Variant Bands Research Articles

Identification and Activity Study of an Impurity Band Observed in the nrSDS-PAGE of Aflibercept.

Aflibercept is a biopharmaceutical targeting vascular endothelial growth factor (VEGF) that has shown promise in the treatment of neovascular age-related macular degeneration (nAMD) and diabetic macular edema (DME) in adults. Quality control studies of aflibercept employing non-reduced SDS-PAGE (nrSDS-PAGE) have shown that a significant variant band (IM1) is consistently present below the main band. Considering the quality control strategy of biopharmaceuticals, structural elucidation and functional studies are required. In this study, the variant bands in nrSDS-PAGE were collected through electroelution and identified by peptide mass fingerprinting based on liquid chromatography-tandem MS (LC-MS/MS). This variant was expressed using knob-into-hole (KIH) design transient transfection for the detection of ligand affinity, binding activity and biological activity. The variant band was formed by C-terminal truncation at position N99 of one chain in the aflibercept homodimer. Then, this variant was successfully expressed using KIH design transient transfection. The ligand affinity of the IM1 truncated variant was reduced by 18-fold, and neither binding activity nor biological activity were detected. The efficacy of aflibercept is influenced by the loss of biological activity of the variant. Therefore, this study supports the development of a quality control strategy for aflibercept.

Ozone stress-induced DNA methylation variations and their transgenerational inheritance in foxtail millet.

Elevated near-surface ozone (O3) concentrations have surpassed the tolerance limits of plants, significantly impacting crop growth and yield. To mitigate ozone pollution, plants must evolve a rapid and effective defense mechanism to alleviate ozone-induced damage. DNA methylation, as one of the most crucial epigenetic modifications, plays a pivotal role in maintaining gene stability, regulating gene expression, and enhancing plant resilience to environmental stressors. However, the epigenetic response of plants to O3 stress, particularly DNA methylation variations and their intergenerational transmission, remains poorly understood. This study aims to explore the epigenetic mechanisms underlying plant responses to ozone stress across generations and to identify potential epigenetic modification sites or genes crucial in response to ozone stress. Using Open Top Chambers (OTCs), we simulated ozone conditions and subjected foxtail millet to continuous ozone stress at 200 nmol mol-1 for two consecutive generations (S0 and S1). Results revealed that under high-concentration ozone stress, foxtail millet leaves exhibited symptoms ranging from yellowing and curling to desiccation, but the damage in the S1 generation was not more severe than that in the S0 generation. Methylation Sensitive Amplified Polymorphism (MSAP) analysis of the two generations indicated that ozone stress-induced methylation variations ranging from 10.82% to 13.59%, with demethylation events ranged from 0.52% to 5.58%, while hypermethylation occurred between 0.35% and 2.76%. Reproductive growth stages were more sensitive to ozone than vegetative stages. Notably, the S1 generation exhibited widespread demethylation variations, primarily at CNG sites, compared to S0 under similar stress conditions. The inheritance pattern between S0 and S1 generations was mainly of the A-A-B-A type. By recovering and sequencing methylation variant bands, we identified six stress-related differential amplification sequences, implicating these variants in various biological processes. These findings underscore the potential significance of DNA methylation variations as a critical mechanism in plants' response to ozone stress, providing theoretical insights and references for a comprehensive understanding of plant adaptation mechanisms to ozone stress and the epigenetic role of DNA methylation in abiotic stress regulation.

Prospective Determination of Orbital Perfusion Dominance before Intra-Arterial Chemotherapy for Retinoblastoma Using Time-of-Flight Magnetic Resonance Angiography

AbstractIntra-arterial chemotherapy (IAC) represents a mainstay in the treatment of retinoblastoma. In a minority of cases, the external carotid artery (ECA) serves as the dominant supply to the central retinal artery and is associated with prolonged fluoroscopy times and higher intraprocedural radiation doses. The aim of this study was to evaluate the utility of time-of-flight (TOF) magnetic resonance angiography (MRA) for prospective determination of internal (ICA) versus ECA dominance for procedural planning. Between April 2017 and December 2020 (44 months), staging MR prior to IAC for retinoblastoma included variant spatial saturation band position TOF angiography. Exams were then retrospectively reviewed for concordance of ICA versus ECA dominance between the two modalities. Eight consecutive patients were included in the study. Mean patient age at time of diagnosis was 20.3 ± 10.7 months (range: 2.7–33.2 months). Ten affected eyes were included (2 cases of bilateral disease), with stage D disease in eight eyes and stage B disease in two eyes. MRA techniques demonstrated antegrade ophthalmic artery (OA) flow in 9/10 (90%) of affected eyes. Subsequent catheter angiography confirmed ICA dominant supply in 9/9 (100%). For a single affected eye (10%), the OA was demonstrated as orthotopic by T2 flow void, nonvisualized on anterior saturation TOF sequences, and faintly visualized on posterior saturation TOF sequences. Aggregate MRA to catheter angiographic concordance was 10/10 (100%). Variant saturation TOF MRA predicts ICA versus ECA dominant supply to the central retinal artery in retinoblastoma.

Understanding the Temperature Dependence of the Seebeck Coefficient from First-Principles Band Structure Calculations for Organic Thermoelectric Materials

Understanding the Temperature Dependence of the Seebeck Coefficient from First-Principles Band Structure Calculations for Organic Thermoelectric Materials

New sequence variants in patients affected by amyloidosis show transthyretin instability by isoelectric focusing

The plasma protein transthyretin (TTR) can aggregate into insoluble amyloid fibrils causing systemic amyloidosis (ATTR amyloidosis) in patients carrying a variant TTR protein. If new variants arise, it is crucial to clarify whether they are disease-associated or benign. In this study, we further functionally characterize three new and unclassified TTR variants (Thr40Asn, Phe64Val and the described but not functionally assessed variant Leu12Val), using a simplified, fast isoelectric focusing (IEF) approach. After validating the system with known TTR variants, we assessed the sera of five patients carrying these new TTR variants in a heterozygous state. All three variants showed aberrant banding patterns that were similar to those of other well-characterized TTR variants, including the common Val30Met variant that causes ATTR amyloidosis. In addition to a clear band corresponding to monomeric wild-type TTR, we observed an additional variant band at the cathodal side of the IEF gel. These results indicate conformational instability of the new Thr40Asn, Phe64Val and Leu12Val variants. Together with the clinical and immunohistological data of these patients and affected family members, as well as the absence of these variants in human genetic mutation databases, our results strongly hint that these variants are amyloidogenic and therefore probably disease-associated. These findings have implications for patient therapy and for genetic counselling of family members.

Analysis of DNA methylation alterations in rice seeds induced by different doses of carbon-ion radiation.

To investigate the mechanism underlying differences in biological effects induced by low- versus high-dose heavy-ion radiation (HIR) in rice plants, two-dimensional gel electrophoresis (2-DE) coupled with methylation-sensitive amplification polymorphism (MSAP) analysis were used to check the expression changes in rice leaf proteome profiles and the changes in DNA methylation after exposure of seeds to ground-based carbon-ion radiation at various cumulative doses (0, 0.01, 0.02, 0.1, 0.2, 1, 2, 5 or 20 Gy; 12C6+; energy, 165 MeV/u; mean linear energy transfer, 30 KeV/μm). In this study, principal component analysis (PCA) and gene ontology (GO) functional analysis of differentially expressed proteins of rice at tillering stage showed that proteins expressed in rice samples exposed to 0.01, 0.02, 0.1, 0.2 or 1 Gy differed from those exposed to 2, 5 or 20 Gy. Correspondingly, the proportion of hypermethylation was higher than that of hypomethylation at CG sites following low-dose HIR (LDR; 0.01, 0.2 or 1 Gy), whereas this was reversed at high-dose HIR (HDR; 2, 5 or 20 Gy). The hypomethylation changes tended to occur at CHG sites with both low- and high-dose HIR. Furthermore, sequencing of MSAP variant bands indicated that the plants might activate more metabolic processes and biosynthetic pathways on exposure to LDR, but activate stress resistance on exposure to HDR. This study showed that radiation induced different biological effects with low- and high-dose HIR, and that this may have been caused by different patterns of hyper- and hypomethylation at the CG sites.

Shear-Induced Changes of Electronic Properties in Gallium Nitride.

We show that sliding on the surface of GaN can permanently change the surface band structure, resulting in an increased degree of band bending by more than 0.5 eV. We hypothesize that shear and contact stresses introduce vacancies that cause a spatially variant band bending. Band bending is observed by shifts and broadening of core-level binding energies toward lower values in X-ray photoelectron spectroscopy. The extent of band bending is controlled by humidity, number of sliding cycles and applied load, presenting opportunities for scalable tuning of the degree of band bending on a GaN surface. Scanning transmission electron microscopy revealed that the epitaxy of GaN was preserved up to the surface with regions of defects near the surface. The hypothesized mechanism of band bending is shear-induced defect generation, which has been shown to affect the surface states. The ability to introduce band bending at the GaN surface is promising for applications in photovoltaics, photocatalysis, gas sensing, and photoelectrochemical processes.

DETERMINATION AND DISTRIBUTION OF CRY1-TYPE GENES IN Bacillus thuringiensis ISOLATED FROM NORTH INDIA

Bacillus thuringiensis (Bt) is a spore-forming bacterium which produces insecticidal crystal protein in the sporulation phase. Polymerase chain reaction (PCR)-based identification of Bacillus thuringiensis toxin genes has become a routine step in most B. thuringiensis isolation and characterization initiatives. In the present study, eighteen native Bt isolates from diverse habitats of North India were taken for the presence of cry1 type genes. The distribution of cry1 gene families in native Bt isolates was examined by PCR amplification of genes with three sets of corresponding PCR primers. In native Bt isolates many variant bands were also observed in addition to expected bands on PCR amplification. Different sets of primers for the same gene give different results due to different sites of primer binding. Maximum number of isolates showed expected bands when primers designed by Ceron were used compared to other primers. However, with other primers, the numbers of variant bands was larger. The isolates, SK-13, 63 and 105 showed the maximum number of cry1-type genes, followed by SK-20, 28, 48, 88, 94, 301, 304 and 307, whereas SK-3 showed the presence of only 3 cry1-type genes. RFLP analysis of 1.6 kb fragment indicated the presence of variant bands from the reference strains. Several promising isolates with predicted toxicity towards lepidoptera have been observed in this study.

In-situ observation of deformation induced α″ phase transformation in a β-titanium alloy

Transmission electron microscopy observations during in situ tensile tests revealed α″ phase transformation in a β phase Ti-24Nb-4Zr-7.9Sn (wt%) alloy. Single variant α″ phase is formed in deformation bands under tensile stress and the deformation bands vanish after the applied stress is released. With increasing strain, two variants of the α″ phase are observed in deformation bands along different directions, respectively. When these deformation bands intersected with each other, these two variant α″ phases could remain after unloading of the external stress. The α″ phase transformation in single variant deformation bands likely contributes to the nonlinear pseudo-elasticity of this alloy.

Spatially variant periodic structures in electromagnetics.

Spatial transforms are a popular technique for designing periodic structures that are macroscopically inhomogeneous. The structures are often required to be anisotropic, provide a magnetic response, and to have extreme values for the constitutive parameters in Maxwell's equations. Metamaterials and photonic crystals are capable of providing these, although sometimes only approximately. The problem still remains about how to generate the geometry of the final lattice when it is functionally graded, or spatially varied. This paper describes a simple numerical technique to spatially vary any periodic structure while minimizing deformations to the unit cells that would weaken or destroy the electromagnetic properties. New developments in this algorithm are disclosed that increase efficiency, improve the quality of the lattices and provide the ability to design aplanatic metasurfaces. The ability to spatially vary a lattice in this manner enables new design paradigms that are not possible using spatial transforms, three of which are discussed here. First, spatially variant self-collimating photonic crystals are shown to flow unguided waves around very tight bends using ordinary materials with low refractive index. Second, multi-mode waveguides in spatially variant band gap materials are shown to guide waves around bends without mixing power between the modes. Third, spatially variant anisotropic materials are shown to sculpt the near-field around electric components. This can be used to improve electromagnetic compatibility between components in close proximity.

Contact‐Induced Mechanisms in Organic Photovoltaics: A Steady‐State and Transient Study

The role of the contacts in thin‐film, blended heterojunctions (<100 nm thick) organic photovoltaics is explored, specifically considering concepts of carrier selectivity, injection, and extraction efficiency, relative to recombination. Contact effects are investigated by comparing two hole‐collecting interlayers: a phosphonic acid monolayer on indium tin oxide (ITO) and a nickel oxide thin film. The interlayers have equivalent work functions (≈5.4 eV) but widely variant energy band offsets relative to the lowest unoccupied molecular orbital of the acceptor (electron blocking versus not), which are coupled to large differences in carrier density. Trends in open‐circuit voltages (VOC) as a function of light intensity and temperature are compared and it is concluded that the dominant mechanism limiting VOC for high density of states contacts is free carrier injection, not surface recombination or extraction barriers. Transient photocurrent decay measurements confirm excess reinjected carriers decrease the extraction efficiency via increased recombination and decrease free carrier lifetime, even at high internal electric fields, due to space charge accumulation. These results demonstrate that the energetics and injection dynamics of the interface between interlayers and high carrier density electrodes (typically ITO and metals) must be considered with fabrication and processing of interlayers, in addition to possible carrier selectivity and the interface with the active layer.

Assessment of genetic variation and changes in protein subunits induced by petroleum oil in three species of Red Sea fishes using SDS-PAGE and ISSRs markers

Genetic variability among three species of red sea fishes (Lethrinus borbonicus, Siganus rivulatus and Mulloidichthys flavolineatus) were determined to evaluate the effect of petroleum oil component pollutions on the fishes using Inter-simple sequence repeats (ISSRs) and sodium dodecyl sulfate-polyacrlamide gel electrophoresis (SDS-PAGE) markers. According to ISSR analysis of DNA, nineteen (19) ISSR primers generated a total of 465 bands with an average 24.5 bands per primer. Analyses of SDS-PAGE protein, 137 bands were identified variant bands from 44, 43 and 50 in sample Lethrinus borbonicus, Siganus rivulatus and Mulloidichthys flavolineatus respectively. Analysis of SDS-PAGE protein provided more precise information concerning of the effect of petroleum oil components on protein subunits and genetic variation in Siganus rivulatus species than ISSR-PCR of DNA. Whereas ISSR technique showed more polymorphism or relatively a close to the percentage of SDS-PAGE result among the tow species Lethrinus borbonicus and Mulloidichthys flavolineatus compared to there controls. A remarkable result from this study was identifying that petroleum oil components pollution have distinct effect in genetic structure of fishes and lead to disappear of some protein subunits or appear new some protein subunits in fish muscle tissues.

Hemoglobin Variants That Produce Abnormal Elevations In Hb A1c In The Clinical Laboratory: Electrophoretic Patterns, HPLC Analysis and Globin Gene Sequencing

Introduction There are two commonly-used methods for measuring Hemoglobin A1c (Hb A1c) – chromatographic and immunochemical. Immunochemical analysis depends on a specific antibody directed towards an epitope that consists of the linked glucose as well as the N-terminal betachain. High-performance liquid chromatography (HPLC)-determined Hb A1c concentrations that are unusually high (above 18%) may be a consequence of a hemoglobinopathy. Standard alkaline and acid gel electrophoresis and even an extended HPLC chromatogram may not fully resolve the abnormal hemoglobin. Globin gene analysis is an invaluable aid in such a situation. Over the course of a 12-month period, 4 individuals were identified in whom the HbA1c fraction exceeded 20%. All cases were encountered as part of our routine clinical chemistry hemoglobin A1c service. All cases were shown to be a result of heterozygosity for an uncommon or rare hemoglobin variant. This resulted in an early-eluting variant that was close to, or coincident with, the Hb A1c peak. Only one of these 4 subjects had anemia and microcytosis, apparently related to iron deficiency and chronic kidney disease. Methods & Materials Hb A1c quantitation was carried out using EDTA-anticoagulated blood on a Bio-Rad Variant II HPLC analyzer (Bio-Rad Laboratories, Hercules CA). Gel electrophoresis was performed on a SPIFE 3000 system (Helena Laboratories, Beaumont TX) using spun, washed red cell pellets. Extended-run HPLC analysis utilized a BioRad D-10™ Hemoglobin Analyzer in the A2/F mode. Globin gene analysis was carried out at Boston University Medical Center using the residual washed whole blood pellet. The globin genes were amplified by PCR, the amplicons were purified, and nucleotide sequencing was performed. Case Descriptions Case 1. Female 58 y. Hb A1c23.8%. o Hb 13.6 g/dL, Hct 41.2%, MCV 94 fL, RBC 4.39 x 106 per mm3, RDW 13.4% o Alkaline gel electrophoresis: Very fast variant band, 23% of total Hb. o Acid Gel electrophoresis: Normal. o Globin gene analysis: Heterozygous for Hb I (α2 codon 16 AAG>GAG or Lys16Glu). Case 2. Female 66 y. Hb A1c46%. o Hb 7.3 g/dL, Hct 24.9 %, MCV 70 fL, RBC 3.5 x 106 per mm3, RDW 16.5%. Iron deficient with low serum ferritin of 8.3. The patient also had a history of diabetes and chronic kidney disease. o Alkaline gel electrophoresis: “Fast” hemoglobin, but migrating very close to & slightly anodal of Hb A. o Acid Gel electrophoresis: Variant hemoglobin migrates with Hb F o D-10 HPLC A2/F Mode: 45% A1c o Globin gene analysis: Heterozygous for Hb Hope, β chain codon 136 GGT>GAT or Gly136Asp Case 3. Female 47 y. Hb A1c 43%. o Hb 12.6 g/dL, Hct 41.7%, MCV 96.2 fL, RBC 4.33x 106 per mm3, RDW 22.9% o Alkaline gel electrophoresis: Normal. o D-10 HPLC A2/F Mode: Variant peak, 46% eluting between Hb A1c and A0 o Globin gene analysis: Heterozygous for Hb Moriguchi, β codon 97 CAC>TAC or His97Tyr. Case 4. Female 83 y. Hb A1c 41% as “labile Hb A1c” or 51% as Hb A1c by extended run in A2/F mode. o Hb 13.8 g/dL, Hct 43.4%, MCV 88.5 fL, RBC 4.9 x 106 per mm3, RDW 14% o Alkaline gel electrophoresis: Normal. o Acid Gel electrophoresis: Variant band, slightly anodal to Hb F o D-10 HPLC A2/F Mode: 51% Hb A1c o Globin gene analysis: Heterozygous for a novel variant hemoglobin, β codon 2 CAC>AAC or Histidine to Asparagine Conclusions Hb A1c analysis by HPLC frequently reveals the presence of a variant hemoglobin in asymptomatic individuals who have the S, C or E trait. In such cases, Hb A1c can be accurately determined despite the presence of the abnormal hemoglobin. We describe 4 individuals with marked elevation of Hb A1c. All these individuals were heterozygous for uncommon globin chain variants due to single point mutations. One subject had a Hb variant that had not been previously recognized. It is unclear whether the immunochemical assays might experience interference from these unusual variants. Disclosures: No relevant conflicts of interest to declare.

Automated Analysis of Hemoglobin Variants Using NanoLC–MS and Customized Databases

Unambiguous analysis of hemoglobin variants is critical in the diagnosis of hemoglobinopathies. In diagnostic laboratories, alkaline gel electrophoresis and automated HPLC are used in identifying variants. In specific instances, comigration of hemoglobin variant bands in gel and coelution of different variants or elution of variants with unmatched library information in HPLC can result in ambiguities in interpretation. Hemoglobin variants mostly arise from point mutations leading to very high sequence homology between normal and variant hemoglobin. In addition, unavailability of a variant database compatible with proteomics data analysis software makes mass spectrometry based variant analysis very challenging. In the present study, we standardized a nanoLC-MS based method for variant analysis to achieve substantially high sequence coverage. We developed three hemoglobin variant databases, specific to three different proteolytic enzymes, compatible with proteomics search engine software. The above nanoLC-MS method and the compatibility of the customized databases were validated by analysis of a sickle hemoglobin variant. Six other hemoglobin variants were characterized wherein diagnosis reports based on conventional tools were ambiguous. The novelty of our method lies in its simplicity and accuracy of the analysis with minimal manual intervention. The presently described method may be used in the future for the routine hemoglobin variant diagnosis.

SSR-Marker Analysis of the Intracultivar Phenotypic Variation Discovered within 3 Soybean Cultivars

Genetic variation within homogeneous gene pools in various crops is assumed to be very limited. One objective of this study was to use 144 simple sequence repeat (SSR) markers to determine if the single-plant lines selected at ultra-low plant density in honeycomb designs within the soybean cultivars Benning, Haskell, and Cook had unique SSR genetic fingerprints. Another objective was to investigate if the variation found was the result of residual genetic heterozygosity that could be detected in the original gene pool where selection initiated. Our results showed that the phenotypic variation for seed protein content and seed weight has a genotypic component identified by the SSR band variation. The 7 lines from Haskell had a total of 63 variant alleles, the 5 lines from Benning had 34 variant alleles, and the 7 lines from Cook had 34 variant alleles, therefore, possessing unique genetic fingerprints. Most of the intracultivar SSR band variation discovered was the result of residual heterozygosity in the initial plant selected to become the cultivar. More specifically, 82% of the SSR variant alleles were traced in the Benning Foundation seed source, 93% in the Haskell seed source, and 82% in the Cook seed source. The remaining variant bands (18% for Benning, 7% for Haskell, and 18% for Cook) could not be detected in the Foundation seed source and were likely the result of mutation or some other mechanism generating de novo variation. These results provide evidence that genetic variation among individual plants is present even in homogeneous gene pools and can be further utilized in breeding programs.

Homozygous bisalbuminemia or homozygous mutant albumin?

IntroductionBisalbuminemia is a genetic condition in which an albumin variant is found in serum in addition to normal albumin. Methods and materialsSerum protein electrophoresis using the Sebia HYDRASYS electrophoresis system was performed on an 84year old male. ResultsSerum protein electrophoresis showed a single albumin band migrating faster than normal albumin. ConclusionThe presence of a homozygous albumin variant band, albumin Naskapi, is noted.

The Quest for Hb Questembert: A Challenging Case of An Unstable Hb Variant

AbstractAbstract 4814Diagnosis of unstable hemoglobins can be challenging, especially those involving the α globin chain. We present here an extremely rare unstable hemoglobin variant in a black female. A 30 y/o female from Haiti presented in the third trimester of pregnancy with anemia. She reported a low Hb in 2007 prior to a myomectomy and dark urine post exercise and in the morning but was otherwise entirely asymptomatic. Her mother and sister had sickle cell trait, but she tested negative; no other familial blood disorder history was elicited. Physical exam was remarkable only for mild pallor; splenomegaly was not appreciated with advanced pregnancy. Laboratory data revealed a Coombs negative hemolytic anemia with Hb values between 9–11g/dl, MCV of 97, reticulocyte count of 7.3%, LDH between 600–800, bilirubin of 1.2 –mostly indirect and a mild elevation of AST at 46. Haptoglobin was undetectable. Peripheral blood smear review showed [Display omitted] moderate basophilic stippling and polychromasia (Figure 1). Heinz body preparation and testing for PNH were negative. RBC enzyme panel was normal including 5′ nucleotidase levels. IEF was performed on a thin-layer agarose gel (RESOLVE Hemoglobin kit) with a pH range of 6.0–8.0, according to the manufacturer's instructions (Perkin Elmer Life and Analytical Sciences, Norton, OH, USA). Quantitative analyses of hemoglobin was done by Cation Exchange HPLC by using a Synchropak CM-300, 250mm × 4.6mm, 6u column (Eprogen Inc., Downers Grove, IL, USA) and the globin chains were separated on Reverse Phase HPLC with a Discovery BIO Wide Pore C5, 250mm × 4.6mm, 5um column (Supleco, Bellefonte, PA, USA). Isopropanal stability test was negative. DNA was isolated from peripheral blood leukocytes using QIAamp DNA Blood Mini Kit, according to manufacturer's instructions (QIAGEN Inc, Valencia, CA, USA). Segments of the genomic DNA were amplified by polymerase chain reaction (PCR) using target specific primers flanking Exons I, II, and III of the a1 and a2 genes. IEF revealed Hb A, A 2, and a variant “S” like band (<1%) (Figure 2). However, Cation exchange HPLC revealed 97.0% Hb A, 2.5% Hb A2 and 0.5% Hb F without detection of variant band. Reverse phase HPLC showed only βA and αA [Display omitted] chains without any detectable abnormal chains. Sequencing of the PCR amplified a2 gene revealed heterozygosity for TCTàCCT mutation (Ser131Pro) at codon 131 (Hb Questembert), Exon III (Figure 3). The a1 gene was normal. Hb Questembert is an extremely rare unstable Hb having been reported in French and French-Yugoslavian families. Although it is highly unstable, since the amount of variant hemoglobin is <1.0% and therefore not detectable on cation exchange HPLC, reverse phase HPLC, or unstable hemoglobin test. Our patient was entirely asymptomatic with periods of increased hemolysis during stress; she had never been transfused including during prior pregnancies. The mechanism by which this variant is unstable is highly complex. The substitution of Proline for Serine at a2 131 approximately mid way along the H helix next to heme contact residue 132 (H15) Val, disrupts the a helical structure leading to an unstable a-globin chain. The lack of family history (de novo mutation), a negative stability test, absence of a readily detectable variant hemoglobin do not readily suggest the presence of an unstable hemoglobin in this case. This patient highlights the importance of a molecular diagnostic approach in reaching the correct diagnosis in patients with a chronic, Coombs negative hemolytic anemia. [Display omitted] Alpha-2/Exon III/Codon 131 T à C/Ser131Pro/Hb Questembert/Heterozygous Disclosures:No relevant conflicts of interest to declare.

Rapid genomic alteration in an ‘incompatible’ pair of maize reciprocal F1hybrids - a possible cause for the accumulation of inter-strain genetic diversity

Recent studies have revealed unexpected high levels of genetic variation across maize inbred lines, which led to violation of colinearity that holds even between species of the grass family. Although activity of certain mobile elements is likely a contributing factor for this kind of intra-specific variations in maize, it is conceivable that other mechanisms might be involved. Here, we report that de novo genetic variation occurred instantaneously in a pair of reciprocal maize F(1) hybrids between inbred lines JAU8 and JAUM. Because expected genetic stability was observed in two other pairs of reciprocal hybrids in which each of these two lines was used as a crossing parent, we consider that the genomic instability in the JAU8/JAUM hybrids is due to specific incompatibilities between the two lines upon hybridization. Pairwise sequence analysis revealed the nature of the genetic changes as predominantly nucleotide substitutions with occasional small indels. At least some of the hybridization-induced genetic variations are likely associated with alteration in cytosine methylation. Given that a substantial portion of the variant bands bear meaningful homology to known or predicted genes, we suspect that the genetic changes and associated epigenetic alterations may have functional consequences.

In Vitro Micropropagation of Freesia hybrida and the Assessment of Genetic and Epigenetic Stability in Regenerated Plantlets

Freesia hybrida is an important worldwide cut flower, especially in America and Europe. For efficient regeneration of this flower from young inflorescence and rachillae in tetraploid, we developed a simple in vitro micropropagation protocol. Explants of Freesia hybrida can regenerate plantlets through somatic embryogenesis via two kinds of pathways, that is, directly from the epidermal cells or indirectly from an embryonic callus, depending on the exogenous plant growth regulators (PGRs) used in the culture media. In direct embryogenesis, when the explants were cultured on Murashige and Skoog (MS) medium supplemented with 11.43 μM indole acetic acid (IAA) and 4.44 μM 6-benzylaminopurine (6-BA), the induction rate was 84% for young inflorescence and 100% for rachillae. After the multishoots were subcultured on the rooting MS medium containing 1.08 μM α-naphthalene acetic acid (NAA), the rooting rate was close to 100%. In indirect embryogenesis, embryonic calluses were formed when the culture medium contained 22.22 μM 6-BA and 4.52 μM 2,4-dichlorophenoxy acetic acid (2,4-D), and the induction rate was 92.4% for young inflorescence and 100% for rachillae. After the embryonic calluses were transferred to the medium supplemented with 11.43 μM IAA and 13.33 μM 6-BA, they could develop into plantlets with roots. In assessing the two regeneration pathways in terms of genetic and epigenetic fidelity of the regenerants, two kinds of molecular markers [amplified fragment length polymorphism (AFLP) and methylation-sensitive amplified polymorphism (MSAP)] were employed. The AFLP analysis used 20 primer pairs that yielded 916 scorable bands among the donor plant and 11 regenerants from direct embryogenesis, of which 8 (0.87%) were polymorphic. The regenerants from indirect embryogenesis had 1075 clear bands of which 3 (0.27%) were polymorphic scorable bands from 18 primer pairs. Moreover, the variant band patterns included two types, that is, loss-of-original and gain-of-novel bands. MSAP analysis revealed that tissue culturing of the flower induced DNA cytosine methylation alterations in both CG and CNG levels and patterns compared with the donor plant. The variation rate was 1.1 and 1.3% for the direct and indirect embryogenesis pathways, respectively. The findings show that tissue culture of flowering plants is a form of stress which can induce some heritable epigenetic variations and should be considered in future long-term genotype preservation programs of Freesia hybrida.

Absence of mutations in the HOXA11 and HOXD11 genes in children with congenital renal malformations

Experimental studies have shown that homeobox genes are essential for the development of the kidney and urinary tract. Hoxa11/Hoxd11 double mutant mice demonstrate renal agenesis or hypoplasia. Since, to our knowledge, these genes have never been examined for alterations in humans with congenital anomalies of the kidney and urinary tract (CAKUT), we investigated whether mutations of HOXA11/HOXD11 genes are associated with non-syndromal congenital renal parenchymal malformations. DNA samples from 26 unrelated children with unilateral renal agenesis (URA), 20 with renal hypodysplasia (RHD) and 13 with multicystic dysplastic kidney (MCDK) were included in the study. Exons 1 and 2 of the HOXA11/HOXD11 genes were amplified individually by polymerase chain reaction (PCR) using 12 unique oligonucleotide primers. Single-strand conformation polymorphism (SSCP) analysis of overlapping polymerase chain reaction products was performed. SSCP analysis revealed no variant band shifts in the samples of the amplified segments of the 59 patients, suggesting lack of either mutation or polymorphisms. Our findings do not support the hypothesis that mutations in the HOXA11/HOXD11 coding regions are involved in the pathogenesis of human non-syndromal congenital renal parenchymal malformations. Further studies are necessary, since other genes known to affect nephrogenesis, as well as genetic and environmental factors, may be involved.