Neurodevelopmental Outcome of Children with Congenital Heart Disease: A Cohort Study from Infancy to Preschool Age.

OBJECTIVE: To characterize the neuropsychological outcome of children with congenital heart disease (CHD) at age 5 years; the stability of cognitive and language abilities across childhood; and to identify early neurodevelopmental markers of neuropsychological outcomes in these children. STUDY DESIGN: Five-year-old children (n = 55) with complex CHD were assessed using standardized and comprehensive neuropsychological measures. Stability of language and cognitive performance was assessed by comparing standardized scores between ages 1, 2, and 5 years old. Association between 5-year-old skills and scores obtained in early childhood was studied to identify potential early markers of preschool performance. Receiver operating characteristic curves were used to evaluate the classification accuracy of Bayley Scales of Infant Development, Third Edition scales in identifying later impairments. RESULTS: At age 5 years, our cohort obtained scores significantly below the norms on most developmental domains, with 35% to 65% of participants showing impaired short-term/working memory, attention, and preacademic skills. Developmental patterns measured between ages 1 and 5 years were different for cognitive and language domains, with a decline with age for cognitive functioning and stable results for expressive language. The Bayley Scales of Infant Development, Third Edition language scores at age 2 years provided a good predictive value in identifying children with impaired language at age 5 years. CONCLUSIONS: In our cohort, we found a high prevalence of impairments affecting higher-order cognitive domains. Although language difficulties can be detected as early as 2 years of age, other neuropsychological impairments, such as attention and pre-academic skills, only appear later during development, which reinforces the need for long-term monitoring and systematic assessment before school entry.

Social support and sleep quality in older benzodiazepine users.

Objective: Benzodiazepines (BZD) are often prescribed to address sleep difficulties but many BZD users report a poor quality of sleep. Although social support was found to be associated with quality of sleep in a recent meta-analysis, this relationship was never studied in older BZD users. This study thus aims to examine how social support is associated with quality of sleep in older BZD users.Method: Seventy-two older adults (age 60-85) using BZD were recruited. Data was collected during the pre-test of the ''PASSE-60+; Support program for a successful withdrawal, NCT02281175'' study. Quality of sleep was measured using the Pittsburgh Sleep Quality Index (PSQI), while social support was evaluated with the Social Support Questionnaire (SSQ-6).Results: When examining the various dimensions of self-reported sleep quality as a whole, we found no significant association with social support, while controlling for daily BZD dose, anxiety and depression. However, we found a significant association between self-reported diurnal dysfunctions (e.g., daytime sleepiness) and satisfaction with social support.Conclusion: Although the results of our study should be replicated with larger samples, they might indicate that social support is not a significant factor influencing sleep quality in older chronic BZD users. Our results could differ from those found in other populations because of the changes in sleep quality associated with long term BZD use. Longitudinal studies should analyse the relationship between diurnal dysfunctions and satisfaction with social support, to examine if social support could help older adults alleviate their diurnal dysfunctions and eventually facilitate BZD tapering.

Lipid environment induces ER stress, TXNIP expression and inflammation in immune cells of individuals with type 2 diabetes.

AIMS/HYPOTHESIS: Obesity and type 2 diabetes are concomitant with low-grade inflammation affecting insulin sensitivity and insulin secretion. Recently, the thioredoxin interacting protein (TXNIP) has been implicated in the activation process of the NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome. In this study, we aim to determine whether the expression of TXNIP is altered in the circulating immune cells of individuals with type 2 vs type 1 diabetes and whether this can be related to specific causes and consequences of inflammation. METHODS: The expression of TXNIP, inflammatory markers, markers of the unfolded protein response (UPR) to endoplasmic reticulum (ER) stress and enzymes involved in sphingolipid metabolism was quantified by quantitative reverse transcription real-time PCR (qRT-PCR) in peripheral blood mononuclear cells (PBMCs) of 13 non-diabetic individuals, 23 individuals with type 1 diabetes and 81 with type 2 diabetes. A lipidomic analysis on the plasma of 13 non-diabetic individuals, 35 individuals with type 1 diabetes and 94 with type 2 diabetes was performed. The effects of ER stress or of specific lipids on TXNIP and inflammatory marker expression were analysed in human monocyte-derived macrophages (HMDMs) and THP-1 cells. RESULTS: The expression of TXNIP and inflammatory and UPR markers was increased in the PBMCs of individuals with type 2 diabetes when compared with non-diabetic individuals or individuals with type 1 diabetes. TXNIP expression was significantly correlated with plasma fasting glucose, plasma triacylglycerol concentrations and specific UPR markers. Induction of ER stress in THP-1 cells or cultured HMDMs led to increased expression of UPR markers, TXNIP, NLRP3 and IL-1ß. Conversely, a chemical chaperone reduced the expression of UPR markers and TXNIP in PBMCs of individuals with type 2 diabetes. The lipidomic plasma analysis revealed an increased concentration of saturated dihydroceramide and sphingomyelin in individuals with type 2 diabetes when compared with non-diabetic individuals and individuals with type 1 diabetes. In addition, the expression of specific enzymes of sphingolipid metabolism, dihydroceramide desaturase 1 and sphingomyelin synthase 1, was increased in the PBMCs of individuals with type 2 diabetes. Palmitate or C2 ceramide induced ER stress in macrophages as well as increased expression of TXNIP, NLRP3 and IL-1ß. CONCLUSIONS/INTERPRETATION: In individuals with type 2 diabetes, circulating immune cells display an inflammatory phenotype that can be linked to ER stress and TXNIP expression. Immune cell ER stress can in turn be linked to the specific exogenous and endogenous lipid environment found in type 2 diabetes.

Recovery Trajectories in Adolescent Girls with Anorexia Nervosa.

Background: This study aimed to document recovery trajectories among adolescents with anorexia nervosa (AN) based on three markers of remission, namely changes in body weight, food restriction, and excessive exercise, and to identify predictors of these trajectories. Methods: One hundred twenty-six adolescent girls (14.7 ± 1.3 years) were recruited during initial assessment visits at specialized eating disorder (ED) programs in five University Health Centers across the province of Quebec, Canada. z-BMI and AN symptom severity (food restriction and excessive exercise) were assessed at initial assessment visits and subsequently reassessed at each quarterly follow-up over a 12-month period to identify recovery trajectories. Results: Considering the three markers of remission, three distinct trajectories emerged: Group 1, rapid responders; Group 2, gradual responders; and Group 3, unstable responders. At initial visits, a difference between groups was found regarding the type of treatment (p = 0.01) and weight suppression (p = 0.02). Group 1 had a higher number of youths hospitalized than Group 2 and Group 3, and a greater weight suppression than Group 3. Furthermore, individuals with atypical AN were more likely to belong to Group 2 than to Group 1 and Group 3 (p < 0.0001). Conclusions: This study contributes to a better understanding of the heterogeneity of recovery trajectories in adolescent girls with AN.

[Interaction between environment and genetic background in type 2 diabetes: lessons from animal models]. / Interaction entre facteurs d'environnement et fonds génétique dans le diabète de type 2 - L'apport des modèles animaux.

The respective roles of predisposing genetic factors and environmental factors in the development of type 2 diabetes (T2D) in obese subjects is poorly documented. Rodent models have been set up in an attempt to better understand of the differential effect of a prolonged metabolic stress induced by a high fat diet on glycaemic control according to the genetic background. In utero growth retardation resulting from a hypocaloric diet in pregnant rats induces a dramatic alteration of the development of islet cells leading to diabetes and insulin secretory defects in adult age. Experimentally induced diabetes in rodents results in hyperglycaemia and hyperinsulinemia in the fetus related to accelerated endocrine pancreas maturation responsible for the onset of diabetes in the adult. Deranged metabolic environment during fetal life may therefore further contribute to the onset of diabetes in the adult. Normal mouse strains with different genetic backgrounds show a wide range of responses to a high fat diet, with strains resistant to the diet and other more or less sensitive to the diet, the most sensitive exhibiting obesity diabetes and, insulin deficiency. The inability of the ß cell to respond to the increased insulin demand related to insulin resistance seems to be pivotal in the pathophysiologic process and a new notion is emerging: "nutritional genetics" which studies the influence of nutrients on gene expression.

Associations between neurological examination at term-equivalent age and cerebral hemodynamics and oxygen metabolism in infants born preterm.

Background: Infants born at 29-36 weeks gestational age (GA) are at risk of experiencing neurodevelopmental challenges. We hypothesize that cerebral hemodynamics and oxygen metabolism measured by bedside optical brain monitoring are potential biomarkers of brain development and are associated with neurological examination at term-equivalent age (TEA). Methods: Preterm infants (N = 133) born 29-36 weeks GA and admitted in the neonatal intensive care unit were enrolled in this prospective cohort study. Combined frequency-domain near infrared spectroscopy (FDNIRS) and diffuse correlation spectroscopy (DCS) were used from birth to TEA to measure cerebral hemoglobin oxygen saturation and an index of microvascular cerebral blood flow (CBF i ) along with peripheral arterial oxygen saturation (SpO2). In combination with hemoglobin concentration in the blood, these parameters were used to derive cerebral oxygen extraction fraction (OEF) and an index of cerebral oxygen metabolism (CMRO2i ). The Amiel-Tison and Gosselin Neurological Assessment was performed at TEA. Linear regression models were used to assess the associations between changes in FDNIRS-DCS parameters from birth to TEA and GA at birth. Logistic regression models were used to assess the associations between changes in FDNIRS-DCS parameters from birth to TEA and neurological examination at TEA. Results: Steeper increases in CBF i (p < 0.0001) and CMRO2i (p = 0.0003) were associated with higher GA at birth. Changes in OEF, CBF i , and CMRO2i from birth to TEA were not associated with neurological examination at TEA. Conclusion: In this population, cerebral FDNIRS-DCS parameters were not associated with neurological examination at TEA. Larger increases in CBF i and CMRO2i from birth to TEA were associated with higher GA. Non-invasive bedside FDNIRS-DCS monitoring provides cerebral hemodynamic and metabolic parameters that may complement neurological examination to assess brain development in preterm infants.

Utility of the Ages and Stages Questionnaires 3rd Edition for Developmental Screening in Children with Surgically Repaired Congenital Heart Disease.

Aim: This study sought to evaluate the accuracy of the Ages and Stages Questionnaires 3rd Edition (ASQ-3) in identifying developmental delay (DD) in children with congenital heart disease (CHD) born at term who underwent surgical repair.Methods: Participants had to complete ASQ-3 and Bayley Scales of Infant and Toddler Development 3rd Edition (BSID-III) at 12 and 24 months. A child was considered at risk of DD for a ASQ-3 domain when he scored below the cutoff (≤-1SD or ≤-2SD). A child had a DD in a BSID-III domain when the score was ≤-1SD. The validity for each ASQ-3 domain and for overall ASQ-3 was measured.Results: At 12 months (n = 64), overall ASQ-3 (≤-2SD) sensitivity was 88%, specificity 74%. At 24 months (n = 82), overall ASQ-3 (≤-2SD) sensitivity was 74%, specificity 88%.Conclusion: The results support the utility of the ASQ-3 for screening the overall risk of DD in children with CHD.

Normal delivery: physiologic support and medical interventions. Guidelines of the French National Authority for Health (HAS) with the collaboration of the French College of Gynecologists and Obstetricians (CNGOF) and the French College of Midwives (CNSF).

OBJECTIVE: To define for women at low obstetric risk methods of management that respect the rhythm and the spontaneous course of giving birth as well as each woman's preferences. METHODS: These clinical practice guidelines were developed through professional consensus based on an analysis of the literature and of the French and international guidelines available on this topic. RESULTS: Labor should be monitored with a partograph (professional consensus). Digital cervical examination should be offered every 4 h during the first stage of labor, hourly during the second. The choice between continuous (cardiotocography) or discontinuous (by cardiotocography or intermittent auscultation) monitoring should be left to the woman (professional consensus). In the active phase of the first stage of labor, dilation speed is considered abnormal if it is less than 1 cm/4 h between 5 and 7 cm or less than 1 cm/2 h after 7 cm. In those cases, an amniotomy is recommended if the membranes are intact, and the administration of oxytocin if the membranes are already broken and uterine contractions are judged insufficient (professional consensus). It is recommended that pushing not begin when full dilation has been reached; rather, the fetus should be allowed to descend (grade A). Umbilical cord clamping should be delayed beyond the first 30 s in newborns who do not require resuscitation (grade C). CONCLUSION: The establishment of these clinical practice guidelines should enable women at low obstetric risk to receive better care in conditions of optimal safety while supporting physiologic birth.

Novel toxic effects associated with a tropical Limnothrix/Geitlerinema-like cyanobacterium.

The presence of a toxic strain of a fine filamentous cyanobacterium belonging to the Oscillatorialean family Pseudanabaenacea was detected during a survey of cyanobacterial taxa associated with the presence of cylindrospermopsin in dams in Central Queensland (Australia). The strain, AC0243, was isolated and cultured, its genomic DNA extracted and 16S RNA gene sequenced. Phylogenetic analysis placed AC0243 with Limnothrix species, although this genus appears polyphyletic. Moreover, not all morphological characters are consistent with this genus but more closely fit the description of Geitlerinema unigranulatum (R.N. Singh) Komárek and Azevedo. The potential toxic effects of AC0243 extract were assessed chemically and biologically. Cell free protein synthesis was inhibited by the extract. Exposure of Vero cells to the extract resulted in a significant reduction in cellular ATP levels following 24-72 h incubation. The presence of cylindrospermopsin was excluded based on the nature of responses obtained in cell and cell-free assays; in addition, (i) it could not be detected by HPLC, LC-MS, or immunological assay, and (ii) no genes currently associated with the production of cylindrospermopsin were found in the genome. Other known cyanobacterial toxins were not detected. The apparent novelty of this toxin is discussed.

MG53 is not a critical regulator of insulin signaling pathway in skeletal muscle.

In type 2 diabetes (T2D), both muscle and liver are severely resistant to insulin action. Muscle insulin resistance accounts for more than 80% of the impairment in total body glucose disposal in T2D patients and is often characterized by an impaired insulin signaling. Mitsugumin 53 (MG53), a muscle-specific TRIM family protein initially identified as a key regulator of cell membrane repair machinery has been suggested to be a critical regulator of muscle insulin signaling pathway by acting as ubiquitin E3 ligase targeting both the insulin receptor and insulin receptor substrate 1 (IRS1). Here, we show using in vitro and in vivo approaches that MG53 is not a critical regulator of insulin signaling and glucose homeostasis. First, MG53 expression is not consistently regulated in skeletal muscle from various preclinical models of insulin resistance. Second, MG53 gene knock-down in muscle cells does not lead to impaired insulin response as measured by Akt phosphorylation on Serine 473 and glucose uptake. Third, recombinant human MG53 does not alter insulin response in both differentiated C2C12 and human skeletal muscle cells. Fourth, ectopic expression of MG53 in HEK293 cells lacking endogenous MG53 expression fails to alter insulin response as measured by Akt phosphorylation. Finally, both male and female mg53 -/- mice were not resistant to high fat induced obesity and glucose intolerance compared to wild-type mice. Taken together, these results strongly suggest that MG53 is not a critical regulator of insulin signaling pathway in skeletal muscle.

Dual regulation of TxNIP by ChREBP and FoxO1 in liver.

TxNIP (Thioredoxin-interacting protein) is considered as a potential drug target for type 2 diabetes. Although TxNIP expression is correlated with hyperglycemia and glucotoxicity in pancreatic ß cells, its regulation in liver cells has been less investigated. In the current study, we aim at providing a better understanding of Txnip regulation in hepatocytes in response to physiological stimuli and in the context of hyperglycemia in db/db mice. We focused on regulatory pathways governed by ChREBP (Carbohydrate Responsive Element Binding Protein) and FoxO1 (Forkhead box protein O1), transcription factors that play central roles in mediating the effects of glucose and fasting on gene expression, respectively. Studies using genetically modified mice reveal that hepatic TxNIP is up-regulated by both ChREBP and FoxO1 in liver cells and that its expression strongly correlates with fasting, suggesting a major role for this protein in the physiological adaptation to nutrient restriction.

Safety and immunogenicity of a plant-derived rotavirus-like particle vaccine in adults, toddlers and infants.

BACKGROUND: This study is the first clinical trial for a parenteral non-replicating rotavirus vaccine developed using virus-like particle (VLP) technology. METHODS: This open-labeled, randomized, placebo-controlled trial was conducted in two parts: Part A (a first-in-human study in Australian adults) and Part B (ascending dose and descending age in South African adults, toddlers and infants). In Part A, two cohorts of 10 adults were assigned to receive a single intramuscular injection of 1 of 2 escalating dose levels of the rotavirus VLP (Ro-VLP) vaccine (7 µg or 21 µg) or placebo. In Part B, one cohort of 10 adults was assigned to receive a single injection of the Ro-VLP vaccine (21 µg) or placebo, two cohorts of 10 toddlers were assigned to receive 2 injections of 1 of 2 escalating dose levels of the Ro-VLP vaccine (7 µg or 21 µg) or placebo 28 days apart, and three cohorts of 20 infants were assigned to receive 3 injections of 1 of 3 escalating dose levels of the Ro-VLP vaccine (2.5 µg, 7 µg or 21 µg) or placebo or 2 doses of oral Rotarix 28 days apart. Safety, reactogenicity and immunogenicity were assessed. RESULTS: There were no safety or tolerability concerns after administration of the Ro-VLP vaccine. The Ro-VLP vaccine induced an anti-G1P[8] IgG response in infants 4 weeks after the second and third doses. Neutralizing antibody responses against homologous G1P[8] rotavirus were higher in all Ro-VLP infant groups than in the placebo group 4 weeks after the third dose. No heterotypic immunity was elicited by the Ro-VLP vaccine. CONCLUSIONS: The Ro-VLP vaccine was well tolerated and induced a homotypic immune response in infants, suggesting that this technology platform is a favorable approach for a parenteral non-replicating rotavirus vaccine. CLINICAL TRIAL REGISTRATION: NCT03507738.

Evaluation of physician burnout at a major trauma centre using the Copenhagen burnout inventory: cross-sectional observational study.

BACKGROUND: Healthcare workers are susceptible to burnout owing to the demanding nature of their profession. The sequela of this is an increased incidence of medical errors and decreased job satisfaction. AIMS: This study aimed to assess the degree of burnout among physicians of different grades and specialties in a major trauma centre. METHODS: This study was performed in a UK tertiary trauma centre (Brighton and Sussex University Hospitals) in which 165 doctors from four medical specialties working with acute admissions were given the Copenhagen burnout inventory questionnaire via email and responses were received anonymously. Mean scores were calculated, and a two-tailed P test was performed to assess for statistically significant difference between patient- and work-related factors. RESULTS: The response rate was 77.57% (n = 165). General surgeons had the highest total burnout mean score of 50.00 with an SD of 12.78 followed by emergency medicine, acute medicine and finally orthopaedics. Junior doctors had an overall score of 53.42 with a standard deviation of 5.21, followed by consultants and registrars. The total burnout scores showed that 7.0% (n = 9) had low burnout scores while 56.3% (n = 72) had moderate burnout and 36.7% (n = 47) had high burnout scores. A two-tailed P test revealed a statistically significant difference between the work-related and patient-related subscales (P < 0.0001). CONCLUSIONS: Ninety-three percent of responders demonstrated either moderate or high levels of burnout in this study. Work-related factors appeared to contribute more to occurrence of burnout rather than the patient-related or doctor-patient interactions.

Surgical Fires and Burns: A 5-Year Analysis of Medico-legal Cases.

Surgical fires and unintended intraoperative burns cause serious patient harm, yet surveillance data are lacking in Canada. Medico-legal data provide unique descriptions of these events which can inform burn prevention strategies. We extracted 5 years of data on closed (2012-2016) medico-legal cases involving surgical fires and burns from the database of our organization which, in 2016, provided medico-legal support to >93,000 Canadian physicians. We performed a retrospective descriptive analysis of contributing factors using an in-house coding system and case reviews. We identified 53 eligible burn cases: 26 from thermal sources (49.1%), 16 from fires (30.2%), 5 from chemical sources (9.4%), and 6 from undetermined sources (11.3%). Common burn sources were electrosurgical equipment, lasers, lighting, and improper temperatures (causing thermal burns), cautery or lasers combined with supplemental oxygen and/or a flammable fuel source (causing fire), and improperly applied solutions including antiseptics (causing chemical burns). Nontechnical factors also contributed to patient outcomes, such as nonadherence to protocols (15 cases, 28.3%), failures in surgical team communication (3 cases, 5.7%), and lost situational awareness leading to delays in recognizing and treating burns (7 cases, 13.2%). This retrospective study highlights a need for improved surgical safety interventions to address surgical fires and burns. These interventions could include: effectively implemented surgical safety protocols, surgical team communication strategies, and raising awareness about preventing, diagnosing, and managing surgical burns.

Molecular phenotyping of multiple mouse strains under metabolic challenge uncovers a role for Elovl2 in glucose-induced insulin secretion.

OBJECTIVE: In type 2 diabetes (T2D), pancreatic ß cells become progressively dysfunctional, leading to a decline in insulin secretion over time. In this study, we aimed to identify key genes involved in pancreatic beta cell dysfunction by analyzing multiple mouse strains in parallel under metabolic stress. METHODS: Male mice from six commonly used non-diabetic mouse strains were fed a high fat or regular chow diet for three months. Pancreatic islets were extracted and phenotypic measurements were recorded at 2 days, 10 days, 30 days, and 90 days to assess diabetes progression. RNA-Seq was performed on islet tissue at each time-point and integrated with the phenotypic data in a network-based analysis. RESULTS: A module of co-expressed genes was selected for further investigation as it showed the strongest correlation to insulin secretion and oral glucose tolerance phenotypes. One of the predicted network hub genes was Elovl2, encoding Elongase of very long chain fatty acids 2. Elovl2 silencing decreased glucose-stimulated insulin secretion in mouse and human ß cell lines. CONCLUSION: Our results suggest a role for Elovl2 in ensuring normal insulin secretory responses to glucose. Moreover, the large comprehensive dataset and integrative network-based approach provides a new resource to dissect the molecular etiology of ß cell failure under metabolic stress.

Structure-Activity Relationship of Azaindole-Based Glucokinase Activators.

7-Azaindole has been identified as a novel bidentate anchor point for allosteric glucokinase activators. A systematic investigation around three principal parts of the new small molecule glucokinase activators led to a robust SAR in agreement with structural data that also helped to assess the conformational flexibility of the allosteric activation site. The increase in glucose uptake resulting from glucokinase activation in hepatocytes in vitro translated into the efficient lowering of glucose levels in vivo with the best compounds.

Adaptative evolution of metallothionein 3 in the Cd/Zn hyperaccumulator Thlaspi caerulescens.

Laboratoire de Physiologie et Génétique Moléculaire des Plantes, Université Libre de Bruxelles, Campus Plaine (CP 242) Bd du triomphe, B-1050 Brussels, Belgium.

Evidence for copper homeostasis function of metallothionein (MT3) in the hyperaccumulator Thlaspi caerulescens.

Metallothioneins chelate metals and consequently may be a control point of metal homeostasis. Homologous to type 3 metallothioneins, TcMT3 cDNA was identified in the Cd/Zn hyperaccumulator, Thlaspi caerulescens. TcMT3 amino acid sequence showed modifications in the Cys positions when compared with its Arabidopsis orthologue. A structural model established that the MT3 carboxyterminal domain is similar to the beta domain of animal metallothioneins and predicts a smaller cavity to chelate metals for A. thaliana than for T. caerulescens. Functional testing in yeast and Northern blot analysis added further evidence for adaptative variations of MT3 for the maintenance of Cu homeostasis in a metal hyperaccumulator.

A novel CPx-ATPase from the cadmium hyperaccumulator Thlaspi caerulescens.

Thlaspi caerulescens exhibits a unique capacity for cadmium tolerance and accumulation. We investigated the molecular basis of this exceptional Cd(2+) tolerance by screening for T. caerulescens genes, which alleviate Cd(2+) toxicity upon expression in Saccharomyces cerevisiae. This allowed for the isolation of a cDNA encoding a peptide with homology to the C-terminal part of a heavy metal ATPase. The corresponding TcHMA4 full-length sequence was isolated from T. caerulescens and compared to its homolog from Arabidopsis thaliana (AtHMA4). Expression of TcHMA4 and AtHMA4 cDNAs conferred Cd sensitivity in yeast, while expression of TcHMA4-C and AtHMA4-C cDNAs encoding the C-termini of, respectively, TcHMA4 and AtHMA4 conferred Cd tolerance. Moreover, heterologous expression in yeast suggested a higher Cd binding capacity of TcHMA4-C compared to AtHMA4-C. In planta, both HMA4 genes were expressed at a higher level in roots than in shoots. However, TcHMA4 shows a much higher constitutive expression than AtHMA4. Our data indicate that HMA4 could be involved in Cd(2+) transport and possibly in the Cd hyperaccumulation character.