Fatty Acids and Their Lipogenic Enzymes in Anorexia Nervosa Clinical Subtypes.

Disordered eating behavior differs between the restricting subtype (AN-R) and the binging and purging subtype (AN-BP) of anorexia nervosa (AN). Yet, little is known about how these differences impact fatty acid (FA) dysregulation in AN. To address this question, we analyzed 26 FAs and 7 FA lipogenic enzymes (4 desaturases and 3 elongases) in 96 women: 25 AN-R, 25 AN-BP, and 46 healthy control women. Our goal was to assess subtype-specific patterns. Lauric acid was significantly higher in AN-BP than in AN-R at the fasting timepoint (p = 0.038) and displayed significantly different postprandial changes 2 h after eating. AN-R displayed significantly higher levels of n-3 alpha-linolenic acid, stearidonic acid, eicosapentaenoic acid (EPA), docosapentaenoic acid, and n-6 linoleic acid and gamma-linolenic acid compared to controls. AN-BP showed elevated EPA and saturated lauric acid compared to controls. Higher EPA was associated with elevated anxiety in AN-R (p = 0.035) but was linked to lower anxiety in AN-BP (p = 0.043). These findings suggest distinct disordered eating behaviors in AN subtypes contribute to lipid dysregulation and eating disorder comorbidities. A personalized dietary intervention may improve lipid dysregulation and enhance treatment effectiveness for AN.

Factors Shaping Public Support for More Carbon Capture and Storage Projects in the United States.

This study contributes to the literature examining public acceptance of carbon capture and storage (CCS) projects in the US. The examination of factors that shape public support for CCS projects provides policymakers with insights to address public concerns, balance CCS development with public sentiments, and make informed decisions about optimal locations and timing. Based on a nationally representative survey on 1850 respondents, the study finds that in the US, there is very low familiarity (6.4%) regarding CCS technology and some limited opposition (11.5%) to increased CCS development. Regression results suggest that support for increased CCS projects in the US is influenced by perceptions of technical and social risks (leakage and community danger, respectively) but not cost of living risks, perceptions of environmental and economic benefits, familiarity with the technology, confidence in government regulations, and a desire for the US to lead in CCS. We fail to find the 'Not-in-My-Backyard' effect, and individuals supporting the development of more CCS in their states also support it at a national level. Understanding these factors helps policymakers anticipate challenges in implementing CCS initiatives and allows for the development of strategies to address concerns.

Soluble Epoxide Hydrolase Is Associated with Postprandial Anxiety Decrease in Healthy Adult Women.

The metabolism of bioactive oxylipins by soluble epoxide hydrolase (sEH) plays an important role in inflammation, and sEH may be a risk modifier in various human diseases and disorders. The relationships that sEH has with the risk factors of these diseases remain elusive. Herein, sEH protein expression and activity in white blood cells were characterized before and after a high-fat meal in healthy women (HW) and women with anorexia nervosa (AN). sEH expression and sEH activity were significantly correlated and increased in both groups two hours after consumption of the study meal. Fasting sEH expression and activity were positively associated with body mass index (BMI) in both groups, while an inverse association with age was found in AN only (p value < 0.05). sEH was not associated with anxiety or depression in either group at the fasting timepoint. While the anxiety score decreased after eating in both groups, a higher fasting sEH was associated with a lower postprandial anxiety decrease in HW (p value < 0.05). sEH characterization using direct measurements verified the relationship between the protein expression and in vivo activity of this important oxylipin modulator, while a well-controlled food challenge study design using HW and a clinical control group of women with disordered eating elucidated sEH's role in the health of adult women.

Generation of CD163-edited pig via electroporation of the CRISPR/Cas9 system into porcine in vitro-fertilized zygotes.

CD163 is a putative fusion receptor for virus of porcine reproductive and respiratory syndrome (PRRS). In this study, we introduced a CRISPR/Cas9 system [guide RNAs (gRNAs) with Cas9 protein] targeting the CD163 gene into in vitro-fertilized porcine zygotes by electroporation to generate CD163-modified pigs. First, we designed four types of gRNAs that targeted distinct sites in exon 7 of the CD163 gene. Cas9 protein with different gRNAs was introduced into in vitro-fertilized zygotes by electroporation. When the electroporated zygotes were allowed to develop to blastocysts in vitro and the genome editing efficiency was evaluated using these blastocysts, three (gRNA1, 2, and 4) of the four gRNAs tested successfully edited the CD163 gene. To generate CD163-knockout pigs, a total of 200 electroporated zygotes using these three gRNAs were transferred into the oviducts of oestrous-synchronized surrogate and the surrogate gave birth to eight piglets. Subsequent sequence analysis revealed that one of the piglets carried no wild-type sequence in CD163 gene. The other seven piglets carried only wild-type sequence. Thus, we successfully generated a CD163-edited pig by electroporation of the CRISPR/Cas9 system into in vitro-fertilized zygotes, although further improvement is required to generate genetically modified pigs with high efficiency.

One-Step Generation of Multiple Gene-Edited Pigs by Electroporation of the CRISPR/Cas9 System into Zygotes to Reduce Xenoantigen Biosynthesis.

Xenoantigens cause hyperacute rejection and limit the success of interspecific xenografts. Therefore, genes involved in xenoantigen biosynthesis, such as GGTA1, CMAH, and B4GALNT2, are key targets to improve the outcomes of xenotransplantation. In this study, we introduced a CRISPR/Cas9 system simultaneously targeting GGTA1, CMAH, and B4GALNT2 into in vitro-fertilized zygotes using electroporation for the one-step generation of multiple gene-edited pigs without xenoantigens. First, we optimized the combination of guide RNAs (gRNAs) targeting GGTA1 and CMAH with respect to gene editing efficiency in zygotes, and transferred electroporated embryos with the optimized gRNAs and Cas9 into recipient gilts. Next, we optimized the Cas9 protein concentration with respect to the gene editing efficiency when GGTA1, CMAH, and B4GALNT2 were targeted simultaneously, and generated gene-edited pigs using the optimized conditions. We achieved the one-step generation of GGTA1/CMAH double-edited pigs and GGTA1/CMAH/B4GALNT2 triple-edited pigs. Immunohistological analyses demonstrated the downregulation of xenoantigens; however, these multiple gene-edited pigs were genetic mosaics that failed to knock out some xenoantigens. Although mosaicism should be resolved, the electroporation technique could become a primary method for the one-step generation of multiple gene modifications in pigs aimed at improving pig-to-human xenotransplantation.

Efficient generation of GGTA1-deficient pigs by electroporation of the CRISPR/Cas9 system into in vitro-fertilized zygotes.

BACKGROUND: Xenoantigens are a major source of concern with regard to the success of interspecific xenografts. GGTA1 encodes α1,3-galactosyltransferase, which is essential for the biosynthesis of galactosyl-alpha 1,3-galactose, the major xenoantigen causing hyperacute rejection. GGTA1-modified pigs, therefore, are promising donors for pig-to-human xenotransplantation. In this study, we developed a method for the introduction of the CRISPR/Cas9 system into in vitro-fertilized porcine zygotes via electroporation to generate GGTA1-modified pigs. RESULTS: We designed five guide RNAs (gRNAs) targeting distinct sites in GGTA1. After the introduction of the Cas9 protein with each gRNA via electroporation, the gene editing efficiency in blastocysts developed from zygotes was evaluated. The gRNA with the highest gene editing efficiency was used to generate GGTA1-edited pigs. Six piglets were delivered from two recipient gilts after the transfer of electroporated zygotes with the Cas9/gRNA complex. Deep sequencing analysis revealed that five out of six piglets carried a biallelic mutation in the targeted region of GGTA1, with no off-target events. Furthermore, staining with isolectin B4 confirmed deficient GGTA1 function in GGTA1 biallelic mutant piglets. CONCLUSIONS: We established GGTA1-modified pigs with high efficiency by introducing a CRISPR/Cas9 system into zygotes via electroporation. Multiple gene modifications, including knock-ins of human genes, in porcine zygotes via electroporation may further improve the application of the technique in pig-to-human xenotransplantation.

Generation of viable PDX1 gene-edited founder pigs as providers of nonmosaics.

Pancreatic duodenal homeobox 1 (PDX1) is a crucial gene for pancreas development during the fetal period. PDX1-modified pigs have the potential to be used as a model of diabetes mellitus. However, the severe health problems caused by the PDX1 mutation limit phenotypic studies of PDX1-modified pigs as diabetes models. In this study, we generated PDX1-modified pigs by the CRISPR/Cas9 system introduced into zygotes via electroporation and investigated the mosaicism, phenotypes, and inheritance of the resulting pigs. After the embryo transfer of PDX1-modified zygotes, nine mutant piglets were delivered. Two piglets were apancreatic biallelic mutants. For the other seven piglets, the ratio of mutant alleles to total alleles was 17.5-79.7%. Two mutant piglets with high mutation rates (67.7% and 79.7%) exhibited hypoplasia of the pancreas, whereas the other five piglets were healthy. One of the male mutant piglets was further analyzed. The ejaculated semen from the pig contained PDX1-mutant spermatozoa and the pig showed normal reproductive ability. In conclusion, the frequency of the PDX1 mutation is presumed to relate to pancreas formation, and PDX1 mutant founder pigs generated from zygotes introduced to the CRISPR/Cas9 system can serve as providers of nonmosaics to contribute to medical research on diabetes mellitus.

Evaluation of multiple gene targeting in porcine embryos by the CRISPR/Cas9 system using electroporation.

The CRISPR/Cas9 system now allows for unprecedented possibilities of genome editing. However, there are some limitations, including achieving efficient one-step multiple genome targeting to save costs, time, and ensure high quality. In the present study, we investigated the efficiency of one-step multiple gene modification by electroporation in porcine zygotes using pooled guide RNAs (gRNAs) targeting CMAH, GHR, GGTA1, and PDX1. We first selected the best-performing gRNA from three different designs for each gene based on the effect on embryo development and mutation efficiency. The three gRNAs showed equivalent effects on the rates of blastocyst formation in each targeted gene; however, gRNAs CMAH #2, GHR #3, GGTA1 #3, and PDX1 #3 showed the highest biallelic mutation rate, although the total mutation rate of PDX1 #3 was significantly lower than that of PDX1 #1. Therefore, CMAH #2, GHR #3, GGTA1 #3, and PDX1 #1 were used as a mixture in electroporation to further clarify whether multiple genes can be targeted simultaneously. Individual sequencing of 43 blastocysts at the target sites of each gene showed mutations in one and two target genes in twenty-four (55.8%) and nine (20.9%) blastocysts, respectively. No mutation was detected in any target gene in ten (23.3%) blastocysts and no blastocysts had a mutation in three or more target genes. These results indicate that electroporation could effectively deliver multiple gRNAs and Cas9 protein into porcine zygotes to target multiple genes in a one-step process. However, the technique requires further development to increase the success rate of multiple gene modification.

Curcumin supplementation in the maturation medium improves the maturation, fertilisation and developmental competence of porcine oocytes.

This study was conducted to determine the effects of supplementing the maturation medium with the antioxidant curcumin on the in vitro maturation (IVM), fertilisation and development of porcine oocytes. Curcumin supplementation was performed at concentrations of 0, 5, 10, 20, and 40 µM. At concentrations of 5-20 µM, curcumin had significant positive effects (P < 0.05) on maturation and fertilisation rates compared to the non-treated group. Of the groups cultured with 5-20 µM curcumin, the number of oocytes with DNA-fragmented nuclei after IVM was significantly lower than in groups matured without curcumin. Moreover, curcumin supplementation at 10 µM also gave a significantly higher rate of blastocyst formation compared with oocytes matured without curcumin. Increasing the curcumin concentration to 40 µM yielded negative effects on fertilisation and embryonic development compared with the groups treated with lower concentrations of curcumin. Supplementation with 10 µM curcumin had beneficial effects on the oocyte maturation rate and DNA fragmentation index compared to the non-treated group both in the presence and absence of hydrogen peroxide. These results indicate that curcumin supplementation at a suitable concentration (10 µM) is potentially useful for porcine oocyte culture systems, in terms of protecting oocytes from various forms of oxidative stress.

Effects of concentration of CRISPR/Cas9 components on genetic mosaicism in cytoplasmic microinjected porcine embryos.

Cytoplasmic microinjection (CI) of the CRISPR/Cas9 system enabled the induction of site-specific mutations in porcine zygotes and resulting pigs. However, mosaicism is a serious problem for genetically modified pigs. In the present study, we investigated suitable timing and concentration of CRISPR/Cas9 components for introduction into oocytes/zygotes by CI, to reduce mosaicism in the resulting blastocysts. First, we introduced 20 ng/µl of Cas9 protein and guide RNA (gRNA), targeting the α-1,3-galactosyltransferase (GalT) gene in oocytes before in vitro fertilization (IVF), in zygotes after IVF, or in oocytes/zygotes before and after IVF, twice. CI treatment had no detrimental effects on blastocyst formation rates. The highest value of the rate of mutant blastocysts was observed in zygotes injected after IVF. Next, we injected Cas9 protein and gRNA into zygotes after IVF at a concentration of 20 ng/µl each (20 ng/µl group) or 100 ng/µl each (100 ng/µl group). The ratio of the number of blastocysts that carried mutations to the total number of blastocysts examined in the 100 ng/µl group was significantly higher (P < 0.05) than that in the 20 ng/µl group. Although no blastocysts from the 20 ng/µl group carried a biallelic mutation, 16.7% of blastocysts from the 100 ng/µl group carried a biallelic mutation. In conclusion, increasing the concentration of Cas9 protein and gRNA is effective in generating biallelic mutant blastocysts. To reduce mosaicism, however, further optimization of the timing of CI, and the concentration of CRISPR/Cas9 components, is needed.

The effects of electroporation on viability and quality of in vivo-derived bovine blastocysts.

The introduction of exogenous molecules into embryos is required for analyses of molecular dynamics and specific gene functions during early embryonic development. Electroporation is an effective method to transport exogenous molecules into cells, but is rarely used in bovine embryos. First, we evaluated the viability of in vivo-derived bovine blastocysts after electroporation with fluorescein (FAM) labeled-oligonucleotides with varying pulse numbers (3, 5, 7, and 10), while keeping the pulse duration at 1 msec and the electric field of 20 V/mm. Next, we examined the effects of zona pellucida status on blastocyst quality after electroporation, by comparing the average diameter of blastocysts before and after electroporation using blastocysts with intact zona pellucida and hatching/hatched blastocysts. Electroporation successfully introduced exogenous molecules into in vivo-derived bovine blastocysts without loss of viability. Moreover, the status of the zona pellucida may be associated with the quality of blastocysts after electroporation.

Hypothermic storage of porcine zygotes in serum supplemented with chlorogenic acid.

The current study was conducted to investigate the effects of 100% foetal bovine serum (FBS) and 100% porcine follicular fluid (pFF) as a storage medium on the developmental competence of porcine zygotes stored at 25°C for 24 hr. Moreover, we evaluated the additive effects of chlorogenic acid (CGA) in the storage medium. When in vitro-produced zygotes were stored at 25°C for 24 hr in tubes containing either tissue culture medium (TCM) 199 supplemented with 1 mg/ml bovine serum albumin (BSA), 100% of FBS or 100% of pFF, the rate of blastocyst formation was significantly higher in 100% of FBS than in BSA-containing TCM 199. When the effects of CGA supplementation in 100% of FBS on the development of zygotes stored at 25°C for 24 hr was evaluated, more zygotes stored with 50 µM CGA developed to blastocysts compared with the other concentrations of CGA. When the formation date and quality of blastocysts derived from zygotes stored in 100% of FBS supplemented with 50 µM CGA were investigated, the highest ratio of blastocysts formation in the storage group appeared 1 day later than in the non-stored control group. However, a higher proportion of blastocysts with apoptotic nuclei was observed in the stored group as compared to the non-stored group. In conclusion, 100% of FBS is available for a short storage medium of porcine zygotes. The supplementation of 50 µM CGA into the storage medium improves the rates of blastocyst formation of zygotes after storage, but the quality of embryos from the stored zygotes remains to be improved.

Effects of Tris (hydroxymethyl) aminomethane on the quality of frozen-thawed boar spermatozoa.

Tris (hydroxymethyl) aminomethane (Tris) has been used as a pH regulator for buffering the pH of dilution extenders for boar semen, such as the Modena extender. The purpose of the present study was to assess the effects of Tris supplementation at different concentrations (0, 8, 24 and 72 µM) into the freezing extender on the quality and fertilising capacity of frozen-thawed boar spermatozoa. The results showed that the supplementation of 24 µM of Tris gave significantly higher percentages of sperm viability and plasma membrane integrity than those of the control group at any time point of assessment (0 h and 3 h post-thawing) (P < 0.05). However, there were no significant differences in the acrosome integrity parameter among the groups. Higher percentages of sperm motility were observed in the spermatozoa cryopreserved with 24 µM of Tris compared to the control groups when the samples were analysed 0 h after thawing (P < 0.05). However, an increase of the Tris concentration to 72 µM did not enhance the sperm motility parameters. The total numbers of fertilised oocytes and blastocysts obtained with spermatozoa frozen with 24 µM Tris were significantly higher than those of the control group without Tris (P < 0.05). In conclusion, the supplementation of 24 µM Tris into the freezing extender contributes to a better boar sperm quality and fertilising capacity after the process of freezing and thawing.

Effects of chlorogenic acid and caffeic acid on the quality of frozen-thawed boar sperm.

Chlorogenic acid (CGA) and caffeic acid (CA) are potent antioxidants that are mostly found in coffee beans. This study aimed to investigate the effects of CGA and CA supplementation during semen freezing on the quality of frozen-thawed boar spermatozoa. The antioxidants CGA and CA were added to a semen extender to achieve final concentrations of 50, 100, 200 and 400 µM. Supplementation of 100 µM CGA and CA yielded a significantly higher percentage of sperm viability (increased by 8%-10%) and plasma membrane integrity (increased by 4%-6%) than the control groups without the antioxidants at 0 and 3 hr after thawing (p < 0.05). At a concentration of 100 µM, CGA and CA also yielded beneficial effects on total and progressive sperm motility. Increases of CGA and CA concentrations to more than 200 µM did not enhance any sperm quality parameters. When the sperm penetrability and oocyte development by spermatozoa frozen with CGA and CA were evaluated, CGA and CA supplementations had no positive effects on the percentages of total fertilization, monospermic fertilization, cleavage and blastocyst formation. In conclusion, the supplementation of 100 µM CGA and CA during sperm freezing improved certain sperm parameters including motility, viability and plasma membrane integrity.

Optimal health and disease management using spatial uncertainty: a geographic characterization of emergent artemisinin-resistant Plasmodium falciparum distributions in Southeast Asia.

BACKGROUND: Artemisinin-resistant Plasmodium falciparum malaria parasites are now present across much of mainland Southeast Asia, where ongoing surveys are measuring and mapping their spatial distribution. These efforts require substantial resources. Here we propose a generic 'smart surveillance' methodology to identify optimal candidate sites for future sampling and thus map the distribution of artemisinin resistance most efficiently. METHODS: The approach uses the 'uncertainty' map generated iteratively by a geostatistical model to determine optimal locations for subsequent sampling. RESULTS: The methodology is illustrated using recent data on the prevalence of the K13-propeller polymorphism (a genetic marker of artemisinin resistance) in the Greater Mekong Subregion. CONCLUSION: This methodology, which has broader application to geostatistical mapping in general, could improve the quality and efficiency of drug resistance mapping and thereby guide practical operations to eliminate malaria in affected areas.

Towards a better future for DNA barcoding: Evaluating monophyly- and distance-based species identification using COI gene fragments of Dacini fruit flies.

The utility of a universal DNA 'barcode' fragment (658 base pairs of the Cytochrome C Oxidase I [COI] gene) has been established as a useful tool for species identification, and widely criticized as one for understanding the evolutionary history of a group. Large amounts of COI sequence data have been produced that hold promise for rapid species identification, for example, for biosecurity. The fruit fly tribe Dacini holds about a thousand species, of which 80 are pests of economic concern. We generated a COI reference library for 265 species of Dacini containing 5601 sequences that span most of the COI gene using circular consensus sequencing. We compared distance metrics versus monophyly assessments for species identification and although we found a 'soft' barcode gap around 2% pairwise distance, the exceptions to this rule dictate that a monophyly assessment is the only reliable method for species identification. We found that all fragments regularly used for Dacini fruit fly identification >450 base pairs long provide similar resolution. 11.3% of the species in our dataset were non-monophyletic in a COI tree, which is mostly due to species complexes. We conclude with recommendations for the future generation and use of COI libraries. We revise the generic assignment of Dacus transversus stat. rev. Hardy 1982, and Dacus perpusillus stat. rev. Drew 1971 and we establish Dacus maculipterus White 1998 syn. nov. as a junior synonym of Dacus satanas Liang et al. 1993.

Use of a Smartphone-Based Medication Adherence Platform to Improve Outcomes in Uncontrolled Type 2 Diabetes Among Veterans: Prospective Case-Crossover Study.

BACKGROUND: Medication nonadherence is a problem that impacts both the patient and the health system. OBJECTIVE: The objective of this study was to evaluate the impact of a novel smartphone app with patient-response-directed clinical intervention on medication adherence and blood glucose control in noninsulin-dependent patients with type 2 diabetes mellitus (T2DM). METHODS: We enrolled 50 participants with T2DM not on insulin with smartphones from a rural health care center in Northern Nevada for participation in this case-crossover study. Participants underwent a standard of care arm and an intervention arm. Each study arm was 3 months long, for a total of 6 months of follow-up. Participants had a hemoglobin A1c (HbA1c) lab draw at enrollment, 3 months, and 6 months. Participants had monthly "medication adherence scores" (MAS) and "Self-Efficacy for Appropriate Medication Use Scale" (SEAMS) questionnaires completed at baseline and monthly for the duration of the study. Our primary outcomes of interest were the changes in HbA1c between study arms. Secondary outcomes included the evaluation of the difference in the proportion of participants achieving a clinically meaningful reduction in HbA1c and the difference in the number of participants requiring diabetes therapy escalation between study arms. Exploratory outcomes included the analysis of the variation in medication possession ratio (MPR), MAS, and SEAMS during each study arm. RESULTS: A total of 30 participants completed both study arms and were included in the analysis. Dropouts were higher in participants enrolled in the standard of care arm first (9/25, 36% vs 4/25, 16%). Participants had a median HbA1c of 9.1%, had been living with T2DM for 6 years, had a median age of 66 years, and had a median of 8.5 medications. HbA1c reduction was 0.69% in the intervention arm versus 0.35% in the standard of care arm (P=.30). A total of 70% (21/30) of participants achieved a clinically meaningful reduction in HbA1c of 0.5% in the app intervention arm versus 40% (12/30) in the standard of care arm (odds ratio 2.29, 95% CI 0.94-5.6; P=.09). Participants had higher odds of a therapy escalation while in the standard of care arm (18/30, 60% vs 5/30, 16.7%, odds ratio 4.3, 95% CI 1.2-15.2; P=.02). The median MPR prior to enrollment was 109%, 112% during the study's intervention arm, and 102% during the standard of care arm. The median real-time MAS was 93.2%. The change in MAS (1 vs -0.1; P=.02) and SEAMS (1.9 vs -0.2; P<.001) from baseline to month 3 was higher in the intervention arm compared to standard of care. CONCLUSIONS: A novel smartphone app with patient-response-directed provider intervention holds promise in the ability to improve blood glucose control in complex non-insulin-dependent T2DM and is worthy of additional study.

Skin penetration of caffeine from commercial eye creams and eye creams designed and optimized based on Hansen solubility parameters.

Computer-aided formulation design can streamline and speed up product development. In this study, ingredient screening and optimizing software, Formulating for Efficacy® (FFE), was used to design and optimize creams for the topical delivery of caffeine. FFE was set up to optimize lipophilic active ingredients, therefore, this study challenged the program's capabilities. The effect of two chemical penetration enhancers, including dimethyl isosorbide (DMI) and ethoxydiglycol (EDG), were studied based on their favorable Hansen Solubility Parameter physicochemical input parameters for the skin delivery of caffeine in the FFE® software application. Four oil-in-water emulsions containing 2% caffeine were formulated, one without a chemical penetration enhancer, one with five percent of DMI, one with five percent of EDG, and one with 2.5% of DMI and EDG each (DMI + EDG). Additionally, three commercial products were used as reference products. The cumulative amount of caffeine released and permeated, and the flux across Strat-M® membranes were determined using Franz diffusion cells. The eye creams had skin-compatible pH, excellent spreadability for the application area, were opaque emulsions with 14-17 µm droplet size, and were stable at 25 °C for 6 months. All four eye creams formulated released over 85% of caffeine in 24 h, outperforming the commercial products. DMI + EDG cream provided the highest permeation in vitro in 24 h, which was significantly higher than the commercial products (p < 0.05). FFE proved to be a valuable and quick tool to aid in the topical delivery of caffeine.

Improving Agrobacterium tumefaciens-Mediated Genetic Transformation for Gene Function Studies and Mutagenesis in Cucumber (Cucumis sativus L.).

In the post-genomics era, Agrobacterium tumefaciens-mediated genetic transformation is becoming an increasingly indispensable tool for characterization of gene functions and crop improvement in cucumber (Cucumis sativus L.). However, cucumber transformation efficiency is still low. In this study, we evaluated the effects of several key factors affecting the shoot-regeneration rate and overall transformation efficiency in cucumber including genotypes, the age and sources of explants, Agrobacterium strains, infection/co-cultivation conditions, and selective agents. We showed that in general, North China cucumbers exhibited higher shoot-regeneration rate than US pickling or slicing cucumbers. The subapical ground meristematic regions from cotyledons or the hypocotyl had a similar shoot-regeneration efficiency that was also affected by the age of the explants. Transformation with the Agrobacterium strain AGL1 yielded a higher frequency of positive transformants than with GV3101. The antibiotic kanamycin was effective in selection against non-transformants or chimeras. Optimization of various factors was exemplified with the development of transgenic plants overexpressing the LittleLeaf (LL) gene or RNAi of the APRR2 gene in three cucumber lines. The streamlined protocol was also tested in transgenic studies in three additional genes. The overall transformation efficiency defined by the number of verified transgenic plants out of the number of seeds across multiple experiments was 0.2-1.7%. Screening among T1 OE transgenic plants identified novel, inheritable mutants for leaf or fruit color or size/shape, suggesting T-DNA insertion as a potential source of mutagenesis. The Agrobacterium-mediated transformation protocol from this study could be used as the baseline for further improvements in cucumber transformation.

Abnormal Wall Shear Stress Area is Correlated to Coronary Artery Bypass Graft Remodeling 1 Year After Surgery.

Coronary artery bypass graft surgery is a common intervention for coronary artery disease; however, it suffers from graft failure, and the underlying mechanisms are not fully understood. To better understand the relation between graft hemodynamics and surgical outcomes, we performed computational fluid dynamics simulations with deformable vessel walls in 10 study participants (24 bypass grafts) based on CT and 4D flow MRI one month after surgery to quantify lumen diameter, wall shear stress (WSS), and related hemodynamic measures. A second CT acquisition was performed one year after surgery to quantify lumen remodeling. Compared to venous grafts, left internal mammary artery grafts experienced lower abnormal WSS (< 1 Pa) area one month after surgery (13.8 vs. 70.1%, p = 0.001) and less inward lumen remodeling one year after surgery (- 2.4% vs. - 16.1%, p = 0.027). Abnormal WSS area one month post surgery correlated with percent change in graft lumen diameter one year post surgery (p = 0.030). This study shows for the first time prospectively a correlation between abnormal WSS area one month post surgery and graft lumen remodeling 1 year post surgery, suggesting that shear-related mechanisms may play a role in post-operative graft remodeling and might help explain differences in failure rates between arterial and venous grafts.