A newly identified ferritin L-subunit variant results in increased proteasomal subunit degradation, impaired complex assembly, and severe hypoferritinemia.

Ferritin is a hetero-oligomeric nanocage, composed of 24 subunits of two types, FTH1 and FTL. It protects the cell from excess reactive iron, by storing iron in its cavity. FTH1 is essential for the recruitment of iron into the ferritin nanocage and for cellular ferritin trafficking, whereas FTL contributes to nanocage stability and iron nucleation inside the cavity. Here we describe a female patient with a medical history of severe hypoferritinemia without anemia. Following inadequate heavy IV iron supplementation, the patient developed severe iron overload and musculoskeletal manifestations. However, her serum ferritin levels rose only to normal range. Genetic analyses revealed an undescribed homozygous variant of FTL (c.92A > G), which resulted in a Tyr31Cys substitution (FTLY31C ). Analysis of the FTL structure predicted that the Y31C mutation will reduce the variant's stability. Expression of the FTLY31C variant resulted in significantly lower cellular ferritin levels compared with the expression of wild-type FTL (FTLWT ). Proteasomal inhibition significantly increased the initial levels of FTLY31C , but could not protect FTLY31C subunits from successive degradation. Further, variant subunits successfully incorporated into hetero-polymeric nanocages in the presence of sufficient levels of FTH1. However, FTLY31C subunits poorly assembled into nanocages when FTH1 subunit levels were low. These results indicate an increased susceptibility of unassembled monomeric FTLY31C subunits to proteasomal degradation. The decreased cellular assembly of FTLY31C -rich nanocages may explain the low serum ferritin levels in this patient and emphasize the importance of a broader diagnostic approach of hypoferritinemia without anemia, before IV iron supplementation.

Doubled Haploid Production in High- and Low-Response Genotypes of Rapeseed (Brassica napus) Through Isolated Microspore Culture.

Rapeseed (Brassica napus) is one of the most important oilseed crops worldwide. It is also a model system to study the process of microspore embryogenesis, due to the high response of some B. napus lines, and to the refinements of the protocols. This chapter presents a protocol for the induction of haploid and DH embryos in B. napus through isolated microspore culture in two specific backgrounds widely used in DH research, the high response DH4079 line and the low response DH12075 line. We also present methods to identify the best phenological window to identify buds with microspores/pollen at the right developmental stage to induce this process. Methods to determine microspore/pollen viability and to check the ploidy by flow cytometry are also described.

Anther and Isolated Microspore Culture in Eggplant (Solanum melongena L.).

Eggplant is one of the five important, worldwide-distributed solanaceous crops. The use of anther culture technology to produce pure, 100% homozygous doubled haploid lines for hybrid seed production is possible since 1982, where the first protocol of wide application to different eggplant materials was published. From then on, different improvements and adaptations to different materials have been made. In parallel, protocols to implement isolated microspore culture technology in eggplant have been developed principally in the last decade, which opens the door for a more efficient DH production in this species. In this chapter, two protocols, one for anther and other for isolated microspore culture in eggplant, are described. Some steps and materials are common to both approaches. A detailed description of each step from is provided.

Characterization of starches obtained from several native potato varieties grown in Cusco (Peru).

Ten native potato varieties grown in Cusco (at 3,672 m above sea level) were used for starches extraction (at a pilot scale), and their physicochemical, functional, morphological, and structural characteristics were assessed. The content of protein, apparent amylose and phosphorus ranged from 0.1% to 0.44%, 23.42% to 35.5%, and 0.07 to 0.10%, respectively. Starch granules revealed smooth surface, with ellipsoidal and spherical shapes, particle size analysis exhibited bimodal or multimodal distribution, while the averaged crystallinity was 27.7% assessed by XRD. Gelatinization temperatures of the starches ranged from 57.4 to 60.1 °C, 60.8 to 64.3 °C, and 68.4 to 71.1°C for To, Tp, and Tc, respectively; and the gelatinization enthalpies ranged from 15.4 to 17.1 J/g. Respect to pasting properties, the peak viscosity and setback viscosity ranged from 12,970 to 16,970 mPa⋅s and from 968 to 2498 mPa⋅s, respectively. Thermogravimetric analysis (TGA) revealed no significant relationship between apparent amylose content and thermal stability. Therefore, the results reveal subtle differences in the functional characteristics of the starches from the native varieties of potato studied, which can be recommended for food industry applications. PRACTICAL APPLICATION: This study contributes to show several varieties of native potatoes from Cusco and their valorization as nonconventional starch source. Describing the physicochemical, functional, and structural characteristics of these starches could be useful for food industry applications.

Live Imaging of embryogenic structures in Brassica napus microspore embryo cultures highlights the developmental plasticity of induced totipotent cells.

KEY MESSAGE: In vitro embryo development is highly plastic; embryo cell fate can be re-established in tissue culture through different pathways. In most angiosperms, embryo development from the single-celled zygote follows a defined pattern of cell divisions in which apical (embryo proper) and basal (root and suspensor) cell fates are established within the first cell divisions. By contrast, embryos that are induced in vitro in the absence of fertilization show a less regular initial cell division pattern yet develop into histodifferentiated embryos that can be converted into seedlings. We used the Brassica napus microspore embryogenesis system, in which the male gametophyte is reprogrammed in vitro to form haploid embryos, to identify the developmental fates of the different types of embryogenic structures found in culture. Using time-lapse imaging of LEAFY COTYLEDON1-expressing cells, we show that embryogenic cell clusters with very different morphologies are able to form haploid embryos. The timing of surrounding pollen wall (exine) rupture is a major determinant of cell fate in these clusters, with early exine rupture leading to the formation of suspensor-bearing embryos and late rupture to suspensorless embryos. In addition, we show that embryogenic callus, which develops into suspensor-bearing embryos, initially expresses transcripts associated with both basal- and apical-embryo cell fates, suggesting that these two cell fates are fixed later in development. This study reveals the inherent plasticity of in vitro embryo development and identifies new pathways by which embryo cell fate can be established.

Anther Culture in Eggplant (Solanum melongena L.).

For a long time, conventional breeding methods have been used to obtain pure, 100% homozygous lines for hybrid seed production in crops of agronomic interest. However, by doubled haploid technology, it is possible to produce 100% homozygous plants derived from precursors of male gametophytes (androgenesis), to accelerate the production of pure lines, which implies important time and cost savings. In this chapter, a protocol for anther culture in eggplant is described, from donor plant growth conditions to regeneration and acclimation of doubled haploid plants, as well as a description of how to analyze ploidy levels of regenerated plants.

Isolated Microspore Culture in Brassica napus.

Isolated microspore culture is the most efficient technique among those used to induce microspore embryogenesis. In the particular case of Brassica napus, it is also the most widely used and optimized. In this chapter, we describe a protocol for microspore culture in B. napus which includes the steps necessary to isolate and culture microspores, to induce microspore-derived embryos, to produce doubled haploid plants from them, as well as to check for the developmental stage of the microspores isolated, their viability, and the ploidy level of regenerated plantlets.

Embryogenic competence of microspores is associated with their ability to form a callosic, osmoprotective subintinal layer.

Microspore embryogenesis is an experimental morphogenic pathway with important applications in basic research and applied plant breeding, but its genetic, cellular, and molecular bases are poorly understood. We applied a multidisciplinary approach using confocal and electron microscopy, detection of Ca2+, callose, and cellulose, treatments with caffeine, digitonin, and endosidin7, morphometry, qPCR, osmometry, and viability assays in order to study the dynamics of cell wall formation during embryogenesis induction in a high-response rapeseed (Brassica napus) line and two recalcitrant rapeseed and eggplant (Solanum melongena) lines. Formation of a callose-rich subintinal layer (SL) was common to microspore embryogenesis in the different genotypes. However, this process was directly related to embryogenic response, being greater in high-response genotypes. A link could be established between Ca2+ influx, abnormal callose/cellulose deposition, and the genotype-specific embryogenic competence. Callose deposition in inner walls and SLs are independent processes, regulated by different callose synthases. Viability and control of internal osmolality are also related to SL formation. In summary, we identified one of the causes of recalcitrance to embryogenesis induction: a reduced or absent protective SL. In responding genotypes, SLs are markers for changes in cell fate and serve as osmoprotective barriers to increase viability in imbalanced in vitro environments. Genotype-specific differences relate to different responses against abiotic (heat/osmotic) stresses.

Inherited or acquired modifiers of iron status may dramatically affect the phenotype in dehydrated hereditary stomatocytosis.

Severe iron overload is frequent in dehydrated hereditary stomatocytosis (DHSt) despite well-compensated hemolysis and no or little transfusion requirement. We investigated 4 patients with proven DHSt, in whom the degree of hemolysis was closely related to iron status. Genetic modifiers increasing iron stores (HFE:pCys282Tyr, HAMP:c-153C>T mutations) were accompanied with high liver iron concentrations and increased hemolysis, whereas therapeutic phlebotomies alleviated the hemolytic phenotype. There were no manifestations of hemolysis in one patient with low iron stores. Hemolysis reappeared when iron supplementation was given. The search for genetic or acquired modifiers of iron status and the modulation of iron stores may help in the management of these patients.

Fallo hepático por intoxicación con producto casero elaborado con anís estrellado y anís verde en lactante de 4 meses / Liver failure secondary to poisoning by a homemade product made of star and green anise in a 4-month-old infant

Las intoxicaciones en edad pediátrica representan una causa frecuente de consulta en urgencias hospitalarias. Los productos elaborados con hierbas pueden resultar tóxicos para el lactante. Se han descrito ampliamente las propiedades neurotóxicas del anís estrellado (Illicium verum), producto clásicamente empleado para el tratamiento del cólico del lactante. La presentación de fallo hepático agudo por consumo de infusiones elaboradas con hierbas de anís es excepcional en nuestro entorno. Se describe el caso de un lactante de 4 meses con hipertransaminasemia, coagulopatía grave, hipoglucemia no cetósica, acidosis metabólica moderada y síntomas neurológicos con crisis convulsivas y nistagmo. Tras descartar etiología infecciosa, metabólica y autoinmune y realizar una anamnesis cuidadosa, la familia refería haber administrado al paciente durante los últimos dos meses una infusión diaria con anís estrellado y anís verde (Pimpinella anisum). Es de gran importancia resaltar el grave riesgo de administrar infusiones de hierbas caseras en el lactante (AU)

Intoxications in pediatric age represent a frequent cause of visit to the hospital emergency unit. Herb-made products can be toxic for the infant. The neurotoxic properties of the star anise (Illicium verum) have been widely described, although it is a classic product used to treat the infantile colic. Hepatic failure due to the consumption of anise herb elaborated infusions is presented as an exceptional finding in our environment. A case of a 4-month-old infant with hypertransaminasemia, severe coagulopathy, non ketotic hypoglycemia, moderated metabolic acidosis and neurologic symptoms such as seizures and nistagmus is described. After discarding infectious, metabolic and autoimmune etiology and through a meticulous anamnesis, the family referred having administered in the last two months a daily star anise and green anise (Pimpinella anisum) infusion to the patient. It is important to emphasize the serious risk of administering homemade herb infusions to infants (AU)

Ultrastructural Immunolocalization of Arabinogalactan Protein, Pectin and Hemicellulose Epitopes Through Anther Development in Brassica napus.

In this work, we performed an extensive and detailed analysis of the changes in cell wall composition during Brassica napus anther development. We used immunogold labeling to study the spatial and temporal patterns of the composition and distribution of different arabinogalactan protein (AGP), pectin, xyloglucan and xylan epitopes in high-pressure-frozen/freeze-substituted anthers, quantifying and comparing their relative levels in the different anther tissues and developmental stages. We used the following monoclonal antibodies: JIM13, JIM8, JIM14 and JIM16 for AGPs, LM5, LM6, JIM7, JIM5 and LM7 for pectins, CCRC-M1, CCRC-M89 and LM15 for xyloglucan, and LM11 for xylan. Each cell wall epitope showed a characteristic temporal and spatial labeling pattern. Microspore, pollen and tapetal cells showed similar patterns for each epitope, whereas the outermost anther layers (epidermis, endothecium and middle layers) presented remarkably different patterns. Our results suggested that AGPs, pectins, xyloglucan and xylan have specific roles during anther development. The AGP epitopes studied appeared to belong to AGPs specifically involved in microspore differentiation, and contributed first by the tapetum and then, upon tapetal dismantling, by the endothecium and middle layers. In contrast, the changes in pectin and hemicellulose epitopes suggested a specific role in anther dehiscence, facilitating anther wall weakening and rupture. The distribution of the different cell wall constituents is regulated in a tissue- and stage-specific manner, which seems directly related to the role of each tissue at each stage.

Liver failure secondary to poisoning by a homemade product made of star and green anise in a 4-month-old infant.

Intoxications in pediatric age represent a frequent cause of visit to the hospital emergency unit. Herb-made products can be toxic for the infant. The neurotoxic properties of the star anise (Illicium verum) have been widely described, although it is a classic product used to treat the infantile colic. Hepatic failure due to the consumption of anise herb elaborated infusions is presented as an exceptional finding in our environment. A case of a 4-month-old infant with hypertransaminasemia, severe coagulopathy, non ketotic hypoglycemia, moderated metabolic acidosis and neurologic symptoms such as seizures and nistagmus is described. After discarding infectious, metabolic and autoimmune etiology and through a meticulous anamnesis, the family referred having administered in the last two months a daily star anise and green anise (Pimpinella anisum) infusion to the patient. It is important to emphasize the serious risk of administering homemade herb infusions to infants.

Novel features of Brassica napus embryogenic microspores revealed by high pressure freezing and freeze substitution: evidence for massive autophagy and excretion-based cytoplasmic cleaning.

Induction of embryogenesis from isolated microspore cultures is a complex experimental system where microspores undergo dramatic changes in developmental fate. After ~40 years of application of electron microscopy to the study of the ultrastructural changes undergone by the induced microspore, there is still room for new discoveries. In this work, high pressure freezing and freeze substitution (HPF/FS), the best procedures known to date for ultrastructural preservation, were used to process Brassica napus microspore cultures covering all the stages of microspore embryogenesis. Analysis of these cultures by electron microscopy revealed massive processes of autophagy exclusively in embryogenic microspores, but not in other microspore-derived structures also present in cultures. However, a significant part of the autophagosomal cargo was not recycled. Instead, it was transported out of the cell, producing numerous deposits of extracytoplasmic fibrillar and membranous material. It was shown that commitment of microspores to embryogenesis is associated with both massive autophagy and excretion of the removed material. It is hypothesized that autophagy would be related to the need for a profound cytoplasmic cleaning, and excretion would be a mechanism to avoid excessive growth of the vacuolar system. Together, the results also demonstrate that the application of HPF/FS to the study of the androgenic switch is the best option currently available to identify the complex and dramatic ultrastructural changes undergone by the induced microspore. In addition, they provide significant insights to understand the cellular basis of induction of microspore embryogenesis, and open a new door for the investigation of this intriguing developmental pathway.

[Hereditary hemochromatosis, the clinician point of view]. / L'hémochromatose génétique, le point de vue du clinicien.

Hereditary hemochromatosis (HH) is an autosomal recessive disease whose most common form is due to homozygosity for the C282Y mutation of the HFE gene. Its prevalence is estimated between 1/200 and 1/600 in France. This represents potentially several thousands of affected people. The disease is characterized by progressive iron overload, which can lead to irreversible parenchymal tissue damage. When clinical signs become evocative the disease is already at an advanced stage and years of life are probably lost. It is therefore important to detect the disease early. The clinician, either general practitioner or specialist, plays a pivotal role in the diagnostic process: he/she receives complaints of patients, prescribes complementary investigations, conducts the treatment and must organize of the family screening. Based on the French recommendations and on literature data, this paper presents the main lines of management of the patient as responses to the eight following questions: 1) Under what circumstances should the clinician suspect HH? 2) How to conduct investigations in the presence of high ferritin levels? 3) What manifestations must be feared with the worsening of iron overload? 4) What medical evaluation should be performed when the diagnosis of HH has been made? 5) How to conduct iron depletive treatment in practice? 6) How to monitor the treated patient? 7) What is the place of iron-chelating drugs in the treatment of HH? 8) How to take charge of the family members?

Androgenesis in recalcitrant solanaceous crops.

Tomato, eggplant, and pepper are three solanaceous crops of outstanding importance worldwide. For hybrid seed production in these species, a fast and cheap method to obtain pure (homozygous) lines is a priority. Traditionally, pure lines are produced by classical inbreeding and selection techniques, which are time consuming (several years) and costly. Alternatively, it has become possible to accelerate the production of homozygous lines through a biotechnological approach: the induction of androgenesis to generate doubled haploid (homozygous) plants. This biotechnological in vitro tool reduces the process to only one generation, which implies important time and costs savings. These facts make androgenic doubled haploids the choice in a number of important crops where the methodology is well set up. Unfortunately, recalcitrant solanaceous crops such as tomato, eggplant, and pepper are still far from an efficient and reliable technology to be applied on a routine basis to different genotypes in breeding programs. In eggplant and pepper, only anther cultures are known to work relatively well. Unfortunately, a more efficient and promising technique, the culture of isolated microspores, is not sufficiently developed yet. In tomato, none of these methods is available nowadays. However, recent advances in the knowledge of embryo development are filling the gaps and opening new ways to achieve the final goal of an efficient protocol in these three recalcitrant species. In this review, we outline the state of the art on androgenic induction in tomato, eggplant, and pepper, and postulate new experimental ways in order to overcome current limitations.

Severe hemochromatosis in a Portuguese family associated with a new mutation in the 5'-UTR of the HAMP gene.

Juvenile hereditary hemochromatosis is a genetically heterogeneous disorder transmitted as an autosomal recessive trait. It is most often caused by mutations in the HJV gene and rarely in the HAMP gene. Hepcidin is considered to constitute a negative regulator of iron absorption, and its production is increased in inflammatory states and iron overload. We report the detection of a new mutation in the HAMP gene leading to juvenile hemochromatosis in 2 members of a Portuguese family. The mutation lies in the 5'-UTR (untranslated region) of the gene and creates a new initiation codon in the context of a Kozak sequence. We found no trace of hepcidin protein in the patients' urine, suggesting that ribosomes select the mutant initiation codon for translation. The decrease of hepcidin production would thus lead to increased iron absorption, resulting in iron deposition in parenchymal tissues. Phlebotomy therapy of the 2 patients resulted in impressive clinical improvement.

Retroalimentación biológica y su aplicación clínica / Clinical applications of biofeedback

Antecedentes: La incontinencia fecal constituye un serio problema social que afecta a todos los grupos de edad. Su tratamiento es tan complejo como desconocido. Métodos: Efectuamos un estudio longitudinal, prospectivo, experimental, entre 47 pacientes consecutivos que presentaban incontinencia total por un periodo de 55.4 ñ 7.6 meses (intervalo: seis meses a 21 años), con 2.4 ñ 0.2 episodios de incontinencia (intervalo: 1-7 por día). A todos se les efectuó cuestionario para validar el diagnóstico de incontinencia, historia clínica completa, laboratorio, colon por enema, rectosigmoidoscopia, sensibilidad rectal, manometría rectoanal, y retroalimentación biológica sin instrumentación electrónica. Veintiún sujetos normales sirvieron como grupo control. Resultados: Los pacientes con incontinencia fecal presentaron disminución de la sensibilidad rectal (p<0.01) y alteraciones en el reflejo recto anal inhibitorio espontáneo. Todos obtuvieron curación completa en un periodo de 4 ñ 0.5 meses (intervalo: 15 días a 15 meses) y fueron seguidos por uno a ocho años. Conclusiones: La nueva modalidad de retroalimentación biológica aquí descrita no utiliza ningún instrumento electrónico. La curación y el tiempo de curación de los pacientes no solamente es comparable, sino superior a lo hasta ahora informados. Puede ser reproducido, con el entrenamiento adecuado, por mayor número de médicos en cualquier centro y condicionar, por tanto, un beneficio significativo en la calidad de vida de un mayor número de pacientes