Phosphate deprivation-induced changes in tomato are mediated by an interaction between brassinosteroid signaling and zinc.

Inorganic phosphate (Pi) is a necessary macronutrient for basic biological processes. Plants modulate their root system architecture (RSA) and cellular processes to adapt to Pi deprivation albeit with a growth penalty. Excess application of Pi fertilizer, on the contrary, leads to eutrophication and has a negative environmental impact. We compared RSA, root hair elongation, acid phosphatase activity, metal ion accumulation, and brassinosteroid hormone levels of Solanum lycopersicum (tomato) and Solanum pennellii, which is a wild relative of tomato, under Pi sufficiency and deficiency conditions to understand the molecular mechanism of Pi deprivation response in tomato. We showed that S. pennellii is partially insensitive to phosphate deprivation. Furthermore, it mounts a constitutive response under phosphate sufficiency. We demonstrate that activated brassinosteroid signaling through a tomato BZR1 ortholog gives rise to the same constitutive phosphate deficiency response, which is dependent on zinc overaccumulation. Collectively, these results reveal an additional strategy by which plants can adapt to phosphate starvation.

Ruptured Median Raphe Cyst Mimicking a Vascular Penile Mass on Ultrasound.

Median raphe cysts are uncommon benign cysts thought to occur due to improper fusion of the genital tubercle and can occur anywhere along the median raphe, from the glans to the anus, most commonly occurring along the ventral penile shaft. Limited information is available in the literature about the common imaging features of median raphe cysts with available reports highlighting an avascular cystic lesion. Our case demonstrates a 10-year-old male patient presenting with a ventral penile mass that demonstrated interval growth in the absence of trauma without overlying skin changes. Doppler ultrasound examination demonstrated a solid vascular mass measuring up to 1.6 cm at the ventral aspect of the penis with arterial and venous waveforms. The patient underwent elective resection of the mass which revealed a 2.0 cm inflamed glandular subtype median raphe cyst. This report demonstrates an atypical imaging presentation of an inflamed median raphe cyst, particularly that of a heterogeneous solid mass with arterial and venous blood flow on ultrasound.

Accurate measurements of phase refractive index of soft contact lenses.

We describe a measurement approach for the bulk phase refractive index of hydrogel and silicone hydrogel (soft) contact lenses in solution at multiple wavelengths with an accuracy of +/- 0.001, and sensitivity to change of 0.0002. The method uses time-domain low-coherence interferometry to obtain group refractive index (GRI) for contact lenses between 530 to 670 nm in 10 nm increments. The measured GRI dispersion curve is then mathematically converted to the phase refractive index values. The approach is based on a point measurement using focused beam, and therefore does not require flattening of the lens for the measurements. We discuss practical implications of the method for the quality control in manufacturing of ophthalmic optics.

Regulation of Root Angle and Gravitropism.

Regulation of plant root angle is critical for obtaining nutrients and water and is an important trait for plant breeding. A plant's final, long-term root angle is the net result of a complex series of decisions made by a root tip in response to changes in nutrient availability, impediments, the gravity vector and other stimuli. When a root tip is displaced from the gravity vector, the short-term process of gravitropism results in rapid reorientation of the root toward the vertical. Here, we explore both short- and long-term regulation of root growth angle, using natural variation in tomato to identify shared and separate genetic features of the two responses. Mapping of expression quantitative trait loci mapping and leveraging natural variation between and within species including Arabidopsis suggest a role for PURPLE ACID PHOSPHATASE 27 and CELL DIVISION CYCLE 73 in determining root angle.

Nitrogen fixation in a landrace of maize is supported by a mucilage-associated diazotrophic microbiota.

Plants are associated with a complex microbiota that contributes to nutrient acquisition, plant growth, and plant defense. Nitrogen-fixing microbial associations are efficient and well characterized in legumes but are limited in cereals, including maize. We studied an indigenous landrace of maize grown in nitrogen-depleted soils in the Sierra Mixe region of Oaxaca, Mexico. This landrace is characterized by the extensive development of aerial roots that secrete a carbohydrate-rich mucilage. Analysis of the mucilage microbiota indicated that it was enriched in taxa for which many known species are diazotrophic, was enriched for homologs of genes encoding nitrogenase subunits, and harbored active nitrogenase activity as assessed by acetylene reduction and 15N2 incorporation assays. Field experiments in Sierra Mixe using 15N natural abundance or 15N-enrichment assessments over 5 years indicated that atmospheric nitrogen fixation contributed 29%-82% of the nitrogen nutrition of Sierra Mixe maize.

Velopharyngeal videofluoroscopy: Providing useful clinical information in the era of reduced dose radiation and safety.

BACKGROUND: The state of the art for correcting velopharyngeal insufficiency (VPI) is a surgical procedure which is customized according to findings on imaging procedures: multiplanar videofluoroscopy (MPVF) and flexible videonasopharyngoscopy (FVNP). Recently, the use of MPVF has been challenged because of the potential risk of using ionizing radiation, especially in children. OBJECTIVE: To study whether using a protocol for performing MPVF can effectively decrease radiation dose in patients with VPI while providing useful information for planning surgical correction of VPI in combination with FVNP. The methodology used for performing the imaging procedures is described as well as the effectiveness of the surgical procedure. MATERIAL AND METHODS: Eighty - nine patients (Age range = 3-17 years; median = 5.5 years) with VPI resulting from multiple etiologies were studied. All patients underwent MPVF and FVNP for planning surgical correction of VPI. Radiation dosage data in each case was recorded. Forty of the 89 patients also completed a postoperative evaluation. Eleven out of the remaining 49 patients have not completed a postoperative evaluation and 38 patients are still pending surgical correction. RESULTS: Radiation dosage ranged from 1.00 to 8.75 miliSieverts (mSv); Mean = 2.88 mSv; SD = 1.575 mSv. Preoperative nasometry demonstrated mean nasalance ranging from 41%-95%; Mean = 72.30; SD = 4.54. Postoperatively mean nasalance was within normal limits in 36 (90%) out of 40 cases, ranging from 21% to 35%; Mean = 28.10; SD = 5.40. Nasal emission was eliminated postoperatively in all cases. CONCLUSION: MPVF provides useful information for planning the surgical procedure aimed at correcting VPI. The combination of MPVF and FVNP is a reliable procedure for assessing velopharyngeal closure and to surgically correcting VPI with a highly successful outcome.

Pneumatosis intestinalis associated with Henoch-Schönlein purpura.

Henoch-Schönlein purpura (HSP) is the most common vasculitis in children. It is a disorder of the inflammatory cascade leading to immunoglobulin A deposition and leukocytoclastic vasculitis of small vessels of skin, kidneys, joints, and gastrointestinal (GI) tract. A wide variety of GI manifestations are seen in ∼50% to 75% of patients with HSP. Diffuse colicky abdominal pain is the most common GI symptom. The small bowel is the most frequently involved GI site. Intussusception is rare but is the most common surgical complication. We report the case of a 2-year-old girl with a 5-day history of abdominal pain followed by a palpable purpuric rash. Her urinalysis, complete blood cell count, and tests of renal function were normal. An acute abdominal series was unremarkable initially, and abdominal ultrasound imaging showed ascites and thickened small bowel loops. She was diagnosed with HSP. The abdominal pain worsened, and an abdominal computed tomography scan demonstrated distal small bowel wall thickening and pneumatosis intestinalis in the descending colon. She was started on total parenteral nutrition and antibiotics and placed on bowel rest. She was given 2 mg/kg of intravenous immunoglobulin. Her abdominal pain gradually improved over the next week, and a repeat computed tomography scan showed significant improvement of the small bowel wall thickening and pneumatosis. The purpuric rash improved, and her abdominal pain resolved. We report a case of HSP and pneumatosis intestinalis, an association that has not been reported previously.

22q11.2 deletion detected by endoscopic observation of pharyngeal pulsations in a child with submucous cleft palate and persistent velopharyngeal insufficiency.

22q11.2 microdeletion syndrome (22q11.2DS) is the most common syndrome associated with cleft palate and velopharyngeal insufficiency (VPI). Over 180 clinical features have been described. Most common features include: cardiac malformations, cleft palate, velopharyngeal insufficiency, characteristic facial features, hypotonia, behavioral disorders, and musculoskeletal disorders among several other fenotipical features. A case of 22q11.2DS confirmed by cytogenomic analysis is presented with review of the literature. Main clinical features were a submucous cleft palate (SMCP) with persistent VPI after palatoplasty, an ectopic left internal carotid artery and a prominent aortic root. VPI was corrected with a pharyngeal flap, tailored according to findings of videonasopharyngoscopy, videofluoroscopy and neck CT scan with contrast.

Argonaute2 complexes carry a population of circulating microRNAs independent of vesicles in human plasma.

MicroRNAs (miRNAs) circulate in the bloodstream in a highly stable, extracellular form and are being developed as blood-based biomarkers for cancer and other diseases. However, the mechanism underlying their remarkable stability in the RNase-rich environment of blood is not well understood. The current model in the literature posits that circulating miRNAs are protected by encapsulation in membrane-bound vesicles such as exosomes, but this has not been systematically studied. We used differential centrifugation and size-exclusion chromatography as orthogonal approaches to characterize circulating miRNA complexes in human plasma and serum. We found, surprisingly, that the majority of circulating miRNAs cofractionated with protein complexes rather than with vesicles. miRNAs were also sensitive to protease treatment of plasma, indicating that protein complexes protect circulating miRNAs from plasma RNases. Further characterization revealed that Argonaute2 (Ago2), the key effector protein of miRNA-mediated silencing, was present in human plasma and eluted with plasma miRNAs in size-exclusion chromatography. Furthermore, immunoprecipitation of Ago2 from plasma readily recovered non-vesicle-associated plasma miRNAs. The majority of miRNAs studied copurified with the Ago2 ribonucleoprotein complex, but a minority of specific miRNAs associated predominantly with vesicles. Our results reveal two populations of circulating miRNAs and suggest that circulating Ago2 complexes are a mechanism responsible for the stability of plasma miRNAs. Our study has important implications for the development of biomarker approaches based on capture and analysis of circulating miRNAs. In addition, identification of extracellular Ago2-miRNA complexes in plasma raises the possibility that cells release a functional miRNA-induced silencing complex into the circulation.

Paratesticular rhabdomyosarcoma with metastatic encasement of the abdominal aorta.

Paratesticular rhabdomyosarcoma is a rare but aggressive malignancy in children and adolescents. Prognosis is related to initial tumor resectability as well as staging of the disease based on tumor invasiveness, tumor bulk, nodal disease and metastases. We report the unusual presentation of paratesticular rhabdomyosarcoma with metastatic extension through the inguinal canal and encasement of the abdominal aorta. These features portend a poor prognosis given their association with a greater stage of disease and unresectable nature at presentation. Delayed surgical resection follows a regimen of chemotherapy and radiation therapy in such cases of extensive disease. Encasement of the abdominal aorta has been shown to increase presurgical risk for intraoperative vascular injury when related to other malignancies, but its role in relation to metastatic paratesticular rhabdomyosarcoma has not been investigated. Also, rhabdomyosarcoma should be considered in the differential diagnoses of tumors that demonstrate encasement of the abdominal aorta.

Characterization of a recombinant form of annexin VI for detection of apoptosis.

We developed a recombinant form of human annexin VI called annexin VI-601 (M(r) 76,224) with the N-terminal extension of Ala-Gly-Gly-Cys-Gly-His to allow ready attachment of fluorescent or radioactive labels. The protein was produced by expression in E. coli and was purified by calcium-dependent membrane binding, anion-exchange chromatography, and heparin-Sepharose affinity chromatography. The protein could be readily labeled with iodoacetamidofluorescein and with (99m)Tc. The protein bound with high affinity to PS-containing phospholipid vesicles and to erythrocytes with exposed phosphatidylserine. Fluorescent annexin VI-601 readily detected apoptosis of Jurkat cells by flow cytometry at much lower calcium concentrations than those required for equivalent detection by annexin V. In vivo administration of radiolabeled protein showed that blood clearance was much slower than annexin V. In conclusion, annexin VI may have advantages over annexin V in certain situations for both in vitro and in vivo detection of apoptosis and therapeutic targeting of PS due to its lower calcium requirement for membrane binding and its higher molecular weight.

Transmembrane voltage regulates binding of annexin V and lactadherin to cells with exposed phosphatidylserine.

BACKGROUND: Cells expose phosphatidylserine during apoptosis. The voltage across the plasma membrane also decreases or disappears during apoptosis, but the physiological significance of this is unknown. RESULTS: Here we show that transmembrane potential regulates membrane binding of two unrelated proteins that recognize exposed phosphatidylserine on apoptotic cells. In Jurkat T leukemia cells and K562 promyelocytic leukemia cells undergoing apoptosis, extracellular binding of annexin V was increased by decreasing membrane potential in a dose-dependent manner. Studies with phospholipid vesicles showed that the effect was mediated via an increase in binding affinity. The effect was independent of the apoptotic stimulus. The same phenomenon occurred with lactadherin, a structurally unrelated protein that also binds to apoptotic cells via phosphatidylserine and is essential for in vivo clearance of dying cells. CONCLUSION: Alterations in membrane potential regulate the binding of annexin V and lactadherin to cell membranes, and may also influence the membrane binding of other classes of phosphatidylserine-binding proteins.

Entropic and enthalpic contributions to annexin V-membrane binding: a comprehensive quantitative model.

Annexin V binds to membranes with very high affinity, but the factors responsible remain to be quantitatively elucidated. Analysis by isothermal microcalorimetry and calcium titration under conditions of low membrane occupancy showed that there was a strongly positive entropy change upon binding. For vesicles containing 25% phosphatidylserine at 0.15 m ionic strength, the free energy of binding was -53 kcal/mol protein, whereas the enthalpy of binding was -38 kcal/mol. Addition of 4 m urea decreased the free energy of binding by about 30% without denaturing the protein, suggesting that hydrophobic forces make a significant contribution to binding affinity. This was confirmed by mutagenesis studies that showed that binding affinity was modulated by the hydrophobicity of surface residues that are likely to enter the interfacial region upon protein-membrane binding. The change in free energy was quantitatively consistent with predictions from the Wimley-White scale of interfacial hydrophobicity. In contrast, binding affinity was not increased by making the protein surface more positively charged, nor decreased by making it more negatively charged, ruling out general ionic interactions as major contributors to binding affinity. The affinity of annexin V was the same regardless of the head group present on the anionic phospholipids tested (phosphatidylserine, phosphatidylglycerol, phosphatidylmethanol, and cardiolipin), ruling out specific interactions between the protein and non-phosphate moieties of the head group as a significant contributor to binding affinity. Analysis by fluorescence resonance energy transfer showed that multimers did not form on phosphatidylserine membranes at low occupancy, indicating that annexin-annexin interactions did not contribute to binding affinity. In summary, binding of annexin V to membranes is driven by both enthalpic and entropic forces. Dehydration of hydrophobic regions of the protein surface as they enter the interfacial region makes an important contribution to overall binding affinity, supplementing the role of protein-calcium-phosphate chelates.

Epithelial internalization of superparamagnetic nanoparticles and response to external magnetic field.

Superparamagnetic magnetite nanoparticles (MNP) coated with silica were synthesized and chronically implanted into the middle ear epithelial tissues of a guinea pig model (n=16) for the generation of force by an external magnetic field. In vivo limitations of biocompatibility include particle morphology, size distribution, composition and mode of internalization. Synthesis of MNP was performed using a modified precipitation technique and they were characterized by transmission electron microscopy, X-ray diffractometry and energy dispersive spectroscopy, which verified size distribution, composition and silica encapsulation. The mechanism for internalizing 16+/-2.3 nm diameter MNP was likely endocytosis, enhanced by magnetically force. Using sterile technique, middle ear epithelia of tympanic membrane or ossicles was exposed and a suspension of particles with fluoroscein isothiocyanate (FITC) label applied to the surface. A rare earth, NdFeBo magnet (0.35 T) placed under the animal, was used to pull the MNP into the tissue. After 8 days, following euthanasia, tissues were harvested and confocal scanning laser interferometry was used to verify intracellular MNP. Displacements of the osscicular chain in response to an external sinusoidal electromagnetic field were also measured using laser Doppler interferometry. We showed for the first time a physiologically relevant, biomechanical function, produced by MNP responding to a magnetic field.

Essential role of B-helix calcium binding sites in annexin V-membrane binding.

Crystal structures of annexin V have shown up to 10 bound calcium ions in three different types of binding sites, but previous work concluded that only one of these sites accounted for nearly all of the membrane binding affinity of the molecule. In this study we mutated residues contributing to potential calcium binding sites in the AB and B helices in each of the four domains (eight sites in total) and in DE helices in the first, second, and third domains (three sites in total). We measured the affinity of each protein for phospholipid vesicles and cell membranes by quantitative calcium titration under low occupancy conditions (< 1% saturation of available membrane binding sites). Affinity was calculated from the midpoint and slope of the calcium titration curve and the concentration of membrane binding sites. The results showed that all four AB sites were essential for high affinity binding, as were three of the four B sites (in domains 1, 2, and 3); the DE site in the first domain made a slight contribution to affinity. Multisite mutants showed that each domain contributed additively and independently to binding affinity; in contrast, AB and B sites within the same domain were interdependent. The number of functionally important sites identified was consistent with the Hill coefficient observed in calcium titrations. This study shows an essential and previously unappreciated role for B-helix calcium binding sites in the membrane binding of annexins and indicates that all four domains of the molecule are required for maximum membrane binding affinity.

Measurement of the affinity and cooperativity of annexin V-membrane binding under conditions of low membrane occupancy.

We developed a method for measuring the binding affinity of annexin V for phospholipid vesicles and cells at very low levels of membrane occupancy. The annexin V-117 mutant was labeled with fluorescein iodoacetamide on its single N-terminal cysteine residue; binding to phospholipid vesicles containing phosphatidylserine (PS) and 2% rhodamine-phosphatidylethanolamine was measured by fluorescence quenching due to resonance energy transfer; binding to cells with exposed PS was measured by fluorometry after elution of bound protein. The equilibrium constant was calculated as a function of the midpoint of the calcium titration curve, the Hill coefficient, and the concentration of membrane binding sites. Calcium titrations at very low ratios of protein to membrane revealed Hill coefficients of approximately 8 for both vesicles and cells, far higher than previously measured, but as the protein-membrane ratio was increased above 3% of maximum membrane occupancy, the value of the Hill coefficient progressively decreased to a limiting value of about 2. High Hill coefficients were also observed for measurements performed at different ionic strengths and with membrane PS content varied over the range from 20 to 50%. This method allows the accurate determination of the affinity and cooperativity of annexin V-membrane binding and will be useful for the evaluation of modified annexin V derivatives intended for diagnostic and therapeutic applications.

Development of annexin V mutants suitable for labeling with Tc(i)-carbonyl complex.

(99m)Tc-annexin V can be used to image organs undergoing cell death during cancer chemotherapy and organ transplant rejection. We investigated whether the novel Tc-carbonyl labeling method would be suitable for annexin V. Two mutant molecules of annexin V, called annexin V-122 and annexin V-123, were constructed with N-terminal extensions containing either three or six histidine residues. These molecules were expressed cytoplasmically in E. coli and purified with a final yield of 33 mg of protein/L of culture. Analysis by SDS-PAGE, isoelectric focusing, gel filtration chromatography, and mass spectrometry confirmed the purity and homogeneity of the protein preparations. Both mutant proteins retained full binding affinity for cell membranes with exposed phosphatidylserine. Using the Tc-carbonyl reagent, both proteins could be labeled with (99m)Tc to specific activities of at least 10-20 microCi/microg with full retention of bioactivity. The radiolabeled proteins were stable when incubated with phosphate-buffered saline or serum in vitro, and there was no transchelation of label to serum proteins during in vitro incubation. In conclusion, annexin V can be modified near its N-terminus to incorporate sequences that form specific chelation sites for (99m)Tc-carbonyl without altering its high affinity for cell membranes.