HOMEOBOX2, the paralog of SIX-ROWED SPIKE1/HOMEOBOX1, is dispensable for barley spikelet development.

The HD-ZIP class I transcription factor Homeobox 1 (HvHOX1), also known as Vulgare Row-type Spike 1 (VRS1) or Six-rowed Spike 1, regulates lateral spikelet fertility in barley (Hordeum vulgare L.). It was shown that HvHOX1 has a high expression only in lateral spikelets, while its paralog HvHOX2 was found to be expressed in different plant organs. Yet, the mechanistic functions of HvHOX1 and HvHOX2 during spikelet development are still fragmentary. Here, we show that compared with HvHOX1, HvHOX2 is more highly conserved across different barley genotypes and Hordeum species, hinting at a possibly vital but still unclarified biological role. Using bimolecular fluorescence complementation, DNA-binding, and transactivation assays, we validate that HvHOX1 and HvHOX2 are bona fide transcriptional activators that may potentially heterodimerize. Accordingly, both genes exhibit similar spatiotemporal expression patterns during spike development and growth, albeit their mRNA levels differ quantitatively. We show that HvHOX1 delays the lateral spikelet meristem differentiation and affects fertility by aborting the reproductive organs. Interestingly, the ancestral relationship of the two genes inferred from their co-expressed gene networks suggested that HvHOX1 and HvHOX2 might play a similar role during barley spikelet development. However, CRISPR-derived mutants of HvHOX1 and HvHOX2 demonstrated the suppressive role of HvHOX1 on lateral spikelets, while the loss of HvHOX2 does not influence spikelet development. Collectively, our study shows that through the suppression of reproductive organs, lateral spikelet fertility is regulated by HvHOX1, whereas HvHOX2 is dispensable for spikelet development in barley.

INTERMEDIUM-C mediates the shade-induced bud growth arrest in barley.

Tiller formation is a key agronomic determinant for grain yield in cereal crops. The modulation of this trait is controlled by transcriptional regulators and plant hormones, tightly regulated by external environmental conditions. While endogenous (genetic) and exogenous (environmental factors) triggers for tiller formation have mostly been investigated separately, it has remained elusive how they are integrated into the developmental program of this trait. The transcription factor gene INTERMEDIUM-C (INT-C), which is the barley ortholog of the maize domestication gene TEOSINTE BRANCHED1 (TB1), has a prominent role in regulating tiller bud outgrowth. Here we show that INT-C is expressed in tiller buds, required for bud growth arrest in response to shade. In contrast to wild-type plants, int-c mutant plants are impaired in their shade response and do not stop tiller production after shading. Gene expression levels of INT-C are up-regulated under light-limiting growth conditions, and down-regulated after decapitation. Transcriptome analysis of wild-type and int-c buds under control and shading conditions identified target genes of INT-C that belong to auxin and gibberellin biosynthesis and signaling pathways. Our study identifies INT-C as an integrator of the shade response into tiller formation, which is prerequisite for implementing shading responses in the breeding of cereal crops.

Identification of QTL hot spots for malting quality in two elite breeding lines with distinct tolerance to abiotic stress.

BACKGROUND: Barley (Hordeum vulgare) is an important crop cultivated across the world. Drought is a major abiotic factor compromising barley yield worldwide, therefore in modern spring barley cultivars superior seed and malting quality characteristics should be combined with reasonable level of drought tolerance. Previously we have identified a number of barley lines demonstrating the superior yield performance under drought conditions. The aim of this work was to perform a QTL analysis of malting quality traits in a doubled haploid (DH) mapping population of two elite barley lines that differ in their reaction pattern to drought stress. RESULTS: A population of DH lines was developed by crossing two drought-tolerant elite breeding lines, Victoriana and Sofiara, exploiting distinct mechanism of drought tolerance, sustaining assimilation vs remobilization. The mapping population was assayed under field conditions at four distinct locations that differed in precipitation rate. DH lines were genotyped with the Illumina 9 K iSelect assay, and linkage map including 1782 polymorphic markers and covering a total map length of 1140 cM was constructed. The result of quantitative trait loci (QTL) analysis showed that majority of the traits were affected by several main effect QTL and/or QTL x environment (QE) interactions. In total, 57, 41, and 5 QTL were associated with yield-related traits, malting quality traits and seed quality traits, respectively. 11 and 29 of mapped QTL explained more than 10 and 5% of phenotypic variation, respectively. In several chromosomal regions co-localization between QTL for various traits were observed. The largest clusters were detected on chromosomes 3H and 4H. CONCLUSIONS: Our QTL mapping results revealed several novel consistent genomic regions controlling malting quality which could be exploited in marker assisted selection. In this context, the complex QTL region on chromosome 3H seems of particular interest, as it harbors several large effect QTL.

Abscisic acid influences tillering by modulation of strigolactones in barley.

Strigolactones (SLs) represent a class of plant hormones that are involved in inhibiting shoot branching and in promoting abiotic stress responses. There is evidence that the biosynthetic pathways of SLs and abscisic acid (ABA) are functionally connected. However, little is known about the mechanisms underlying the interaction of SLs and ABA, and the relevance of this interaction for shoot architecture. Based on sequence homology, four genes (HvD27, HvMAX1, HvCCD7, and HvCCD8) involved in SL biosynthesis were identified in barley and functionally verified by complementation of Arabidopsis mutants or by virus-induced gene silencing. To investigate the influence of ABA on SLs, two transgenic lines accumulating ABA as a result of RNAi-mediated down-regulation of HvABA 8'-hydroxylase 1 and 3 were employed. LC-MS/MS analysis confirmed higher ABA levels in root and stem base tissues in these transgenic lines. Both lines showed enhanced tiller formation and lower concentrations of 5-deoxystrigol in root exudates, which was detected for the first time as a naturally occurring SL in barley. Lower expression levels of HvD27, HvMAX1, HvCCD7, and HvCCD8 indicated that ABA suppresses SL biosynthesis, leading to enhanced tiller formation in barley.

Six-rowed spike4 (Vrs4) controls spikelet determinacy and row-type in barley.

Inflorescence architecture of barley (Hordeum vulgare L.) is common among the Triticeae species, which bear one to three single-flowered spikelets at each rachis internode. Triple spikelet meristem is one of the unique features of barley spikes, in which three spikelets (one central and two lateral spikelets) are produced at each rachis internode. Fertility of the lateral spikelets at triple spikelet meristem gives row-type identity to barley spikes. Six-rowed spikes show fertile lateral spikelets and produce increased grain yield per spike, compared with two-rowed spikes with sterile lateral spikelets. Thus, far, two loci governing the row-type phenotype were isolated in barley that include Six-rowed spike1 (Vrs1) and Intermedium-C. In the present study, we isolated Six-rowed spike4 (Vrs4), a barley ortholog of the maize (Zea mays L.) inflorescence architecture gene RAMOSA2 (RA2). Eighteen coding mutations in barley RA2 (HvRA2) were specifically associated with lateral spikelet fertility and loss of spikelet determinacy. Expression analyses through mRNA in situ hybridization and microarray showed that Vrs4 (HvRA2) controls the row-type pathway through Vrs1 (HvHox1), a negative regulator of lateral spikelet fertility in barley. Moreover, Vrs4 may also regulate transcripts of barley SISTER OF RAMOSA3 (HvSRA), a putative trehalose-6-phosphate phosphatase involved in trehalose-6-phosphate homeostasis implicated to control spikelet determinacy. Our expression data illustrated that, although RA2 is conserved among different grass species, its down-stream target genes appear to be modified in barley and possibly other species of tribe Triticeae.

Abscisic acid flux alterations result in differential abscisic acid signaling responses and impact assimilation efficiency in barley under terminal drought stress.

Abscisic acid (ABA) is a central player in plant responses to drought stress. How variable levels of ABA under short-term versus long-term drought stress impact assimilation and growth in crops is unclear. We addressed this through comparative analysis, using two elite breeding lines of barley (Hordeum vulgare) that show senescence or stay-green phenotype under terminal drought stress and by making use of transgenic barley lines that express Arabidopsis (Arabidopsis thaliana) 9-cis-epoxycarotenoid dioxygenase (AtNCED6) coding sequence or an RNA interference (RNAi) sequence of ABA 8'-hydroxylase under the control of a drought-inducible barley promoter. The high levels of ABA and its catabolites in the senescing breeding line under long-term stress were detrimental for assimilate productivity, whereas these levels were not perturbed in the stay-green type that performed better. In transgenic barley, drought-inducible AtNCED expression afforded temporal control in ABA levels such that the ABA levels rose sooner than in wild-type plants but also subsided, unlike as in the wild type , to near-basal levels upon prolonged stress treatment due to down-regulation of endogenous HvNCED genes. Suppressing of ABA catabolism with the RNA interference approach of ABA 8'-hydroxylase caused ABA flux during the entire period of stress. These transgenic plants performed better than the wild type under stress to maintain a favorable instantaneous water use efficiency and better assimilation. Gene expression analysis, protein structural modeling, and protein-protein interaction analyses of the members of the PYRABACTIN RESISTANCE1/PYRABACTIN RESISTANCE1-LIKE/REGULATORY COMPONENT OF ABA RECEPTORS, TYPE 2C PROTEIN PHOSPHATASE Sucrose non-fermenting1-related protein kinase2, and ABA-INSENSITIVE5/ABA-responsive element binding factor family identified specific members that could potentially impact ABA metabolism and stress adaptation in barley.

Integrated phenotyping of root and shoot growth dynamics in maize reveals specific interaction patterns in inbreds and hybrids and in response to drought.

In recent years, various automated methods for plant phenotyping addressing roots or shoots have been developed and corresponding platforms have been established to meet the diverse requirements of plant research and breeding. However, most platforms are only either able to phenotype shoots or roots of plants but not both simultaneously. This substantially limits the opportunities offered by a joint assessment of the growth and development dynamics of both organ systems, which are highly interdependent. In order to overcome these limitations, a root phenotyping installation was integrated into an existing automated non-invasive high-throughput shoot phenotyping platform. Thus, the amended platform is now capable of conducting high-throughput phenotyping at the whole-plant level, and it was used to assess the vegetative root and shoot growth dynamics of five maize inbred lines and four hybrids thereof, as well as the responses of five inbred lines to progressive drought stress. The results showed that hybrid vigour (heterosis) occurred simultaneously in roots and shoots and was detectable as early as 4 days after transplanting (4 DAT; i.e., 8 days after seed imbibition) for estimated plant height (EPH), total root length (TRL), and total root volume (TRV). On the other hand, growth dynamics responses to progressive drought were different in roots and shoots. While TRV was significantly reduced 10 days after the onset of the water deficit treatment, the estimated shoot biovolume was significantly reduced about 6 days later, and EPH showed a significant decrease even 2 days later (8 days later than TRV) compared with the control treatment. In contrast to TRV, TRL initially increased in the water deficit period and decreased much later (not earlier than 16 days after the start of the water deficit treatment) compared with the well-watered plants. This may indicate an initial response of the plants to water deficit by forming longer but thinner roots before growth was inhibited by the overall water deficit. The magnitude and the dynamics of the responses were genotype-dependent, as well as under the influence of the water consumption, which was related to plant size.

ABA biosynthesis and degradation contributing to ABA homeostasis during barley seed development under control and terminal drought-stress conditions.

Drought is one of the most severe environmental stress factors limiting crop yield especially when occurring during anthesis and seed filling. This terminal drought is characterized by an excess production of the phytohormone abscisic acid (ABA) which plays an important role during seed development and dormancy. All the genes putatively involved in ABA biosynthesis and inactivation in barley were identified and their expression studied during plant ontogeny under standard and drought-stress conditions to learn more about ABA homeostasis and the possible mode of cross-talk between source and sink tissues. Out of 41 genes related to ABA biosynthesis and inactivation 19 were found to be differentially regulated under drought stress in both flag leaves and developing seed during seed filling. Transcripts of plastid-located enzymes are regulated similarly in flag leaf and seed under terminal drought whereas transcripts of cytosolic enzymes are differentially regulated in the two tissues. Detailed information on the expression of defined gene family members is supplemented by measurements of ABA and its degradation and conjugation products, respectively. Under drought stress, flag leaves in particular contain high concentrations of both ABA and the ABA degradation products phaseic acid (PA) and diphaseic acid (DPA); whereas, in seeds, besides ABA, DPA was mainly found. The measurements also revealed a positive correlation between ABA level and starch content in developing seeds for the following reasons: (i) genes of the ABA controlled SnRK2.6 and RCAR/PP2C-mediated signal transduction pathway to the ABF transcription factor HvABI5 are activated in the developing grain under drought, (ii) novel ABA- and dehydration-responsive cis-elements have been found in the promoters of key genes of starch biosynthesis (HvSUS1, HvAGP-L1) and degradation (HvBAM1) and these transcripts/activity are prominently induced in developing seeds during 12 and 16 DAF, (iii) spraying of fluridone (an ABA biosynthesis inhibitor) to drought-stressed plants results in severely impaired starch content and thousand grain weight of mature seeds.

Fully-automated root image analysis (faRIA).

High-throughput root phenotyping in the soil became an indispensable quantitative tool for the assessment of effects of climatic factors and molecular perturbation on plant root morphology, development and function. To efficiently analyse a large amount of structurally complex soil-root images advanced methods for automated image segmentation are required. Due to often unavoidable overlap between the intensity of fore- and background regions simple thresholding methods are, generally, not suitable for the segmentation of root regions. Higher-level cognitive models such as convolutional neural networks (CNN) provide capabilities for segmenting roots from heterogeneous and noisy background structures, however, they require a representative set of manually segmented (ground truth) images. Here, we present a GUI-based tool for fully automated quantitative analysis of root images using a pre-trained CNN model, which relies on an extension of the U-Net architecture. The developed CNN framework was designed to efficiently segment root structures of different size, shape and optical contrast using low budget hardware systems. The CNN model was trained on a set of 6465 masks derived from 182 manually segmented near-infrared (NIR) maize root images. Our experimental results show that the proposed approach achieves a Dice coefficient of 0.87 and outperforms existing tools (e.g., SegRoot) with Dice coefficient of 0.67 by application not only to NIR but also to other imaging modalities and plant species such as barley and arabidopsis soil-root images from LED-rhizotron and UV imaging systems, respectively. In summary, the developed software framework enables users to efficiently analyse soil-root images in an automated manner (i.e. without manual interaction with data and/or parameter tuning) providing quantitative plant scientists with a powerful analytical tool.

Delineating the structural, functional and evolutionary relationships of sucrose phosphate synthase gene family II in wheat and related grasses.

BACKGROUND: Sucrose phosphate synthase (SPS) is an important component of the plant sucrose biosynthesis pathway. In the monocotyledonous Poaceae, five SPS genes have been identified. Here we present a detailed analysis of the wheat SPSII family in wheat. A set of homoeologue-specific primers was developed in order to permit both the detection of sequence variation, and the dissection of the individual contribution of each homoeologue to the global expression of SPSII. RESULTS: The expression in bread wheat over the course of development of various sucrose biosynthesis genes monitored on an Affymetrix array showed that the SPS genes were regulated over time and space. SPSII homoeologue-specific assays were used to show that the three homoeologues contributed differentially to the global expression of SPSII. Genetic mapping placed the set of homoeoloci on the short arms of the homoeologous group 3 chromosomes. A resequencing of the A and B genome copies allowed the detection of four haplotypes at each locus. The 3B copy includes an unspliced intron. A comparison of the sequences of the wheat SPSII orthologues present in the diploid progenitors einkorn, goatgrass and Triticum speltoides, as well as in the more distantly related species barley, rice, sorghum and purple false brome demonstrated that intronic sequence was less well conserved than exonic. Comparative sequence and phylogenetic analysis of SPSII gene showed that false purple brome was more similar to Triticeae than to rice. Wheat - rice synteny was found to be perturbed at the SPS region. CONCLUSION: The homoeologue-specific assays will be suitable to derive associations between SPS functionality and key phenotypic traits. The amplicon sequences derived from the homoeologue-specific primers are informative regarding the evolution of SPSII in a polyploid context.

Obtaining High-Quality Transcriptome Data from Cereal Seeds by a Modified Method for Gene Expression Profiling.

The characterization of gene expression is dependent on RNA quality. In germinating, developing and mature cereal seeds, the extraction of high-quality RNA is often hindered by high starch and sugar content. These compounds can reduce both the yield and the quality of the extracted total RNA. The deterioration in quantity and quality of total RNA can subsequently have a significant impact on the downstream transcriptomic analyses, which may not accurately reflect the spatial and/or temporal variation in the gene expression profile of the samples being tested. In this protocol, we describe an optimized method for extraction of total RNA with sufficient quantity and quality to be used for whole transcriptome analysis of cereal grains. The described method is suitable for several downstream applications used for transcriptomic profiling of developing, germinating, and mature cereal seeds. The method of transcriptome profiling using a microarray platform is shown. This method is specifically designed for gene expression profiling of cereals with described genome sequences. The detailed procedure from microarray handling to final quality control is described. This includes cDNA synthesis, cRNA labelling, microarray hybridization, slide scanning, feature extraction, and data quality validation. The data generated by this method can be used to characterize the transcriptome of cereals during germination, in various stages of grain development, or at different biotic or abiotic stress conditions. The results presented here exemplify high-quality transcriptome data amenable for downstream bioinformatics analyses, such as the determination of differentially expressed genes (DEGs), characterisation of gene regulatory networks, and conducting transcriptome-wide association study (TWAS).

Quantification of DNA Methylation as Biomarker for Grain Quality.

DNA methylation is an important biomarker for gene activity. It contributes to gene silencing and is involved in regulating various seed developmental processes in plants. Many of these processes are involved in important traits associated with aspects of grain quality. A reliable, fast, and cheap method is the estimation of DNA methylation utilizing methylation sensitive restriction enzymes (MSRE) and quantitative real-time PCR (qPCR) for selected candidate regions. The presented method can be used to confirm an effect of RNAi constructs on their target genes or trans-activity. Analysis of promoter regions can contribute to estimation of gene activity and related traits.

Semi-automated Root Image Analysis (saRIA).

Quantitative characterization of root system architecture and its development is important for the assessment of a complete plant phenotype. To enable high-throughput phenotyping of plant roots efficient solutions for automated image analysis are required. Since plants naturally grow in an opaque soil environment, automated analysis of optically heterogeneous and noisy soil-root images represents a challenging task. Here, we present a user-friendly GUI-based tool for semi-automated analysis of soil-root images which allows to perform an efficient image segmentation using a combination of adaptive thresholding and morphological filtering and to derive various quantitative descriptors of the root system architecture including total length, local width, projection area, volume, spatial distribution and orientation. The results of our semi-automated root image segmentation are in good conformity with the reference ground-truth data (mean dice coefficient = 0.82) compared to IJ_Rhizo and GiAroots. Root biomass values calculated with our tool within a few seconds show a high correlation (Pearson coefficient = 0.8) with the results obtained using conventional, pure manual segmentation approaches. Equipped with a number of adjustable parameters and optional correction tools our software is capable of significantly accelerating quantitative analysis and phenotyping of soil-, agar- and washed root images.

Analysis of Developing Rice Grain Transcriptome Using the Agilent Microarray Platform.

Transcriptome analysis reflects the status quo of transcribed genetic code present in the form of mRNA, which helps to infer biological processes and unravel metabolic status. Despite the increasing adoption of RNA-Seq technique in recent years, transcriptome analysis using the microarray platform remains the gold standard technique, which offers a simpler, more cost-effective, and efficient method for high-throughput gene expression profiling. In this chapter, we described a streamlined transcriptomic analyses pipeline employed to study developing rice grains that can also be applied to other tissue samples and species. We described a novel RNA extraction method that obviates the problem introduced by high-starch content during rice grain development that usually leads to reduction in RNA yield and quality. The detailed procedure of microarray analysis involved in cDNA synthesis, cRNA labeling, microarray hybridization, slide scanning, feature extraction to QC validation has been described. The description of a newly developed Indica- and Japonica-specific microarray slides developed from the genome information of subpopulation to study gene expression of 60,000 genes has been highlighted. The downstream bioinformatics analyses including expression QTL mapping and gene regulatory network analyses were mentioned.

Phenotyping roots in darkness: disturbance-free root imaging with near infrared illumination.

Root systems architecture (RSA) and size properties are essential determinants of plant performance and need to be assessed in high-throughput plant phenotyping platforms. Thus, we tested a concept that involves near-infrared (NIR) imaging of roots growing along surfaces of transparent culture vessels using special long pass filters to block their exposure to visible light. Two setups were used to monitor growth of Arabidopsis, rapeseed, barley and maize roots upon exposure to white light, filter-transmitted radiation or darkness: root growth direction was analysed (1) through short-term cultivation on agar plates, and (2) using soil-filled transparent pots to monitor long-term responses. White light-triggered phototropic responses were detected for Arabidopsis in setup 1, and for rapeseed, barley and maize roots in setups 1 and 2, whereas light effects could be avoided by use of the NIR filter thus confirming its suitability to mimic darkness. NIR image-derived 'root volume' values correlated well with root dry weight. The root system fractions visible at the different pot sides and in different zones revealed species- and genotype-dependent variation of spatial root distribution and other RSA traits. Following this validated concept, root imaging setups may be integrated into shoot phenotyping facilities in order to enable root system analysis in the context of whole-plant performance investigations.

Investigating glycemic potential of rice by unraveling compositional variations in mature grain and starch mobilization patterns during seed germination.

Rice lines with slower starch digestibility provide opportunities in mitigating the global rise in type II diabetes and related non-communicable diseases. However, screening for low glycemic index (GI) in rice breeding programs is not possible due to time and cost constraints. This study evaluated the feasibility of using in vitro cooked grain amylolysis, starch mobilization patterns during seed germination, and variation in starch structure and composition in the mature seed to differentiate patterns of starch digestibility. Mobilization patterns of total starch, resistant starch, amylose and amylopectin chains, and free sugars during seed germination revealed that the process is analogous to digestion in the human gastrointestinal tract. The combination of these biochemical markers can be used as an alternative measure to predict GI. Additionally, transcriptome analysis of stored mRNA transcripts in high and low GI lines detected differences in starch metabolism and confirmed the importance of seed storage pathways in influencing digestibility. Pathway analyses supported by metabolomics data revealed that resistant starch, cell wall non-starch polysaccharides and flavonoids potentially contribute to slower digestibility. These new insights can guide precision breeding programs to produce low GI rice with acceptable cooking quality to help mitigate the burden of diet-associated lifestyle diseases.

A Potential Role of Flag Leaf Potassium in Conferring Tolerance to Drought-Induced Leaf Senescence in Barley.

Terminal drought stress decreases crop yields by inducing abscisic acid (ABA) and premature leaf senescence. As potassium (K) is known to interfere with ABA homeostasis we addressed the question whether there is genetic variability regarding the role of K nutrition in ABA homeostasis and drought tolerance. To compare their response to drought stress, two barley lines contrasting in drought-induced leaf senescence were grown in a pot experiment under high and low K supply for the analysis of flag leaves from the same developmental stage. Relative to the drought-sensitive line LPR, the line HPR retained more K in its flag leaves under low K supply and showed delayed flag leaf senescence under terminal drought stress. High K retention was further associated with a higher leaf water status, a higher concentration of starch and other primary carbon metabolites. With regard to ABA homeostasis, HPR accumulated less ABA but higher levels of the ABA degradation products phaseic acid (PA) and dehydro-PA. Under K deficiency this went along with higher transcript levels of ABA8'-HYDROXYLASE, encoding a key enzyme in ABA degradation. The present study provides evidence for a positive impact of the K nutritional status on ABA homeostasis and carbohydrate metabolism under drought stress. We conclude that genotypes with a high K nutritional status in the flag leaf show superior drought tolerance by promoting ABA degradation but attenuating starch degradation which delays flag leaf senescence. Flag leaf K levels may thus represent a useful trait for the selection of drought-tolerant barley cultivars.

Aberrant acinar cell CA 19-9 expression and peri-insular acinar cell alterations in an adult human pancreas.

We herein report on a 23-year-old female patient who suffered from Crohn's disease and anorexia nervosa and died after long-term malnutrition and a perforated colitis. At autopsy, her pancreas displayed two peculiar findings. First, there was a constant and aberrant expression of CA 19-9 in the acinar cells. The expression of the carbohydrate antigen CA 19-9 is normally confined to duct-like epithelia, including centroacinar cells, while islet and acinar cells have repeatedly been reported to be immunohistochemically negative. Thus, to the best of our knowledge, our case is the first to show aberrant acinar CA 19-9 expression, and its potential meaning is discussed. Second, the pancreas showed a heterogeneity in acinar morphology, with peri-insular acini being considerably larger than tele-insular acini. The existence of enlarged peri-insular acini, mainly in animals, has occasionally been reported. Its origin, however, is still unclear. Some authors have proposed an influence of high insulin concentrations, exerted via the insulo-acinar capillary axis. We agree with the concept of an islet-derived mechanism. However, as we have observed a similar heterogeneity in streptozotocin- and autoimmune-diabetic rats, we presume that other islet hormones, in particular glucagon, might be more important for this phenomenon in the animals, as well as in the present case.

Gastric adenocarcinoma with leptomeningeal carcinomatosis as the presenting manifestation: an autopsy case report.

Leptomeningeal carcinomatosis is a clinically important and severe complication in patients with cancer. Leptomeningeal involvement as a secondary event in gastric carcinoma is rarely reported and usually occurs late in advanced disease. Herein, we report a case of leptomeningeal carcinomatosis as the initial manifestation of a previously asymptomatic gastric adenocarcinoma. The clinical features and the appropriate diagnostic procedures are discussed.