Nitrogen use efficiency in bread wheat: Genetic variation and prospects for improvement.

Nitrogen (N) is one of the primary macronutrients required for crop growth and yield. This nutrient is especially limiting wheat yields in the dry and low fertile agro-ecologies having low N in the root zone soil strata. Moreover, majority of farmers in India and South Asia are small to marginal with meagre capacity to invest in costly nitrogen fertilizers. Therefore, there is an immense need to identify lines that use nitrogen efficiently. A set of 50 diverse wheat genotypes consisting of indigenous germplasm lines (05), cultivars released for commercial cultivation (23) and selected elite lines from CIMMYT nurseries (22) were evaluated in an alpha-lattice design with two replications, a six-rowed plot of 2.5m length for 24 agro morphological, physiological and NUE related traits during two consecutive crop seasons in an N-depleted precision field under two different N levels of 50%-N50 (T1) and 100%-N100 (T2) of recommended N, i.e., 100 kg/ha. Analysis of variance revealed significant genetic variation among genotypes for all the traits studied. About 11.36% yield reduction was observed at reduced N levels. Significant correlations among NUE traits and yield component traits were observed which indicated pivotal role of N remobilization to the grain in enhancing yield levels. Among N-insensitive genotypes identified based on their yielding ability at low N levels, UASBW13356, UASBW13358, UASBW13354, UASBW13357 and KRL1-4 showed their inherent genotypic plasticity toward N application. The genotypes with more yield and high to moderate NUtE can be used as parents for the breeding of N efficient genotypes for marginal agro-ecologies. Low N tolerant genotypes identified from the current investigation may be further utilized in the identification of genomic regions responsible for NUE and its deployment in wheat breeding programs. The comprehensive data of 24 traits under different nitrogen levels for diverse genotypes from India and global sources (mainly CIMMYT) should be useful for supporting breeding for NUE and thus will be of great help for small and marginal farmers in India and South Asia.

Malaria parasite proteins involved in nutrient channels at the host erythrocyte membrane: advances and questions for future research.

Erythrocytes infected malaria parasites have increased permeability to nutrients and other solutes, as mediated by an unusual ion channel known as the plasmodial surface anion channel (PSAC). Although the increased permeability of infected erythrocytes was identified more than 70 years ago and subsequently characterized with tracer studies, its mechanism and role in parasite biology remained unclear until the introduction of patch-clamp methods and high-throughput screening technologies. These methods discovered and implicated PSAC as the primary mechanism, determined that this channel is essential for parasite development, led to identification of the channel's genes, and stimulated antimalarial drug discovery against this target. Despite these advances, many questions remain about this unusual parasite channel. Our review highlights some recent advances and describes important questions for future research.

FACS-style detection for real-time cell viscoelastic cytometry.

Cell mechanical properties have been established as a label-free biophysical marker of cell viability and health; however, real-time methods with significant throughput for accurately and non-destructively measuring these properties remain widely unavailable. Without appropriate labels for use with fluorescence activated cell sorters (FACS), easily implemented real-time technology for tracking cell-level mechanical properties remains a current need. Employing modulated optical forces and enabled by a low-dimensional FACS-style detection method introduced here, we present a viscoelasticity cytometer (VC) capable of real-time and continuous measurements. We demonstrate the utility of this approach by tracking the high-frequency cell physical properties of populations of chemically-modified cells at rates of ~ 1 s-1 and explain observations within the context of a simple theoretical model.

A simple microfluidic dispenser for single-microparticle and cell samples.

Non-destructive isolation of single-cells has become an important need for many biology research laboratories; however, there is a lack of easily employed and inexpensive tools. Here, we present a single-particle sample delivery approach fabricated from simple, economical components that may address this need. In this, we employ unique flow and timing strategies to bridge the significant force and length scale differences inherent in transitioning from single particle isolation to delivery. Demonstrating this approach, we use an optical trap to isolate individual microparticles and red blood cells that are dispensed within separate 50 µl droplets off a microfluidic chip for collection into microscope slides or microtiter plates.

Extraction of p-coumaric acid and ferulic acid using surfactant-based aqueous two-phase system.

Ferulic acid (FA) and p-coumaric acid (pCA) are high-value products that can be obtained by alkaline hydrolysis of lignocellulose. Present work explores the potential of surfactant-based cloud-point extraction (CPE) for FA and pCA extraction from corn cob hydrolysate. More than 90 % (w/w) extraction of both FA and pCA was achieved from model system with L92. The partition coefficient of FA and pCA in L92 aqueous phase system was 35 and 55, respectively. A significant enrichment (8-10-fold) of both FA and pCA was achieved in surfactant-rich phase. Furthermore, the downstream process volume was reduced by 10 to 13 times. Optimized conditions (5 % v/v L92 and pH 3.0) resulted into 85 and 89 % extraction of FA and p-CA, respectively, from alkaline corn cob hydrolysate. Biocompatibility tests were carried out for L92 for ethanol fermentation and found to be biocompatible. Thus, the new surfactant-based CPE system not only concentrated FA and pCA but also reduced the process volume significantly. Further, aqueous phase containing sugars can be used for ethanol fermentation.

Single-cell isolation using a DVD optical pickup.

A low-cost single-cell isolation system incorporating a digital versatile disc burner (DVD RW) optical pickup has been developed. We show that these readily available modules have the required laser power and focusing optics to provide a steady Gaussian beam capable of optically trapping micron-sized colloids and red blood cells. Utility of the pickup is demonstrated through the non-destructive isolation of such particles in a laminar-flow based microfluidic device that captures and translates single microscale objects across streamlines into designated channel exits. In this, the integrated objective lens focusing coils are used to steer the optical trap across the channel, resulting in the isolation of colloids and red blood cells using a very inexpensive off-the-shelf optical component.

Lung epithelial permeability and inhaled furosemide: added dimensions in asthmatics.

OBJECTIVE: Lung clearance rates of inhaled (99m)Tc-DTPA aerosols constitute a sensitive index to evaluate the permeability changes characteristic of airway epithelial damage. It was thought that edema of the airway wall which is reported in asthma could be relieved with a diuretic like furosemide, helping to relieve the symptoms. We intended to study the effect of inhaled furosemide on lung epithelial permeability in asthmatics and smokers with the help of (99m)Tc-DTPA lung clearance test (LCT). METHODS: The study included three groups (n = 15), viz. normal healthy controls, asymptomatic chronic smokers, and chronic persistent asthmatics. Each subject underwent the LCT twice, baseline and post-furosemide (Lasix) study, within a week's interval. The post-furosemide study was carried out 15 min after inhalation of 10 mg of lasix. Lung epithelial permeability was determined in terms of clearance half-life (T (1/2)). RESULTS: The baseline mean T (1/2) values for controls, smokers, and asthmatics were 50.95 +/- 16.58, 20.81 +/- 5.47, 24.06 +/- 6.19 min, respectively. Post-lasix T (1/2) values were 50.83 +/- 15.84, 20.70 +/- 5.65, 41.27 +/- 15.07 min, respectively. There was a significant difference (P < 0.001) in baseline and post-lasix clearance values in asthmatics only. CONCLUSION: Baseline lung epithelial permeability was altered in smokers and asthmatics compared to the controls. Furosemide was effective only in asthmatics in reverting the permeability almost back to the normal range. Inhaled furosemide was effective even in moderate and severe asthmatics. Furosemide has multiple mechanisms of action. It possibly acts at bronchial level in view of the pathology in asthmatics lying in the airways.

Efficacy of 99mTc-DTPA lung clearance test in the diagnosis of PCP in HIV-positive patients.

The study aims to evaluate the efficacy of technetium-99m diethylenetriaminepentaacetic acid ((99m)Tc-DTPA) lung clearance test in the diagnosis of pneumocystis carinii pneumonia (PCP) in HIV-positive paediatric patients. Twenty HIV-negative patients with no chest symptoms constituted Group A, 25 HIV antibody positive asymptomatic children formed Group B, while 45 HIV antibody positive children with respiratory infections comprised Group C. Group C was subdivided into C(1) (n = 20, documented PCP on microbiology), C(2) (n = 10, tuberculosis) and C(3) (n = 15, bacterial pneumonias). The mean age group of patients in Group A, Group B and Group C was 4.7 +/- 1.9, 4.2 +/- 1.5 and 4.8 +/- 1.7 years, respectively. All patients were subjected to complete blood count, blood culture, chest radiographs, microscopic staining of sputum (PCP stains, Ziehl-Nielsen staining, Gram staining), ABG and Mantoux test. All these patients underwent dynamic lung scans using (99m)Tc-DTPA aerosols and lung clearance was calculated in terms of half-time transfer value (T(1/2)) value. T(1/2) was compared between different groups and lung scan findings were correlated with radiological and microbiological results. Patients with PCP had T(1/2) in the range of 9.02 +/- 1.35, TB 28.2 +/- 3.03 min and other bacterial pneumonias in the range of 20.5 +/- 3.1 min (range for normal individuals was 49.8 +/- 6.13 min). T(1/2) in patients with PCP was found to be significantly lower when compared with T(1/2) in other groups. Patients with PCP had characteristic biphasic curves while the rest had monophasic curves. Some patients with PCP had low T(1/2) values even when chest radiographs and arterial blood gases were normal. (99m)Tc-DTPA lung clearance test is a sensitive, safe and non-invasive diagnostic tool for the early detection of PCP in HIV-positive paediatric patients.

A novel conditional Akt 'survival switch' reversibly protects cells from apoptosis.

The anti-apoptotic Akt kinase is commonly activated by survival factors following plasma membrane relocalization attributable to the interaction of its pleckstrin homology (PH) domain with phosphatidylinositol 3-kinase (PI3K)-generated PI3,4-P(2) and PI3,4,5-P(3). Once activated, Akt can prevent or delay apoptosis by phosphorylation-dependent inhibition or activation of multiple signaling molecules involved in apoptosis, such as BAD, caspase-9, GSK3, and NF-kappaB and forkhead family transcription factors. Here, we describe and characterize a novel, conditional Akt controlled by chemically induced dimerization (CID). In this approach, the Akt PH domain has been replaced with the rapamycin (and FK506)-binding domain, FKBP12, to make F3-DeltaPH.Akt. To effect membrane recruitment, a myristoylated rapamycin-binding domain from FRAP/mTOR, called M-FRB, binds to lipid permeable rapamycin (and non-bioactive synthetic 'rapalogs'), leading to reversible heterodimerization of M-FRB with FKBP-DeltaPH.Akt. Like endogenous c-Akt, we show that the kinase activity of membrane-localized F3-DeltaPH.Akt correlates strongly with phosphorylation at T308 and S473; however, unlike c-Akt, phosphorylation and activation of inducible Akt (iAkt) is largely PI3K independent. CID-mediated activation of iAkt results in phosphorylation of GSK3, and contributes to NF-kappaB activation in vivo in a dose-sensitive manner. Finally, in Jurkat T cells stably expressing iAkt, CID-induced Akt activation rescued cells from apoptosis triggered by multiple apoptotic stimuli, including staurosporine, anti-Fas antibodies, PI3K inhibitors and the DNA damaging agent, etoposide. This novel inducible Akt should be useful for identifying new Akt substrates and for reversibly protecting tissue from apoptosis due to ischemic injury or immunological attack.

Neonatal electroencephalogram does not predict cognitive and academic achievement scores at early school age in survivors of neonatal extracorporeal membrane oxygenation.

Extracorporeal membrane oxygenation is an effective rescue treatment for severe cardiorespiratory failure in term or near-term neonates, although a wide range of neurologic sequelae have been noted in a substantial minority of survivors. The objective of the present study was to determine the value of the neonatal electroencephalogram (EEG) for predicting Wechler Preschool and Primary Scale of Intelligence-Revised (WPPSI-R), Wide Range Achievement Test, and Wide Range Assessment of Memory and Language scores at early school age in 66 testable survivors of extracorporeal membrane oxygenation who were not severely brain damaged. Technically satisfactory EEG recordings were obtained at least twice following admission to our nursery and prior to discharge. The EEGs were classified and graded according to standard criteria. The developmental test results of those who had only normal or mildly abnormal neonatal EEGs (group 1, n = 9) were compared with those who had at least one moderately or markedly abnormal recording (group 2, n = 57). School-age test and subtest scores were not statistically significantly worse in group 2 versus group 1 infants. No child in group 1 and five children in group 2 had WPPSI-R Full-Scale IQ scores of less than 70. Of the nine children in group 2 who had at least one markedly abnormal neonatal EEG recording (graded as burst suppression or as electrographic seizure), only two had abnormally low WPPSI-R Full-Scale IQ scores. We conclude that EEG recordings obtained during the neonatal course of neonates treated with extracorporeal membrane oxygenation do not predict cognitive and academic achievement test results in survivors at early school age who were testable and not severely brain damaged.

Coccidioidomycosis in non-endemic areas: a case series.

Coccidioidomycosis is a systemic infection caused by the soil fungus Coccidioides immitis, which is endemic to the south-western United States. Manifestations range from flu-like illness to pneumonia and septic shock. Diagnosis may be delayed or missed in non-endemic areas because of the low index of suspicion. We describe a series of 23 patients with coccidioidomycosis at one institution in a non-endemic area. Diagnosis was often delayed. In two patients, the route of exposure could not be determined, but 20 patients had a history of residence or travel to endemic areas, and the remaining patient had an occupational history of exposure to fomites from an endemic region. Five patients were immunosuppressed. Most patients responded well to medical therapy, surgery, or both. Although coccidioidomycosis is rare in non-endemic areas, physicians must keep it in mind when evaluating patients who have traveled to endemic areas or who are immunosuppressed.

Palivizumab for respiratory syncytial virus prophylaxis in high-risk infants: a cost-effectiveness analysis.

BACKGROUND: Prophylactic therapy with palivizumab, a humanized monoclonal antibody, has been shown to reduce the number of respiratory syncytial virus (RSV)-related hospitalizations in preterm infants. The cost-effectiveness of this therapy has not been evaluated from the provider's perspective using cost data. OBJECTIVES: The objectives of this study were to determine the cost per RSV infection episode avoided by using prophylactic palivizumab therapy in a high-risk infant population and to determine whether certain subgroups of infants derived greater benefit from prophylactic therapy. METHODS: A decision-analytic model simulating an RSV infection episode was developed to evaluate the cost-effectiveness of palivizumab prophylaxis from the perspective of the health care system (provider). Data to populate the model were gathered from the medical literature (identified through a MEDLINE search of studies on the incidence of RSV infection) and the IMpact-RSV clinical trial. Data included incidence of RSV infection and the associated health care resource use and costs. Costs to the provider were determined using a university-affiliated hospital cost-accounting system. Cost-effectiveness ratios were calculated over a range of RSV infection incidence rates in a control population. Sensitivity analyses were performed for the cost of palivizumab therapy, the cost of RSV-related hospitalization, and the number of emergency department, physician office, and home health care visits. For the subgroup analysis, infants were classified by gestational age (<32 and > or = 32 weeks) and stratified by severity of chronic lung disease. RESULTS: The cost per additional RSV infection episode avoided ranged from dollars 0 (cost savings) to dollars 39,591 for palivizumab prophylaxis costs of dollars 2500 and from dollars 2702 to dollars 79,706 for palivizumab prophylaxis costs of dollars 4500. The model was insensitive to changes in the number of emergency department, physician office, and home health care visits. The difference in RSV incidence between the treatment and control groups was greater among infants > or = 32 weeks' gestational age than among infants <32 weeks' gestational age. onclusions: The incremental cost-effectiveness of palivizumab compared with no prophylactic therapy was sensitive to changes in the incidence of RSV infection in control infants, the average cost of RSV hospitalization, and the cost of palivizumab. Clinicians may use this information along with additional factors to determine whether palivizumab is cost-effective in their clinical setting and geographic area.

Structural relatedness of distinct determinants recognized by monoclonal antibody TP25.99 on beta 2-microglobulin-associated and beta 2-microglobulin-free HLA class I heavy chains.

The association of HLA class I heavy chains with beta2-microglobulin (beta2m) changes their antigenic profile. As a result, Abs react with either beta2m-free or beta2m-associated HLA class I heavy chains. An exception to this rule is the mAb TP25.99, which reacts with both beta2m-associated and beta2m-free HLA class I heavy chains. The reactivity with beta2m-associated HLA class I heavy chains is mediated by a conformational determinant expressed on all HLA-A, -B, and -C Ags. This determinant has been mapped to amino acid residues 194-198 in the alpha3 domain. The reactivity with beta2m-free HLA class I heavy chains is mediated by a linear determinant expressed on all HLA-B Ags except the HLA-B73 allospecificity and on <50% of HLA-A allospecificities. The latter determinant has been mapped to amino acid residues 239-242, 245, and 246 in the alpha3 domain. The conformational and the linear determinants share several structural features, but have no homology in their amino acid sequence. mAb TP25.99 represents the first example of a mAb recognizing two distinct and spatially distant determinants on a protein. The structural homology of a linear and a conformational determinant on an antigenic entity provides a molecular mechanism for the sharing of specificity by B and TCRs.

A voltage-dependent channel involved in nutrient uptake by red blood cells infected with the malaria parasite.

Growth of the malaria parasite in human red blood cells (RBCs) is accompanied by an increased uptake of many solutes including anions, sugars, purines, amino acids and organic cations. Although the pharmacological properties and selectivity of this uptake suggest that a chloride channel is involved, the precise mechanism has not been identified. Moreover, the location of this uptake in the infected RBC is unknown because tracer studies are complicated by possible uptake through fluid-phase pinocytosis or membranous ducts. Here we have studied the permeability of infected RBCs using the whole-cell voltage-clamp method. With this method, uninfected RBCs had ohmic whole-cell conductances of less than 100 pS, consistent with their low tracer permeabilities. In contrast, trophozoite-infected RBCs exhibited voltage-dependent, non-saturating currents that were 150-fold larger, predominantly carried by anions and abruptly abolished by channel blockers. Patch-clamp measurements and spectral analysis confirmed that a small (< 10 pS) ion channel on the infected RBC surface, present at about 1,000 copies per cell, is responsible for these currents. Because its pharmacological properties and substrate selectivities match those seen with tracer studies, this channel accounts for the increased uptake of small solutes in infected RBCs. The surface location of this new channel and its permeability to organic solutes needed for parasite growth indicate that it may have a primary role in a sequential diffusive pathway for parasite nutrient acquisition.

Analysis by NMR spectroscopy of the structural homology between the linear and the cyclic peptide recognized by anti-human leukocyte antigen class I monoclonal antibody TP25.99*.

The anti-human leukocyte antigen (HLA) class I monoclonal antibody (mAb) TP25.99 has a unique specificity since it recognizes both a conformational and a linear determinant expressed on the beta(2)-mu-associated and beta(2)-mu-free HLA class I heavy chains, respectively. Previously, we reported the identification of a cyclic and a linear peptide that inhibits mAb TP25.99 binding to the beta(2)-mu-associated and beta(2)-mu-free HLA class I heavy chains (S. A. Desai, X. Wang, E. J. Noronha, Q. Zhou, V. Rebmann, H. Grosse-Wilde, F. J. Moy, R. Powers, and S. Ferrone, submitted for publication). The linear X(19) and cyclic LX-8 peptides contain sequence homologous to residues 239-242, 245, and 246 and to residues 194-198, respectively, of HLA class I heavy chain alpha(3) domain. Analysis by two-dimensional transfer nuclear Overhauser effect spectroscopy of the induced solution structures of the linear X(19) and cyclic LX-8 peptides in the presence of mAb TP25.99 showed that the two peptides adopt a similar structural motif despite the lack of sequence homology. The backbone fold is suggestive of a short helical segment followed by a tight turn, reminiscent of the determinant loop region (residues 194-198) on beta(2)-mu-associated HLA class I heavy chains. The structural similarity between the linear X(19) and cyclic LX-8 peptides and the lack of sequence homology suggests that mAb TP25.99 predominantly recognizes a structural motif instead of a consensus sequence.

Global T-wave inversion: limited QT dispersion despite QTc prolongation--a correlate of benignity in patients with strikingly abnormal electrocardiograms.

BACKGROUND: The global T-inversion (GTI) electrocardiogram (ECG) is strikingly abnormal with major QTc prolongation, but with a surprisingly good prognosis by Kaplan-Meier curve. This contrasts with most significant QTc prolongations. HYPOTHESIS: This study was undertaken to ascertain QT interval dispersion (QTd) in global T wave inversion, a clinically benign long QTc ECG. METHODS: Longest and shortest QT intervals in all 12 leads in 35 consecutive patients with GTI were determined by two mutually blinded observers. QTd was determined by subtraction (maximum-minimum) and QTc was calculated using the Bazett formula. RESULTS: There was a 2:1 female preponderance QTc was prolonged and equal for men (0.471) and women (0.469). Observer variability of under 2% permitted averaging of QT measurements. Composite mean QTd was 55 ms. The literature revealed a range of QTd in normal subjects of 39 to 59 ms (mostly 49 to 59 ms). Patient series with abnormal QTd were well above this level. CONCLUSION: Despite a strikingly abnormal ECG with marked QTc prolongation, QT dispersion was limited in global T inversion, consistent with its previously demonstrated benignity.

Recurrent enterococcal endophthalmitis following cataract surgery: a case report.

Recurrent post-cataract endophthalmitis is a well-recognized postoperative complication that has been attributed to various organisms and different mechanisms. To our knowledge, there is no case of recurrent postoperative endophthalmitis reported where the organism was found to be sequestered in the posterior capsule, escaping total eradication and thus producing the recurrence. The following is a case report of such recurrent postoperative endophthalmitis caused by Enterococcus faecalis sequestered in the posterior capsule.

Prospective evaluation of medication-induced esophageal injury and its relation to esophageal function.

BACKGROUND: Few prospective studies are available on the incidence of medication-induced esophageal injury (MIEI). AIMS: To prospectively study the occurrence of MIEI with indomethacin and doxycycline and the predictive factors for its development. METHODS: In an operator-blinded study, 51 patients (age 16-65 y) requiring indomethacin (n = 24) or doxycycline (27) underwent symptom evaluation, endoscopy and scintigraphy before and after 7 days of therapy. MIEI was defined as de novo occurrence or worsening of pre-existing esophagitis or development of esophageal ulcer. RESULTS: Pre-therapy endoscopy was normal in 32 patients and revealed esophagitis in 19 (grade I--11, grade II--8). Post-therapy, 16 patients developed esophageal symptoms, which appeared earlier with doxycycline (2.0 [0.8] vs 4.1 [1.7] days, p = 0.016). MIEI developed in 23 patients--de novo esophagitis in 16, worsening of esophagitis in 6; 5 patients developed ulcer. Seven of 12 patients with hiatus hernia developed MIEI. Presence of pre-therapy gastroesophageal reflux disease did not predict MIEI. There was no difference in pre- or post-therapy transit values between patients with and without MIEI; patients who developed ulcers had significantly slower esophageal transit (p < 0.05). There was no difference in esophageal transit or occurrence of MIEI between patients who received indomethacin or doxycycline; however, 5 of 8 patients with hiatus hernia who received doxycycline developed MIEI (p = 0.02; relative risk 3.96 [CI 1.2-12.7]). CONCLUSIONS: 40% of patients receiving doxycycline or indomethacin developed MIEI; 10% developed ulcers. Hiatus hernia increased the risk for MIEI.