strategies and service delivery models. Tobacco interventions targeting American Indians should increase service access, address negative experiences with medications, emphasize empowerment for behavior change, explicitly distinguish ceremonial tobacco from cigarette use, and send culturally-relevant messages. Smoking cessation programs and health promotion efforts may be perceived as more relevant by the target population if they incorporate an understanding of the social and cultural facets of smoking behavior.

Role of magnetic resonance imaging in evaluating the extent of disease.

Preoperative breast imaging evaluation can contribute useful clinical information to the management of the patient with known breast cancer. Breast magnetic resonance imaging (MRI) has been used as part of this imaging evaluation, and the ability of breast MRI to detect otherwise occult multifocal and multicentric disease has been demonstrated in multiple studies. The use of MRI for breast cancer staging remains under debate, however. This article reviews some of the current discussion regarding the use of breast MRI in this patient population. It is important to note that this discussion occurs in an evolving context of surgical and breast conservation therapies.

Effect of topical microbicides on infectious human immunodeficiency virus type 1 binding to epithelial cells.

Topical microbicides (cellulose acetate 1,2 benzene dicarboxylate [CAP], PRO 2000, SPL7013, and UC781) are being investigated to reduce the sexual transmission of human immunodeficiency virus type 1 (HIV-1). These products were shown to prevent the transfer of infectious HIV-1 from urogenital and colorectal epithelial cell lines to peripheral blood mononuclear cells. However, it was unclear if the topical microbicides rendered the virus noninfectious and/or reduced the binding to the epithelial cells. To test this, epithelial cells were cultured with HIV-1 in the presence or absence of topical microbicides or their placebos. The cells were washed, RNA lysates were made, and real-time PCR was performed for HIV-1. PRO 2000 and SPL7013 significantly (P < 0.0001) reduced the amount of bound HIV-1 to the colorectal epithelial cell line across clades A, B, C, and CRF01-AE. While none of the products reduced the binding of HIV-1 clades A and C to the urogenital cell line, CAP, PRO 2000, and SPL7013 significantly (P

Mutagenesis of the La Crosse Virus glycoprotein supports a role for Gc (1066-1087) as the fusion peptide.

The La Crosse Virus (LACV) M segment encodes two glycoproteins (Gn and Gc), and plays a critical role in the neuropathogenesis of LACV infection as the primary determinant of neuroinvasion. A recent study from our group demonstrated that the region comprising the membrane proximal two-thirds of Gc, amino acids 860-1442, is critical in mediating LACV fusion and entry. Furthermore, computational analysis identified structural similarities between a portion of this region, amino acids 970-1350, and the E1 fusion protein of two alphaviruses: Sindbis virus and Semliki Forrest virus (SFV). Within the region 970-1350, a 22-amino-acid hydrophobic segment (1066-1087) is predicted to correlate structurally with the fusion peptides of class II fusion proteins. We performed site-directed mutagenesis of key amino acids in this 22-amino acid segment and determined the functional consequences of these mutations on fusion and entry. Several mutations within this hydrophobic domain affected glycoprotein expression to some extent, but all mutations either shifted the pH threshold of fusion below that of the wild-type protein, reduced fusion efficiency, or abrogated cell-to-cell fusion and pseudotype entry altogether. These results, coupled with the aforementioned computational modeling, suggest that the LACV Gc functions as a class II fusion protein and support a role for the region Gc 1066-1087 as a fusion peptide.

Disorders of extreme stress: The empirical foundation of a complex adaptation to trauma.

Children and adults exposed to chronic interpersonal trauma consistently demonstrate psychological disturbances that are not captured in the posttraumatic stress disorder (PTSD) diagnosis. The DSM-IV (American Psychiatric Association, 1994) Field Trial studied 400 treatment-seeking traumatized individuals and 128 community residents and found that victims of prolonged interpersonal trauma, particularly trauma early in the life cycle, had a high incidence of problems with (a) regulation of affect and impulses, (b) memory and attention, (c) self-perception, (d) interpersonal relations, (e) somatization, and (f) systems of meaning. This raises important issues about the categorical versus the dimensional nature of posttraumatic stress, as well as the issue of comorbidity in PTSD. These data invite further exploration of what constitutes effective treatment of the full spectrum of posttraumatic psychopathology.

The Northern California Chronic Care Network for Dementia.

The Northern California Chronic Care Network for Dementia brings together Northern California's major providers of managed care, community-based care, consumer education, and advocacy in new partnerships to improve the care of persons with dementia enrolled in managed care plans and their family caregivers. These partnerships are part of a national initiative entitled the Chronic Care Network for Alzheimer's Disease (CCN/AD) sponsored by the National Chronic Care Consortium and the Alzheimer's Association. This initiative selected eight promising provider-consumer partnerships across the country to implement and evaluate a new model of coordinated care for people with dementia and their families. This paper describes the Northern California network's partnerships and its intervention and challenges. The intervention is grounded in the key components of the CCN/AD model: "identification of patients with possible dementia, diagnostic assessment, care management and family caregiver information and support." These components, in turn, are translated into protocols and pathways designed to create timely, comprehensive, appropriate, and effective systems of care services that address the unique needs of dementia patients and their caregivers over the course of the disease.

Intracellular human immunodeficiency virus Tat expression in astrocytes promotes astrocyte survival but induces potent neurotoxicity at distant sites via axonal transport.

The human immunodeficiency virus (HIV)-Tat protein has been implicated in the neuropathogenesis of HIV infection. However, its role in modulating astroglial-neuronal relationships is poorly understood. Astrocyte infection with HIV has been associated with rapid progression of dementia. We thus initially transfected astrocytes with HIV proviral DNA and confirmed Tat production in these cells. Subsequently, using stably Tat-producing asytocyte cell lines, we observed that Tat promoted astrocyte survival by causing a prominent antioxidant effect and resistance to cell injury in these cells. Tat was released extracellularly where it could be taken up by other cells. Tat remained functionally active following uptake and caused long terminal repeat (LTR) transactivation in lymphocytic and astrocytic cell lines. Tat released from astrocytes caused mitochondrial dysfunction, trimming of neurites, and cell death in neurons. Tat neurotoxicity was attenuated by anti-Tat antibodies, kynurenate or heparan sulfate. The neurotoxic effects of Tat were caused at concentrations lower than that needed to cause LTR transactivation. When Tat-expressing cells were injected into the rat dentate gyrus, Tat was taken up by granule cells and transported along neuronal pathways to the CA3 region where it caused glial cell activation and neurotoxicity. The arginine-rich domain of Tat was essential for both the LTR transactivation and the neurotoxic properties of Tat. Thus HIV-Tat is a potent neurotoxin that may act at distant sites while at the same time it assures its production by preventing cell death in astrocytes where it is produced.

Biotin dependency due to a defect in biotin transport.

We describe a 3-year-old boy with biotin dependency not caused by biotinidase, holocarboxylase synthetase, or nutritional biotin deficiency. We sought to define the mechanism of his biotin dependency. The child became acutely encephalopathic at age 18 months. Urinary organic acids indicated deficiency of several biotin-dependent carboxylases. Symptoms improved rapidly following biotin supplementation. Serum biotinidase activity and Biotinidase gene sequence were normal. Activities of biotin-dependent carboxylases in PBMCs and cultured skin fibroblasts were normal, excluding biotin holocarboxylase synthetase deficiency. Despite extracellular biotin sufficiency, biotin withdrawal caused recurrent abnormal organic aciduria, indicating intracellular biotin deficiency. Biotin uptake rates into fresh PBMCs from the child and into his PBMCs transformed with Epstein Barr virus were about 10% of normal fresh and transformed control cells, respectively. For fresh and transformed PBMCs from his parents, biotin uptake rates were consistent with heterozygosity for an autosomal recessive genetic defect. Increased biotin breakdown was ruled out, as were artifacts of biotin supplementation and generalized defects in membrane permeability for biotin. These results provide evidence for a novel genetic defect in biotin transport. This child is the first known with this defect, which should now be included in the identified causes of biotin dependency.