Native protein purification of ferroxidase LPR1 from leaf extracts of a transgenic Arabidopsis thaliana line.

LPR1 (LOW PHOSPHATE ROOT 1), a bacterial-type plant ferroxidase, is crucial for local root phosphate (Pi) sensing. Here, we present a detailed protocol for native (tag-free) protein purification of LPR1 from leaf extracts by differential ammonium sulfate precipitation, size exclusion, and cation exchange chromatography of a transgenic Arabidopsis thaliana line overexpressing LPR1. We outline steps for LPR1 purification tracking via immune blot analysis and ferroxidase activity assay. The protocol yields highly pure and active LPR1 protein for biochemical analysis. For complete details on the use and execution of this protocol, please refer to Naumann et al. (2022).

Central Nervous System Xanthoma Disseminatum: Response to 2CdA in an Adolescent.

Xanthoma disseminatum is a normolipemic non-Langerhans cell histiocytosis characterized by red-brown rubbery papules of the skin which coalesce into plaque-like lesions with symmetric involvement of face, flexor, and intertriginous areas. Less commonly, xanthoma disseminatum may affect mucosal linings, abdominal organs, and the central nervous system, leading to endocrinopathies. We report a 12-year-old adolescent with mucosal, central nervous system, and painful cutaneous lesions, further complicated by diabetes insipidus and amenorrhea. Treatment with 2-chlorodeoxyadenosine led to relief of pain and significant improvement of mucosal, central nervous system, and cutaneous lesions, with subsequent restoration of menstrual cycles.

Bacterial-type ferroxidase tunes iron-dependent phosphate sensing during Arabidopsis root development.

Access to inorganic phosphate (Pi), a principal intermediate of energy and nucleotide metabolism, profoundly affects cellular activities and plant performance. In most soils, antagonistic Pi-metal interactions restrict Pi bioavailability, which guides local root development to maximize Pi interception. Growing root tips scout the essential but immobile mineral nutrient; however, the mechanisms monitoring external Pi status are unknown. Here, we show that Arabidopsis LOW PHOSPHATE ROOT 1 (LPR1), one key determinant of Fe-dependent Pi sensing in root meristems, encodes a novel ferroxidase of high substrate specificity and affinity (apparent KM ∼ 2 µM Fe2+). LPR1 typifies an ancient, Fe-oxidizing multicopper protein family that evolved early upon bacterial land colonization. The ancestor of streptophyte algae and embryophytes (land plants) acquired LPR1-type ferroxidase from soil bacteria via horizontal gene transfer, a hypothesis supported by phylogenomics, homology modeling, and biochemistry. Our molecular and kinetic data on LPR1 regulation indicate that Pi-dependent Fe substrate availability determines LPR1 activity and function. Guided by the metabolic lifestyle of extant sister bacterial genera, we propose that Arabidopsis LPR1 monitors subtle concentration differentials of external Fe availability as a Pi-dependent cue to adjust root meristem maintenance via Fe redox signaling and cell wall modification. We further hypothesize that the acquisition of bacterial LPR1-type ferroxidase by embryophyte progenitors facilitated the evolution of local Pi sensing and acquisition during plant terrestrialization.

Intravenous Fluid Management in Critically Ill Adults: A Review.

TOPIC: This article reviews the management of intravenous fluids and the evaluation of volume status in critically ill adults. CLINICAL RELEVANCE: Intravenous fluid administration is one of the most common interventions in the intensive care unit. Critically ill patients have dynamic fluid requirements, making the management of fluid therapy challenging. New literature suggests that balanced salt solutions may be preferred in some patient populations. PURPOSE OF PAPER: The bedside critical care nurse must understand the properties of various intravenous fluids and their corresponding impact on human physiology. The nurse's clinical and laboratory assessments of each patient help define the goals of fluid therapy, which will in turn be used to determine the optimal patient-specific selection and dose of fluid for administration. Nurses serve a vital role in monitoring the safety and efficacy of intravenous fluid therapy. Although this intervention can be lifesaving, inappropriate use of fluids has the potential to yield detrimental effects. CONTENT COVERED: This article discusses fluid physiology and the goals of intravenous fluid therapy, compares the types of intravenous fluids (isotonic crystalloids, including 0.9% sodium chloride and balanced salt solutions; hypotonic and hypertonic crystalloids; and colloids) and their adverse effects and impact on hemodynamics, and describes the critical care nurse's essential role in selecting and monitoring intravenous fluid therapy.

Nondestructive adult age at death estimation: Visualizing cementum annulations in a known age historical human assemblage using synchrotron X-ray microtomography.

OBJECTIVES: Adult age at death estimation continues to challenge physical anthropologists. One estimation method involves counting tooth cementum annulations (TCA). Non-destructively accessing TCA is a critical step to approaching fossil teeth of unknown age and to verifying life history profiles of human ancestors. This pilot study aims to (a) non-destructively image TCA in teeth from a known age archeological human population by propagation phase contrast X-ray synchrotron µCT (PPC-SR-µCT) (b) test the correlation between real and estimated ages, and the accuracy, precision and bias of age estimates. MATERIALS AND METHODS: We examine 20 permanent human canines (aged 20-81 years), from a 18th to 19th century known age collection from St. Luke's Church (London, England). We scanned transverse segments of acellular cementum in the apical portion of the middle root third using PPC-SR-µCT. We generated virtual transverse sections on which two observers perform two sessions of blind TCA counts. We calculate the estimated ages at death by adding 10 years to the TCA counts. RESULTS: A moderately strong positive linear relationship exists between real and estimated ages (r = 0.76, p < .001), with an average inaccuracy of 16.1 years and an average bias towards underestimation of 15.7 years. This difference is lower in individuals <50 years (6.8 and 6.5 years, respectively, n = 10) compared with those >50 years (24.9 years, n = 10). DISCUSSION: We reliably imaged and identified TCA in individuals <50 years from a known-age archeological sample. Scanning refinement will yield a promising alternative to current destructive methods of TCA analyses and to aid access to life history events in adult fossil hominins.

50% dextrose versus normal saline as distension media during cystoscopy for assessment of ureteric patency.

OBJECTIVE: To compare the visualization of ureteric jets when using 50% dextrose (D50) as opposed to normal saline (NS) as distension media during cystoscopy. STUDY DESIGN: Cross sectional study. METHODS: Two patients each had two cystoscopy videos recorded at the time of a ureteric jet; one using NS and the other using D50 resulting in two sets of paired videos (four videos). A fifth cystoscopy video was recorded, as a control, at a time when there was no ureteric-jet. Fifty participants including attending physicians, residents and medical students were recruited at an academic-affiliated community hospital. Participants were blinded to the medium used and viewed each of the five videos. Participants assessed each video for presence of a ureteric-jet, ease of interpretation, and compared the paired D50 and NS videos for clarity of ureteric-jets. MAIN OUTCOME MEASURES: Participant's assessment of clarity of the ureteric jets when D50 was used as compared to when NS was used in the paired videos. RESULTS: All 100 observations of the two D50 videos with jets identified the presence of a jet; for the NS videos, 96/100 observations identified a jet, 2/100 did not identify a jet and 2/100 were unsure. 48/50 observations of the video with no jet were correct, while 2/50 were unsure. Participants rated the ureteric-jets to be clearer in videos with D50 (86% vs 14%, P<0.001); and had difficulty interpreting cystoscopy videos with NS (62% vs 2%, OR: 80, 95% CI: 10.2-627.6). CONCLUSION: Participants preferred the clarity of the ureteric-jet when 50% dextrose was used as the distension medium during cystoscopy as compared to normal saline.

Use of 50% Dextrose as the Distension Medium During Cystoscopy for Visualization of Ureteric Jets.

BACKGROUND: Indigotindisulfonate sodium has been used to color the urine and thereby improve the visualization of ureteric jets during intraoperative cystoscopy. After indigotindisulfonate sodium became unavailable, there has been an ongoing search for an alternate agent to improve visualization of the jets. METHOD: We used 50% dextrose, which is more viscous than urine, as the distension medium during cystoscopy so that the ureteric efflux is seen as a jet of contrasting viscosity. We instilled 100 mL of 50% dextrose into the bladder through an indwelling catheter, which is then removed and cystoscopy is performed as usual. EXPERIENCE: We observed jets of contrasting viscosity in every patient in whom 50% dextrose was used as compared with coloring agents in which the jet is not always colored at the time of cystoscopy. Visualization of the other structures in the bladder and the bladder wall itself is not altered by 50% dextrose, although the volume of 50% dextrose that we typically use may not provide adequate distension for a complete assessment of the bladder. If additional distension is necessary, normal saline may be used in addition to the 50% dextrose once the ureteric jets have been assessed. CONCLUSION: Fifty percent dextrose is an effective alternative to indigotindisulfonate sodium for visualization of ureteric jets during cystoscopy.

Accessing developmental information of fossil hominin teeth using new synchrotron microtomography-based visualization techniques of dental surfaces and interfaces.

Quantification of dental long-period growth lines (Retzius lines in enamel and Andresen lines in dentine) and matching of stress patterns (internal accentuated lines and hypoplasias) are used in determining crown formation time and age at death in juvenile fossil hominins. They yield the chronology employed for inferences of life history. Synchrotron virtual histology has been demonstrated as a non-destructive alternative to conventional invasive approaches. Nevertheless, fossil teeth are sometimes poorly preserved or physically inaccessible, preventing observation of the external expression of incremental lines (perikymata and periradicular bands). Here we present a new approach combining synchrotron virtual histology and high quality three-dimensional rendering of dental surfaces and internal interfaces. We illustrate this approach with seventeen permanent fossil hominin teeth. The outer enamel surface and enamel-dentine junction (EDJ) were segmented by capturing the phase contrast fringes at the structural interfaces. Three-dimensional models were rendered with Phong's algorithm, and a combination of directional colored lights to enhance surface topography and the pattern of subtle variations in tissue density. The process reveals perikymata and linear enamel hypoplasias on the entire crown surface, including unerupted teeth. Using this method, highly detailed stress patterns at the EDJ allow precise matching of teeth within an individual's dentition when virtual histology is not sufficient. We highlight that taphonomical altered enamel can in particular cases yield artificial subdivisions of perikymata when imaged using X-ray microtomography with insufficient resolution. This may complicate assessments of developmental time, although this can be circumvented by a careful analysis of external and internal structures in parallel. We further present new crown formation times for two unerupted canines from South African Australopiths, which were found to form over a rather surprisingly long time (> 4.5 years). This approach provides tools for maximizing the recovery of developmental information in teeth, especially in the most difficult cases.

Application of quality by design (QbD) approach to ultrasonic atomization spray coating of drug-eluting stents.

The drug coating process for coated drug-eluting stents (DES) has been identified as a key source of inter- and intra-batch variability in drug elution rates. Quality-by-design (QbD) principles were applied to gain an understanding of the ultrasonic spray coating process of DES. Statistically based design of experiments (DOE) were used to understand the relationship between ultrasonic atomization spray coating parameters and dependent variables such as coating mass ratio, roughness, drug solid state composite microstructure, and elution kinetics. Defect-free DES coatings composed of 70% 85:15 poly(DL-lactide-co-glycolide) and 30% everolimus were fabricated with a constant coating mass. The drug elution profile was characterized by a mathematical model describing biphasic release kinetics. Model coefficients were analyzed as a DOE response. Changes in ultrasonic coating processing conditions resulted in substantial changes in roughness and elution kinetics. Based on the outcome from the DOE study, a design space was defined in terms of the critical coating process parameters resulting in optimum coating roughness and drug elution. This QbD methodology can be useful to enhance the quality of coated DES.

Survival impact of cytoreduction to microscopic disease for advanced stage cancer of the uterine corpus: a retrospective cohort study.

OBJECTIVE: To assess the impact of cytoreduction to no gross residual disease (R0) on overall survival (OS) in patients with stage III-IV uterine carcinosarcoma (CS), papillary serous/clear cell (UPSC/CC) and endometrioid carcinoma (EC). METHODS: We retrospectively identified 168 patients who underwent primary surgery for advanced uterine cancer between 1984 and 2009 in two teaching hospitals in Brooklyn, New York. Histology, stage, grade, residual disease (RD), adjuvant therapy, age, race and OS were collected. OS was calculated using the Kaplan-Meier method. Predictive factors were compared using the log-rank test and Cox regression analysis. RESULTS: Our cohort included 54 patients with CS (stage III, n = 32; stage IV, n = 22), 54 patients with UPSC/CC (stage III, n = 20; stage IV, n = 34) and 60 patients with EC (stage III, n = 45; stage IV, n = 15). R0 was achieved in 64% of patients with CS, in 53% of patients with UPSC/CC and in 68% of patients with EC. There was no interaction between histologic subtype and feasibility of complete cytoreduction (p = 0.39). R0 was associated with a median OS of 25 months (95% CI [18, 33]) versus 13 months (95% CI [8, 18]) in patients with gross RD (p = 0.03). This effect was uniform among histologic subtypes. On multivariate analysis, predictors of increased mortality were gross residual disease (HR = 2.0, 95% CI [1.1, 3.7], p = 0.01), stage IV (HR = 1.8, 95% CI [1.1, 3.1], p = 0.02) and age (HR = 1.04 per year of age, 95% CI [1.02, 1.07], p = 0.002). CONCLUSION: Cytoreductive surgery to R0 is associated with improved OS in advanced uterine cancer. This effect is uniform among histologies. There is no interaction between histologic subtype and feasibility of complete cytoreduction.

Intra- and interspecific variation in macaque molar enamel thickness.

Enamel thickness has played an important role in studies of primate taxonomy, phylogeny, and functional morphology, although its variation among hominins is poorly understood. Macaques parallel hominins in their widespread geographic distribution, relative range of body sizes, and radiation during the last five million years. To explore enamel thickness variation, we quantified average and relative enamel thickness (AET and RET) in Macaca arctoides, Macaca fascicularis, Macaca fuscata, Macaca mulatta, Macaca nemestrina, and Macaca sylvanus. Enamel area, dentine area, and enamel-dentine junction length were measured from mesial sections of 386 molars scanned with micro-computed tomography, yielding AET and RET indices. Intraspecific sex differences were not found in AET or RET. Macaca fuscata had the highest AET and RET, M. fascicularis showed the lowest AET, and M. arctoides had the lowest RET. The latitudinal distribution of macaque species was associated with AET for these six species. Temperate macaques had thicker molar enamel than did tropical macaques, suggesting that thick enamel may be adaptive in seasonal environments. Additional research is needed to determine if thick enamel in temperate macaques is a response to intensified hard-object feeding, increased abrasion, and/or a broader diet with a greater range of food material properties. The extreme ecological flexibility of macaques may prohibit identification of consistent trends between specific diets and enamel thickness conditions. Such complications of interpretation of ecological variability, dietary diversity, and enamel thickness may similarly apply for fossil Homo species.

Application of the Bang and Ramm age at death estimation method to two known-age archaeological assemblages.

The Bang and Ramm method uses root dentine translucency (RDT) length in sectioned or unsectioned teeth as a sole indicator of chronological age at death in adult human remains. The formulae have been tested on modern remains of known age and on modern and archaeological remains of unknown age. This is the first published study of the method on known-age archaeological specimens and tests whether RDT is a good indicator of chronological age in buried human remains. We applied the Bang and Ramm equations to two 18th and 19th century assemblages excavated from the crypt of Christ Church, Spitalfields, and the cemetery of All Hallows by the Tower. Translucency was defined by shining a light through the external unsectioned root surface and was measured from digital images of 583 and 83 nonmolar roots from 126 Spitalfields and 12 All Hallows individuals, respectively, aged 21-81 years. Average absolute difference between real age and estimated age was 10.7 years and 8.4 years for Spitalfields and All Hallows individuals, respectively, with 58% and 75% estimated within 10 years of known age, and 29% and 33% estimated within five years of known age. These estimations are comparable to results from other ageing methods applied to the Spitalfields collection. Ages from both populations were estimated largely to the middle ranges, with younger individuals overestimated and older individuals underestimated. This is a common occurrence when using inverse calibration, where age is treated as the dependent variable and the ageing feature as the independent variable.

Sensitivity and specificity of new GDx parameters.

PURPOSE: The GDx is a scanning laser polarimeter that assesses peripapillary nerve fiber layer thickness. In addition to the 14 existing outcome parameters, four new parameters have been described recently: the Ellipse Standard Deviation (ESD), the Normalized Superior Area (NSA), the Normalized Inferior Area (NIA) and the Discriminant Analysis (DA). The aim of this study was to investigate the sensitivity and specificity of these four new parameters. METHODS: Only one randomly selected eye of 263 healthy volunteers and 241 glaucoma patients was considered. The healthy group was randomly divided into a reference set (n = 132) to calculate the tenth percentile of the normal distribution and a test set (n = 131) to calculate the specificity against these newly established cut-off points. Sensitivity was calculated for all glaucoma patients (n = 241) and again for three separate subgroups: early glaucoma (n = 90), moderate glaucoma (n = 93), and advanced glaucoma (n = 58). RESULTS: When the tenth percentile of the normal distribution was used as a cut-off point, the sensitivity and specificity pairs of the new parameters were 61.8% and 87.6%, 61.8% and 89.1%, 50.2% and 92.2% and 72.6% and 95.3% for the ESD, NSA, NIA, and the DA, respectively. The Area under the ROC curve was 0.86, 0.86, 0.87, and 0.90, respectively. Among the existing parameters, the Number discriminated best (sensitivity and specificity: 76.8% and 89.1%, respectively; area under the ROC curve: 0.90). When compared with The Number, the DA was equally good, whereas the other three new parameters performed statistically significantly worse. In general, the area under the ROC curve increased from early to moderate to advanced glaucoma. CONCLUSIONS: The new GDx parameters discriminated well between normal subjects and glaucoma patients. None of the new parameters discriminated better than The Number.

Prognostic value of S-100-beta serum concentration in patients with uveal melanoma.

BACKGROUND: In cutaneous melanoma, the S-100-beta serum level is recognized as a marker of metastatic disease. OBJECTIVES: To determine whether S-100-beta is present in the serum of patients with uveal melanoma and to test whether the serum concentration of S-100-beta is related to known clinical and histopathological prognostic factors in these patients. METHODS: The S-100-beta concentration was measured in serum samples collected from 64 patients with uveal melanoma before enucleation and from 58 healthy control subjects. A 2-site immunoluminometric assay was used to quantify the S-100-beta concentration in serum. S-100-beta concentrations in the serum from patients were compared with clinicopathological tumor variables, sex, occurrence of metastasis, and survival. RESULTS: Thirty-seven (57.8%) of 64 patients with uveal melanoma showed detectable levels of serum S-100-beta. There was, however, no significant difference between serum levels of patients and control subjects (P =.71). Statistical analysis showed no significant correlation between S-100-beta concentration and any of the clinicopathological tumor variables, occurrence of metastases, or survival. Only sex was correlated with S-100-beta serum levels, which was not observed in the control group. CONCLUSIONS: In our study on patients with uveal melanoma, the S-100-beta serum concentration was not correlated with any investigated prognostic factor and was not of prognostic value itself. Female patients appeared to have higher S-100-beta concentrations than male patients.