Kaempferol rhamnoside catabolism in rosette leaves of senescing Arabidopsis and postharvest stored radish.

Flavonol rhamnosides including kaempferitrin (i.e., kaempferol 3-O-α-rhamnoside-7-O-α-rhamnoside) occur throughout the plant kingdom. Mechanisms governing flavonol rhamnoside biosynthesis are established, whereas degradative processes occurring in plants are relatively unknown. Here, we investigated the catabolic events affecting kaempferitrin status in the rosette leaves of Arabidopsis thaliana L. Heynh. (Arabidopsis) and Raphanus sativus L. (radish), respectively, in response to developmental senescence and postharvest handling. On a per plant basis, losses of several kaempferol rhamnosides including kaempferitrin were apparent in senescing leaves of Arabidopsis during development and postharvest radish stored at 5 °C. Conversely, small pools of kaempferol 7-O-α-rhamnoside (K7R), kaempferol 3-O-α-rhamnoside (K3R), and kaempferol built up in senescing leaves of both species. Evidence is provided for ⍺-rhamnosidase activities targeting the 7-O-α-rhamnoside of kaempferitrin and K7R in rosette leaves of both species. An HPLC analysis of in vitro assays of clarified leaf extracts prepared from developing Arabidopsis and postharvest radish determined that these metabolic shifts were coincident with respective 237% and 645% increases in kaempferitrin 7-O-⍺-rhamnosidase activity. Lower activity rates were apparent when these ⍺-rhamnosidase assays were performed with K7R. A radish ⍺-rhamnosidase containing peak eluting from a DEAE-Sepharose Fast Flow column hydrolyzed various 7-O-rhamnosylated flavonols, as well as kaempferol 3-O-ß-glucoside. Together it is apparent that the catabolism of 7-O-α-rhamnosylated kaempferol metabolites in senescing plant leaves is associated with a flavonol 7-O-α-rhamnoside-utilizing α-rhamnosidase.

Global analysis of genetic circuitry and adaptive mechanisms enabling resistance to the azole antifungal drugs.

Invasive fungal infections caused by the pathogen Candida albicans have transitioned from a rare curiosity to a major cause of human mortality. This is in part due to the emergence of resistance to the limited number of antifungals available to treat fungal infections. Azoles function by targeting the biosynthesis of ergosterol, a key component of the fungal cell membrane. Loss-of-function mutations in the ergosterol biosynthetic gene ERG3 mitigate azole toxicity and enable resistance that depends upon fungal stress responses. Here, we performed a genome-wide synthetic genetic array screen in Saccharomyces cerevisiae to map ERG3 genetic interactors and uncover novel circuitry important for azole resistance. We identified nine genes that enabled erg3-mediated azole resistance in the model yeast and found that only two of these genes had a conserved impact on resistance in C. albicans. Further, we screened a C. albicans homozygous deletion mutant library and identified 13 genes for which deletion enhances azole susceptibility. Two of the genes, RGD1 and PEP8, were also important for azole resistance acquired by diverse mechanisms. We discovered that loss of function of retrograde transport protein Pep8 overwhelms the functional capacity of the stress response regulator calcineurin, thereby abrogating azole resistance. To identify the mechanism through which the GTPase activator protein Rgd1 enables azole resistance, we selected for mutations that restore resistance in strains lacking Rgd1. Whole genome sequencing uncovered parallel adaptive mechanisms involving amplification of both chromosome 7 and a large segment of chromosome 3. Overexpression of a transporter gene on the right portion of chromosome 3, NPR2, was sufficient to enable azole resistance in the absence of Rgd1. Thus, we establish a novel mechanism of adaptation to drug-induced stress, define genetic circuitry underpinning azole resistance, and illustrate divergence in resistance circuitry over evolutionary time.

An Apoplastic ß-Glucosidase is Essential for the Degradation of Flavonol 3-O-ß-Glucoside-7-O-α-Rhamnosides in Arabidopsis.

Flavonol bisglycosides accumulate in plant vegetative tissues in response to abiotic stress, including simultaneous environmental perturbations (i.e. nitrogen deficiency and low temperature, NDLT), but disappear with recovery from NDLT. Previously, we determined that a recombinant Arabidopsis ß-glucosidase (BGLU), BGLU15, hydrolyzes flavonol 3-O-ß-glucoside-7-O-α-rhamnosides and flavonol 3-O-ß-glucosides, forming flavonol 7-O-α-rhamnosides and flavonol aglycones, respectively. In this study, the transient expression of a BGLU15-Cherry fusion protein in onion epidermal cells demonstrated that BGLU15 was localized to the apoplast. Analysis of BGLU15 T-DNA insertional inactivation lines (bglu15-1 and bglu15-2) revealed negligible levels of BGLU15 transcripts, whereas its paralogs BGLU12 and BGLU16 were expressed in wild-type and bglu15 plants. The recombinant BGLU16 did not hydrolyze quercetin 3-O-ß-glucoside-7-O-α-rhamnoside or rhamnosylated flavonols, but was active with the synthetic substrate, p-nitrophenyl-ß-d-glucoside. In addition, shoots of both bglu15 mutants contained negligible flavonol 3-O-ß-glucoside-7-O-α-rhamnoside hydrolase activity, whereas this activity increased by 223% within 2 d of NDLT recovery in wild-type plants. The levels of flavonol 3-O-ß-glucoside-7-O-α-rhamnosides and quercetin 3-O-ß-glucoside were high and relatively unchanged in shoots of bglu15 mutants during recovery from NDLT, whereas rapid losses were apparent in wild-type shoots. Moreover, losses of two flavonol 3-O-ß-neohesperidoside-7-O-α-rhamnosides and kaempferol 3-O-α-rhamnoside-7-O-α-rhamnoside were evident during recovery from NDLT, regardless of whether BGLU15 was present. A spike in a kaempferol 7-O-α-rhamnoside occurred with stress recovery, regardless of germplasm, suggesting a contribution from hydrolysis of kaempferol 3-O-ß-neohesperidoside-7-O-α-rhamnosides and/or kaempferol 3-O-α-rhamnoside-7-O-α-rhamnoside by hitherto unknown mechanisms. Thus, BGLU15 is essential for catabolism of flavonol 3-O-ß-glucoside-7-O-α-rhamnosides and flavonol 3-O-ß-glucosides in Arabidopsis.

MatOrtho proximal interphalangeal joint arthroplasty: minimum 2-year follow-up.

The MatOrtho proximal interphalangeal replacement is a cementless cobalt-chromium metal-on-polyethylene mobile-bearing surface replacement arthroplasty. The aim of this study is to report the outcome and complications of this implant at a minimum of 2 years follow-up from a single institution. A retrospective case review was performed on all MatOrtho proximal interphalangeal joint replacements performed with a minimum of 2 years follow-up. Patient demographics, diagnosis, implant revision and other surgical interventions were recorded. Subjective and objective outcomes were evaluated at latest follow-up, including pain scores, range of motion, function and radiographic assessment. A total of 109 implants were inserted in 56 patients. Nine implants (six patients) were lost to follow-up. Of the remaining 100 implants, 75 had been undertaken in females. The mean age at time of surgery was 64 years and the principal diagnosis was osteoarthritis in 74%. The mean follow-up was 47 months (range 24-77). Within the group there was a statistically significant diminution in pain. There was also an improvement in functional scores post-operatively. Improvement in range of motion was seen in those joints with a pre-operative range of motion greater than 20°. Radiologically there was no evidence of loosening or of implant subsidence at final follow-up. The revision rate was 13%. Nine joints were revised to the NeuFlex (silicone rubber) prosthesis, three were converted to an arthrodesis and one had exchange of the MatOrtho prosthesis. The survival of the MatOrtho proximal interphalangeal joint arthroplasty was 85% at a minimum of 2-years follow-up. Patients can be advised that the procedure achieves good pain relief, improvement in functional scores and may improve range of motion. We would, however, caution against this implant's use in joints that are either stiff or have significant deformity and/or instability pre-operatively.

CT analysis of fat distribution superficial and deep to the Scarpa's fascial layer in the mid and lower abdomen.

INTRODUCTION: Mismatches in the thickness of subcutaneous fat at the level of the umbilicus and suprapubic region can result in an unsightly bulge and an unfavourable result following standard abdominoplasty. This problem can be avoided by thinning the abdominoplasty flap. This study was carried out to assess the thickness of the subcutaneous fat layer at the level of the umbilicus and the supra-pubic region. Measurements of full thickness fat and the depth of Scarpa's fascia separating superficial and sub-Scarpa fat layers were taken from the CT scans in 69 women; mean age 52 years (range 30-79). RESULTS: The thickness of the skin and abdominal wall fat was an average of 7 mm thicker (max 22 mm; p << 0.05). The thickness of the fat layer superficial to Scarpa's fascia was an average of 19 mm at mid abdomen and 22 mm in the lower abdomen (p << 0.05). The thickness of the fat layer deep to Scarpa's fascia was 14 mm in the mid abdomen and 5 mm in the lower abdomen (p << 0.05). In 55% of patients the difference in thickness of the mid abdominal and lower abdominal fat was greater than 5 mm, a difference that could lead to a noticeable mismatch and therefore an unfavourable outcome. CONCLUSIONS: Results of this study suggest that selectively thinning the fat layer deep to Scarpa's fascia would address potential mismatches and preserve the Scarpa's fascia layer in more than 50% of cases, therefore allowing wounds to be closed with an effective deep tension layer.

Reinforcing outpatient medical student learning using brief computer tutorials: the Patient-Teacher-Tutorial sequence.

BACKGROUND: At present, what students read after an outpatient encounter is largely left up to them. Our objective was to evaluate the education efficacy of a clinical education model in which the student moves through a sequence that includes immediately reinforcing their learning using a specifically designed computer tutorial. METHODS: Prior to a 14-day Pediatric Emergency rotation, medical students completed pre-tests for two common pediatric topics: Oral Rehydration Solutions (ORS) and Fever Without Source (FWS). After encountering a patient with either FWS or a patient needing ORS, the student logged into a computer that randomly assigned them to either a) completing a relevant computer tutorial (e.g. FWS patient + FWS tutorial = "in sequence") or b) completing the non-relevant tutorial (e.g. FWS patient + ORS tutorial = "out of sequence"). At the end of their rotation, they were tested again on both topics. Our main outcome was post-test scores on a given tutorial topic, contrasted by whether done in- or out-of-sequence. RESULTS: Ninety-two students completed the study protocol with 41 in the 'in sequence' group. Pre-test scores did not differ significantly. Overall, doing a computer tutorial in sequence resulted in significantly greater post-test scores (z-score 1.1 (SD 0.70) in sequence vs. 0.52 (1.1) out-of-sequence; 95% CI for difference +0.16, +0.93). Students spent longer on the tutorials when they were done in sequence (12.1 min (SD 7.3) vs. 10.5 (6.5)) though the difference was not statistically significant (95% CI diff: -1.2 min, +4.5). CONCLUSIONS: Outpatient learning frameworks could be structured to take best advantage of the heightened learning potential created by patient encounters. We propose the Patient-Teacher-Tutorial sequence as a framework for organizing learning in outpatient clinical settings.