Bi-allelic TTI1 variants cause an autosomal-recessive neurodevelopmental disorder with microcephaly.

Telomere maintenance 2 (TELO2), Tel2 interacting protein 2 (TTI2), and Tel2 interacting protein 1 (TTI1) are the three components of the conserved Triple T (TTT) complex that modulates activity of phosphatidylinositol 3-kinase-related protein kinases (PIKKs), including mTOR, ATM, and ATR, by regulating the assembly of mTOR complex 1 (mTORC1). The TTT complex is essential for the expression, maturation, and stability of ATM and ATR in response to DNA damage. TELO2- and TTI2-related bi-allelic autosomal-recessive (AR) encephalopathies have been described in individuals with moderate to severe intellectual disability (ID), short stature, postnatal microcephaly, and a movement disorder (in the case of variants within TELO2). We present clinical, genomic, and functional data from 11 individuals in 9 unrelated families with bi-allelic variants in TTI1. All present with ID, and most with microcephaly, short stature, and a movement disorder. Functional studies performed in HEK293T cell lines and fibroblasts and lymphoblastoid cells derived from 4 unrelated individuals showed impairment of the TTT complex and of mTOR pathway activity which is improved by treatment with Rapamycin. Our data delineate a TTI1-related neurodevelopmental disorder and expand the group of disorders related to the TTT complex.

Germline variants in tumor suppressor FBXW7 lead to impaired ubiquitination and a neurodevelopmental syndrome.

Neurodevelopmental disorders are highly heterogenous conditions resulting from abnormalities of brain architecture and/or function. FBXW7 (F-box and WD-repeat-domain-containing 7), a recognized developmental regulator and tumor suppressor, has been shown to regulate cell-cycle progression and cell growth and survival by targeting substrates including CYCLIN E1/2 and NOTCH for degradation via the ubiquitin proteasome system. We used a genotype-first approach and global data-sharing platforms to identify 35 individuals harboring de novo and inherited FBXW7 germline monoallelic chromosomal deletions and nonsense, frameshift, splice-site, and missense variants associated with a neurodevelopmental syndrome. The FBXW7 neurodevelopmental syndrome is distinguished by global developmental delay, borderline to severe intellectual disability, hypotonia, and gastrointestinal issues. Brain imaging detailed variable underlying structural abnormalities affecting the cerebellum, corpus collosum, and white matter. A crystal-structure model of FBXW7 predicted that missense variants were clustered at the substrate-binding surface of the WD40 domain and that these might reduce FBXW7 substrate binding affinity. Expression of recombinant FBXW7 missense variants in cultured cells demonstrated impaired CYCLIN E1 and CYCLIN E2 turnover. Pan-neuronal knockdown of the Drosophila ortholog, archipelago, impaired learning and neuronal function. Collectively, the data presented herein provide compelling evidence of an F-Box protein-related, phenotypically variable neurodevelopmental disorder associated with monoallelic variants in FBXW7.

Narrowing the diagnostic gap: Genomes, episignatures, long-read sequencing, and health economic analyses in an exome-negative intellectual disability cohort.

PURPOSE: Genome sequencing (GS)-specific diagnostic rates in prospective tightly ascertained exome sequencing (ES)-negative intellectual disability (ID) cohorts have not been reported extensively. METHODS: ES, GS, epigenetic signatures, and long-read sequencing diagnoses were assessed in 74 trios with at least moderate ID. RESULTS: The ES diagnostic yield was 42 of 74 (57%). GS diagnoses were made in 9 of 32 (28%) ES-unresolved families. Repeated ES with a contemporary pipeline on the GS-diagnosed families identified 8 of 9 single-nucleotide variations/copy-number variations undetected in older ES, confirming a GS-unique diagnostic rate of 1 in 32 (3%). Episignatures contributed diagnostic information in 9% with GS corroboration in 1 of 32 (3%) and diagnostic clues in 2 of 32 (6%). A genetic etiology for ID was detected in 51 of 74 (69%) families. Twelve candidate disease genes were identified. Contemporary ES followed by GS cost US$4976 (95% CI: $3704; $6969) per diagnosis and first-line GS at a cost of $7062 (95% CI: $6210; $8475) per diagnosis. CONCLUSION: Performing GS only in ID trios would be cost equivalent to ES if GS were available at $2435, about a 60% reduction from current prices. This study demonstrates that first-line GS achieves higher diagnostic rate than contemporary ES but at a higher cost.

Integration of EpiSign, facial phenotyping, and likelihood ratio interpretation of clinical abnormalities in the re-classification of an ARID1B missense variant.

Heterozygous ARID1B variants result in Coffin-Siris syndrome. Features may include hypoplastic nails, slow growth, characteristic facial features, hypotonia, hypertrichosis, and sparse scalp hair. Most reported cases are due to ARID1B loss of function variants. We report a boy with developmental delay, feeding difficulties, aspiration, recurrent respiratory infections, slow growth, and hypotonia without a clinical diagnosis, where a previously unreported ARID1B missense variant was classified as a variant of uncertain significance. The pathogenicity of this variant was refined through combined methodologies including genome-wide methylation signature analysis (EpiSign), Machine Learning (ML) facial phenotyping, and LIRICAL. Trio exome sequencing and EpiSign were performed. ML facial phenotyping compared facial images using FaceMatch and GestaltMatcher to syndrome-specific libraries to prioritize the trio exome bioinformatic pipeline gene list output. Phenotype-driven variant prioritization was performed with LIRICAL. A de novo heterozygous missense variant, ARID1B p.(Tyr1268His), was reported as a variant of uncertain significance. The ACMG classification was refined to likely pathogenic by a supportive methylation signature, ML facial phenotyping, and prioritization through LIRICAL. The ARID1B genotype-phenotype has been expanded through an extended analysis of missense variation through genome-wide methylation signatures, ML facial phenotyping, and likelihood-ratio gene prioritization.

Dominant-negative variants in CBX1 cause a neurodevelopmental disorder.

PURPOSE: This study aimed to establish variants in CBX1, encoding heterochromatin protein 1ß (HP1ß), as a cause of a novel syndromic neurodevelopmental disorder. METHODS: Patients with CBX1 variants were identified, and clinician researchers were connected using GeneMatcher and physician referrals. Clinical histories were collected from each patient. To investigate the pathogenicity of identified variants, we performed in vitro cellular assays and neurobehavioral and cytological analyses of neuronal cells obtained from newly generated Cbx1 mutant mouse lines. RESULTS: In 3 unrelated individuals with developmental delay, hypotonia, and autistic features, we identified heterozygous de novo variants in CBX1. The identified variants were in the chromodomain, the functional domain of HP1ß, which mediates interactions with chromatin. Cbx1 chromodomain mutant mice displayed increased latency-to-peak response, suggesting the possibility of synaptic delay or myelination deficits. Cytological and chromatin immunoprecipitation experiments confirmed the reduction of mutant HP1ß binding to heterochromatin, whereas HP1ß interactome analysis demonstrated that the majority of HP1ß-interacting proteins remained unchanged between the wild-type and mutant HP1ß. CONCLUSION: These collective findings confirm the role of CBX1 in developmental disabilities through the disruption of HP1ß chromatin binding during neurocognitive development. Because HP1ß forms homodimers and heterodimers, mutant HP1ß likely sequesters wild-type HP1ß and other HP1 proteins, exerting dominant-negative effects.

De Novo ZMYND8 variants result in an autosomal dominant neurodevelopmental disorder with cardiac malformations.

PURPOSE: ZMYND8 encodes a multidomain protein that serves as a central interactive hub for coordinating critical roles in transcription regulation, chromatin remodeling, regulation of super-enhancers, DNA damage response and tumor suppression. We delineate a novel neurocognitive disorder caused by variants in the ZMYND8 gene. METHODS: An international collaboration, exome sequencing, molecular modeling, yeast two-hybrid assays, analysis of available transcriptomic data and a knockdown Drosophila model were used to characterize the ZMYND8 variants. RESULTS: ZMYND8 variants were identified in 11 unrelated individuals; 10 occurred de novo and one suspected de novo; 2 were truncating, 9 were missense, of which one was recurrent. The disorder is characterized by intellectual disability with variable cardiovascular, ophthalmologic and minor skeletal anomalies. Missense variants in the PWWP domain of ZMYND8 abolish the interaction with Drebrin and missense variants in the MYND domain disrupt the interaction with GATAD2A. ZMYND8 is broadly expressed across cell types in all brain regions and shows highest expression in the early stages of brain development. Neuronal knockdown of the DrosophilaZMYND8 ortholog results in decreased habituation learning, consistent with a role in cognitive function. CONCLUSION: We present genomic and functional evidence for disruption of ZMYND8 as a novel etiology of syndromic intellectual disability.

Impacts of genomics on the health and social costs of intellectual disability.

BACKGROUND: This study provides an integrated assessment of the economic and social impacts of genomic sequencing for the detection of monogenic disorders resulting in intellectual disability (ID). METHODS: Multiple knowledge bases were cross-referenced and analysed to compile a reference list of monogenic disorders associated with ID. Multiple literature searches were used to quantify the health and social costs for the care of people with ID. Health and social expenditures and the current cost of whole-exome sequencing and whole-genome sequencing were quantified in relation to the more common causes of ID and their impact on lifespan. RESULTS: On average, individuals with ID incur annual costs in terms of health costs, disability support, lost income and other social costs of US$172 000, accumulating to many millions of dollars over a lifetime. CONCLUSION: The diagnosis of monogenic disorders through genomic testing provides the opportunity to improve the diagnosis and management, and to reduce the costs of ID through informed reproductive decisions, reductions in unproductive diagnostic tests and increasingly targeted therapies.

Fetal diagnosis of Mowat-Wilson syndrome by whole exome sequencing.

Mowat-Wilson syndrome (MWS) is a complex genetic disorder associated with heterozygous variation in ZEB2. It is mainly characterized by moderate-to-severe intellectual disability, facial dysmorphism, epilepsy, and various malformations including Hirschsprung disease, corpus callosum anomalies, and congenital heart defects. It is rarely diagnosed prenatally and there is limited information available on the prenatal phenotype associated with MWS. Here we report the detection of a heterozygous de novo nonsense variant in ZEB2 by whole exome sequencing in a fetus with microphthalmia in addition to cardiac defects and typical MWS facial dysmorphism. As the prenatal phenotypic spectrum of MWS expands, the routine addition of fetal genomic testing particularly in the presence of multiple malformations will increase both the sensitivity and specificity of prenatal diagnostics.

Characterisation of a pucBA deletion mutant from Rhodopseudomonas palustris lacking all but the pucBAd genes.

Rhodopseudomonas palustris is a species of purple photosynthetic bacteria that has a multigene family of puc genes that encode the alpha and beta apoproteins, which form the LH2 complexes. A genetic dissection strategy has been adopted in order to try and understand which spectroscopic form of LH2 these different genes produce. This paper presents a characterisation of one of the deletion mutants generated in this program, the pucBAd only mutant. This mutant produces an unusual spectroscopic form of LH2 that only has a single large NIR absorption band at 800 nm. Spectroscopic and pigment analyses on this complex suggest that it has basically a similar overall structure as that of the wild-type HL LH2 complex. The mutant has the unique phenotype where the mutant LH2 complex is only produced when cells are grown at LL. At HL the mutant only produces the LH1-RC core complex.

Elemental distribution in developing rice grains and the effect of flag-leaf arsenate exposure.

We measured the bulk grain concentrations of arsenic (As), along with rubidium (Rb) and strontium (Sr) as indicators of phloem and xylem transport respectively, in rice (Oryza sativa cv. Italica Carolina) pulsed with arsenate at two exposure levels for 5 day periods at progressively later stages of grain fill, between anthesis and maturity, through the cut flag leaf. We compared these to unexposed (negative) controls and positive controls; pulsed with dimethylarsinic acid (DMA). We collected elemental maps of As and micronutrient elements (Fe, Zn, Mn, Cu and Ni) from developing grains of rice. Exposures were either 25 or 100 µg/ml arsenate (As(V)) at various stages of grain development, compared to 25 µg/ml dimethylarsinic acid (DMA); the most efficiently transported As species identified in rice. We used the spatial distribution of arsenic in the grain to infer the presence of As transporters. By exposing grains through the flag leaf rather than via the roots, we were able to measure arsenic transport into the grain during filling under controlled conditions. Exposure to 100 µg/ml As(V) resulted in widespread As localization in both embryo and endosperm, especially in grains exposed to As at later stages of panicle development. This suggests loss of selective transport, likely to be the result of As toxicity. At 25 µg/ml As(V), As colocalized with Mn in the ovular vascular trace (OVT). Exposure to either As(V) or DMA reduced grain Fe, an effect more pronounced when exposure occurred earlier in grain development. The abundance of Cu and Zn were also reduced by As. Arsenic exposure later in grain development caused higher grain As concentrations, indicating the existence of As transporters whose efficiency increases during grain fill. We conclude that localization of As in the grain is a product of both As species and exposure concentration, and that high As(V) translocation from the flag leaf can result in high As concentrations in the endosperm.

Elementary Energy Transfer Pathways in Allochromatium vinosum Photosynthetic Membranes.

Allochromatium vinosum (formerly Chromatium vinosum) purple bacteria are known to adapt their light-harvesting strategy during growth according to environmental factors such as temperature and average light intensity. Under low light illumination or low ambient temperature conditions, most of the LH2 complexes in the photosynthetic membranes form a B820 exciton with reduced spectral overlap with LH1. To elucidate the reason for this light and temperature adaptation of the LH2 electronic structure, we performed broadband femtosecond transient absorption spectroscopy as a function of excitation wavelength in A. vinosum membranes. A target analysis of the acquired data yielded individual rate constants for all relevant elementary energy transfer (ET) processes. We found that the ET dynamics in high-light-grown membranes was well described by a homogeneous model, with forward and backward rate constants independent of the pump wavelength. Thus, the overall B800→B850→B890→ Reaction Center ET cascade is well described by simple triexponential kinetics. In the low-light-grown membranes, we found that the elementary backward transfer rate constant from B890 to B820 was strongly reduced compared with the corresponding constant from B890 to B850 in high-light-grown samples. The ET dynamics of low-light-grown membranes was strongly dependent on the pump wavelength, clearly showing that the excitation memory is not lost throughout the exciton lifetime. The observed pump energy dependence of the forward and backward ET rate constants suggests exciton diffusion via B850→ B850 transfer steps, making the overall ET dynamics nonexponential. Our results show that disorder plays a crucial role in our understanding of low-light adaptation in A. vinosum.

Characterisation of the LH2 spectral variants produced by the photosynthetic purple sulphur bacterium Allochromatium vinosum.

This study systematically investigated the different types of LH2 produced by Allochromatium (Alc.) vinosum, a photosynthetic purple sulphur bacterium, in response to variations in growth conditions. Three different spectral forms of LH2 were isolated and purified, the B800-820, B800-840 and B800-850 LH2 types, all of which exhibit an unusual split 800 peak in their low temperature absorption spectra. However, it is likely that more forms are also present. Relatively more B800-820 and B800-840 are produced under low light conditions, while relatively more B800-850 is produced under high light conditions. Polypeptide compositions of the three different LH2 types were determined by a combination of HPLC and TOF/MS. The B800-820, B800-840 and B800-850 LH2 types all have a heterogeneous polypeptide composition, containing multiple types of both α and ß polypeptides, and differ in their precise polypeptide composition. They all have a mixed carotenoid composition, containing carotenoids of the spirilloxanthin series. In all cases the most abundant carotenoid is rhodopin; however, there is a shift towards carotenoids with a higher conjugation number in LH2 complexes produced under low light conditions. CD spectroscopy, together with the polypeptide analysis, demonstrates that these Alc. vinosum LH2 complexes are more closely related to the LH2 complex from Phs. molischianum than they are to the LH2 complexes from Rps. acidophila.

Antiviral Efficacy of a Respiratory Syncytial Virus (RSV) Fusion Inhibitor in a Bovine Model of RSV Infection.

Respiratory syncytial virus (RSV) is the leading cause of bronchiolitis and pneumonia in infants. Effective treatment for RSV infection is a significant unmet medical need. While new RSV therapeutics are now in development, there are very few animal models that mimic the pathogenesis of human RSV, making it difficult to evaluate new disease interventions. Experimental infection of Holstein calves with bovine RSV (bRSV) causes a severe respiratory infection that is similar to human RSV infection, providing a relevant model for testing novel therapeutic agents. In this model, viral load is readily detected in nasal secretions by quantitative real-time PCR (qRT-PCR), and cumulative symptom scoring together with histopathology evaluations of infected tissue allow for the assessment of disease severity. The bovine RSV model was used to evaluate the antiviral activity of an RSV fusion inhibitor, GS1, which blocks virus entry by inhibiting the fusion of the viral envelope with the host cell membrane. The efficacy of GS1, a close structural analog of GS-5806 that is being developed to treat RSV infection in humans was evaluated in two randomized, blind, placebo-controlled studies in bRSV-infected calves. Intravenous administration of GS1 at 4 mg/kg of body weight/day for 7 days starting 24 h or 72 h postinoculation provided clear therapeutic benefit by reducing the viral load, disease symptom score, respiration rate, and lung pathology associated with bRSV infection. These data support the use of the bovine RSV model for evaluation of experimental therapeutics for treatment of RSV.

The origin of the split B800 absorption peak in the LH2 complexes from Allochromatium vinosum.

The absorption spectrum of the high-light peripheral light-harvesting (LH) complex from the photosynthetic purple bacterium Allochromatium vinosum features two strong absorptions around 800 and 850 nm. For the LH2 complexes from the species Rhodopseudomonas acidophila and Rhodospirillum molischianum, where high-resolution X-ray structures are available, similar bands have been observed and were assigned to two pigment pools of BChl a molecules that are arranged in two concentric rings (B800 and B850) with nine (acidophila) or eight (molischianum) repeat units, respectively. However, for the high-light peripheral LH complex from Alc. vinosum, the intruiging feature is that the B800 band is split into two components. We have studied this pigment-protein complex by ensemble CD spectroscopy and polarisation-resolved single-molecule spectroscopy. Assuming that the high-light peripheral LH complex in Alc. vinosum is constructed on the same modular principle as described for LH2 from Rps. acidophila and Rsp. molischianum, we used those repeat units as a starting point for simulating the spectra. We find the best agreement between simulation and experiment for a ring-like oligomer of 12 repeat units, where the mutual arrangement of the B800 and B850 rings resembles those from Rsp. molischianum. The splitting of the B800 band can be reproduced if both an excitonic coupling between dimers of B800 molecules and their interaction with the B850 manifold are taken into account. Such dimers predict an interesting apoprotein organisation as discussed below.

Ultra-broadband 2D electronic spectroscopy of carotenoid-bacteriochlorophyll interactions in the LH1 complex of a purple bacterium.

We investigate the excitation energy transfer (EET) pathways in the photosynthetic light harvesting 1 (LH1) complex of purple bacterium Rhodospirillum rubrum with ultra-broadband two-dimensional electronic spectroscopy (2DES). We employ a 2DES apparatus in the partially collinear geometry, using a passive birefringent interferometer to generate the phase-locked pump pulse pair. This scheme easily lends itself to two-color operation, by coupling a sub-10 fs visible pulse with a sub-15-fs near-infrared pulse. This unique pulse combination allows us to simultaneously track with extremely high temporal resolution both the dynamics of the photoexcited carotenoid spirilloxanthin (Spx) in the visible range and the EET between the Spx and the B890 bacterio-chlorophyll (BChl), whose Qx and Qy transitions peak at 585 and 881 nm, respectively, in the near-infrared. Global analysis of the one-color and two-color 2DES maps unravels different relaxation mechanisms in the LH1 complex: (i) the initial events of the internal conversion process within the Spx, (ii) the parallel EET from the first bright state S2 of the Spx towards the Qx state of the B890, and (iii) the internal conversion from Qx to Qy within the B890.

Inhibition of hepatitis C virus replication by GS-6620, a potent C-nucleoside monophosphate prodrug.

As a class, nucleotide inhibitors (NIs) of the hepatitis C virus (HCV) nonstructural protein 5B (NS5B) RNA-dependent RNA polymerase offer advantages over other direct-acting antivirals, including properties, such as pangenotype activity, a high barrier to resistance, and reduced potential for drug-drug interactions. We studied the in vitro pharmacology of a novel C-nucleoside adenosine analog monophosphate prodrug, GS-6620. It was found to be a potent and selective HCV inhibitor against HCV replicons of genotypes 1 to 6 and against an infectious genotype 2a virus (50% effective concentration [EC50], 0.048 to 0.68 µM). GS-6620 showed limited activities against other viruses, maintaining only some of its activity against the closely related bovine viral diarrhea virus (EC50, 1.5 µM). The active 5'-triphosphate metabolite of GS-6620 is a chain terminator of viral RNA synthesis and a competitive inhibitor of NS5B-catalyzed ATP incorporation, with Ki/Km values of 0.23 and 0.18 for HCV NS5B genotypes 1b and 2a, respectively. With its unique dual substitutions of 1'-CN and 2'-C-Me on the ribose ring, the active triphosphate metabolite was found to have enhanced selectivity for the HCV NS5B polymerase over host RNA polymerases. GS-6620 demonstrated a high barrier to resistance in vitro. Prolonged passaging resulted in the selection of the S282T mutation in NS5B that was found to be resistant in both cellular and enzymatic assays (>30-fold). Consistent with its in vitro profile, GS-6620 exhibited the potential for potent anti-HCV activity in a proof-of-concept clinical trial, but its utility was limited by the requirement of high dose levels and pharmacokinetic and pharmacodynamic variability.

De novo variants in the non-coding spliceosomal snRNA gene RNU4-2 are a frequent cause of syndromic neurodevelopmental disorders.

Around 60% of individuals with neurodevelopmental disorders (NDD) remain undiagnosed after comprehensive genetic testing, primarily of protein-coding genes1. Increasingly, large genome-sequenced cohorts are improving our ability to discover new diagnoses in the non-coding genome. Here, we identify the non-coding RNA RNU4-2 as a novel syndromic NDD gene. RNU4-2 encodes the U4 small nuclear RNA (snRNA), which is a critical component of the U4/U6.U5 tri-snRNP complex of the major spliceosome2. We identify an 18 bp region of RNU4-2 mapping to two structural elements in the U4/U6 snRNA duplex (the T-loop and Stem III) that is severely depleted of variation in the general population, but in which we identify heterozygous variants in 119 individuals with NDD. The vast majority of individuals (77.3%) have the same highly recurrent single base-pair insertion (n.64_65insT). We estimate that variants in this region explain 0.41% of individuals with NDD. We demonstrate that RNU4-2 is highly expressed in the developing human brain, in contrast to its contiguous counterpart RNU4-1 and other U4 homologs, supporting RNU4-2's role as the primary U4 transcript in the brain. Overall, this work underscores the importance of non-coding genes in rare disorders. It will provide a diagnosis to thousands of individuals with NDD worldwide and pave the way for the development of effective treatments for these individuals.

Carbonate weathering, phosphate fertilizer, and hydrologic controls on dissolved uranium in rivers in the US Corn Belt: Disentangling seasonal geogenic- and fertilizer-derived sources.

Soil and bedrock weathering and phosphate (P) fertilizers may both contribute to the uranium (U) load of rivers in agricultural regions, but controls over their relative influence are not well known. This study investigates the U sources to rivers in Ohio, United States, part of the Eastern Corn Belt in the Mississippi River watershed. We present a regional picture of seasonal U sources to rivers based on four analyses: 1) a spatial analysis of legacy soil and water data, 2) new measurements of U and carbonate weathering products from rivers at 50 locations across the state collected seasonally over two years, 3) a weekly time series with additional 234U/238U (n = 5) and 87Sr/86Sr (n = 5) measurements from an agricultural river, and 4) a mass-balance approach to U addition to the landscape based on reported P fertilizer use. Uranium concentrations in surface waters collected statewide ranged 0.1-21 nM (n = 132), with significantly higher concentrations in the glaciated agricultural portion of the state (mean = 7.3 nM; n = 105) than the non-glaciated portion (mean = 2.0 nM; n = 24). Concentrations in the glaciated region were highest during the spring and summer and decreased during baseflow. In the time-series, concentrations were ~7 nM during baseflow and ~14 nM during intermediate seasonal discharge conditions, indicating a second more surficial endmember source of U in addition to bedrock weathering that is well correlated with other carbonate weathering products. Systematic increases in 87Sr/86Sr and decreases in 234U/238U with increasing discharge confirm a changing source of carbonate and U weathering and a third surficial endmember during high discharge events. Our mass balance approach and geochemical analysis suggest that elevated U concentrations are the result of carbonate weathering deep in the soil column during elevated seasonal flow. Further work on U dynamics in agricultural rivers is required to understand mechanism controlling seasonal changes in U concentrations and 234U/238U in downstream rivers and U flux.

Grain accumulation of selenium species in rice (Oryza sativa L.).

Efficient Se biofortification programs require a thorough understanding of the accumulation and distribution of Se species within the rice grain. Therefore, the translocation of Se species to the filling grain and their spatial unloading were investigated. Se species were supplied via cut flag leaves of intact plants and excised panicle stems subjected to a ± stem-girdling treatment during grain fill. Total Se concentrations in the flag leaves and grain were quantified by inductively coupled plasma mass spectrometry. Spatial accumulation was investigated using synchrotron X-ray fluorescence microtomography. Selenomethionine (SeMet) and selenomethylcysteine (SeMeSeCys) were transported to the grain more efficiently than selenite and selenate. SeMet and SeMeSeCys were translocated exclusively via the phloem, while inorganic Se was transported via both the phloem and xylem. For SeMet- and SeMeSeCys-fed grain, Se dispersed throughout the external grain layers and into the endosperm and, for SeMeSeCys, into the embryo. Selenite was retained at the point of grain entry. These results demonstrate that the organic Se species SeMet and SeMeSeCys are rapidly loaded into the phloem and transported to the grain far more efficiently than inorganic species. Organic Se species are distributed more readily, and extensively, throughout the grain than selenite.

A review of recent developments in the speciation and location of arsenic and selenium in rice grain.

Rice is a staple food yet is a significant dietary source of inorganic arsenic, a class 1, nonthreshold carcinogen. Establishing the location and speciation of arsenic within the edible rice grain is essential for understanding the risk and for developing effective strategies to reduce grain arsenic concentrations. Conversely, selenium is an essential micronutrient and up to 1 billion people worldwide are selenium-deficient. Several studies have suggested that selenium supplementation can reduce the risk of some cancers, generating substantial interest in biofortifying rice. Knowledge of selenium location and speciation is important, because the anti-cancer effects of selenium depend on its speciation. Germanic acid is an arsenite/silicic acid analogue, and location of germanium may help elucidate the mechanisms of arsenite transport into grain. This review summarises recent discoveries in the location and speciation of arsenic, germanium, and selenium in rice grain using state-of-the-art mass spectrometry and synchrotron techniques, and illustrates both the importance of high-sensitivity and high-resolution techniques and the advantages of combining techniques in an integrated quantitative and spatial approach.