UNITY: A low-field magnetic resonance neuroimaging initiative to characterize neurodevelopment in low and middle-income settings.

Measures of physical growth, such as weight and height have long been the predominant outcomes for monitoring child health and evaluating interventional outcomes in public health studies, including those that may impact neurodevelopment. While physical growth generally reflects overall health and nutritional status, it lacks sensitivity and specificity to brain growth and developing cognitive skills and abilities. Psychometric tools, e.g., the Bayley Scales of Infant and Toddler Development, may afford more direct assessment of cognitive development but they require language translation, cultural adaptation, and population norming. Further, they are not always reliable predictors of future outcomes when assessed within the first 12-18 months of a child's life. Neuroimaging may provide more objective, sensitive, and predictive measures of neurodevelopment but tools such as magnetic resonance (MR) imaging are not readily available in many low and middle-income countries (LMICs). MRI systems that operate at lower magnetic fields (< 100mT) may offer increased accessibility, but their use for global health studies remains nascent. The UNITY project is envisaged as a global partnership to advance neuroimaging in global health studies. Here we describe the UNITY project, its goals, methods, operating procedures, and expected outcomes in characterizing neurodevelopment in sub-Saharan Africa and South Asia.

Background levels of neomorphic 2-hydroxyglutarate facilitate proliferation of primary fibroblasts.

Each cell types or tissues contain certain "physiological" levels of R-2-hydroxyglutarate (2HG), as well as enzymes for its synthesis and degradation. 2HG accumulates in certain tumors, possessing heterozygous point mutations of isocitrate dehydrogenases IDH1 (cytosolic) or IDH2 (mitochondrial) and contributes to strengthening their malignancy by inhibiting 2-oxoglutarate-dependent dioxygenases. By blocking histone de-methylation and 5-methyl-cytosine hydroxylation, 2HG maintains cancer cells de-differentiated and promotes their proliferation. However, physiological 2HG formation and formation by non-mutant IDH1/2 in cancer cells were neglected. Consequently, low levels of 2HG might play certain physiological roles. We aimed to elucidate this issue and found that compared to highest 2HG levels in hepatocellular carcinoma HepG2 cells and moderate levels in neuroblastoma SH-SY5Y cells, rat primary fibroblast contained low basal 2HG levels at early passages. These levels increased at late passage and likewise 2HG/2OG ratios dropped without growth factors and enormously increased at hypoxia, reaching levels compared to cancer HepG2 cells. Responses in SH-SY5Y cells were opposite. Moreover, external 2HG supplementation enhanced fibroblast growth. Hence, we conclude that low 2HG levels facilitate cell proliferation in primary fibroblasts, acting via hypoxia-induced factor regulations and epigenetic changes.

Evolutionary origins of the blood vascular system and endothelium.

Every biological trait requires both a proximate and evolutionary explanation. The field of vascular biology is focused primarily on proximate mechanisms in health and disease. Comparatively little attention has been given to the evolutionary basis of the cardiovascular system. Here, we employ a comparative approach to review the phylogenetic history of the blood vascular system and endothelium. In addition to drawing on the published literature, we provide primary ultrastructural data related to the lobster, earthworm, amphioxus, and hagfish. Existing evidence suggests that the blood vascular system first appeared in an ancestor of the triploblasts over 600 million years ago, as a means to overcome the time-distance constraints of diffusion. The endothelium evolved in an ancestral vertebrate some 540-510 million years ago to optimize flow dynamics and barrier function, and/or to localize immune and coagulation functions. Finally, we emphasize that endothelial heterogeneity evolved as a core feature of the endothelium from the outset, reflecting its role in meeting the diverse needs of body tissues.

Methylenetetrahydrofolate reductase genetic polymorphisms and toxicity to 5-FU-based chemoradiation in rectal cancer.

BACKGROUND: There is a large degree of variation in tumour response and host toxicities associated with neoadjuvant chemoradiation for rectal cancer patients. We performed a complimentary pharmacogenetic study to investigate germline polymorphisms of genes involved in 5-fluorouracil (5-FU) and irinotecan pathways and their potential association with clinical outcomes and toxicities from neoadjuvant chemoradiation in patients with rectal cancer treated in a prospective genotype-directed study. METHODS: The germline DNA of 131 patients was genotyped for 10 variants in TYMS, MTHFR, DPYD, UGT1A1, ABCC1 and SLCO1B1 genes. Ninety-six patients were treated with 5-FU/radiotherapy (RT) and 35 received 5-FU/RT/irinotecan. Relationships between genetic variants and adverse events, tumour response, overall and disease-free survivals were assessed. RESULTS: MTHFR 1298A>C and MTHFR diplotypes (for 677C>T and 1298A>C) were associated with chemoradiation-related toxicity when 5-FU was used alone. MTHFR haplotypes (677C-1298C) and diplotypes (CA-TA and TA-TA) showed, respectively, a protective and a negative effect on the incidence of severe diarrhoea or mucositis. No association was observed between genetic markers and drug response. CONCLUSION: MTHFR polymorphisms can potentially predict toxicity in patients treated with 5-FU as a single chemotherapeutic drug.

Why are tumour blood vessels abnormal and why is it important to know?

Tumour blood vessels differ from their normal counterparts for reasons that have received little attention. We report here that they are of at least six distinct types, we describe how each forms, and, looking forward, encourage the targeting of tumour vessel subsets that have lost their vascular endothelial growth factor-A (VEGF-A) dependency and so are likely unresponsive to anti-VEGF-A therapies.

Gender-specific differences in alcoholism: implications for treatment.

Alcohol abuse and alcoholism cut across gender, race and nationality. In general, more men than women are alcohol dependent or have alcohol problems, but women are at greater risk for adverse effects and alcohol-related diseases. Death rates among female alcoholics are 50 to 100 percent higher than those of men. Major physiological impairments, the diagnostic distribution, the psychosocial consequences and their implication on treatment will be outlined.

RNA is closely associated with human mast cell lipid bodies.

Both novel and multiple ultrastructural studies based on different principles show relationships of cytoplasmic lipid bodies and ribonucleic acid (RNA) of potential importance to RNA metabolism in human mast cells. The methods include general ultrastructural morphological observations, imaging of RNA with an EDTA regressive stain, imaging of the incorporation of radio labeled uridine by ultrastructural autoradiography, postembedding immunogold labeling of uridine, ribosomes and small nuclear ribonuclear proteins and ultrastructural in situ hybridization detection of poly(A)-positive messenger RNA. Altogether these studies implicate human mast cell lipid bodies in RNA metabolism and are analogous to earlier similar studies which showed that human mast cell granules also curtain RNA.

Flupenthixol versus risperidone: subjective quality of life as an important factor for compliance in chronic schizophrenic patients.

OBJECTIVE: The primary aim of this paper was to compare the effects of flupenthixol and risperidone on subjective quality of life and attitude towards medication in chronic schizophrenic patients with mainly negative symptoms. In a spectrum ranging from its typical end "haloperidol" to its atypical end "clozapine", flupenthixol has typical and atypical characteristics. METHODS: The effects of flupenthixol versus risperidone were investigated in a multicenter, double-blind trial, whereas subjective quality of life was assessed by means of the EuroQuol-Visual Analogue Scale and the patient satisfaction questionnaire. The attitude towards medication was assessed by means of the Drug Attitude Inventory-30 (DAI-30). RESULTS: Mean daily dose of study medication was 6.6 (SD 2.9) mg/day flupenthixol and 3.6 (SD 1.2) mg/day risperidone. Both groups showed a significant improvement regarding subjective quality of life and positive attitude towards medication. Especially the categories "control of their thoughts", concentration and "feeling better in general" ameliorated in both groups. In the flupenthixol group, the "ability to cope with stress", "feel more relaxed" and the "ability to achieve something" improved significantly more than in the risperidone group. CONCLUSIONS: (1) The spectrum of schizophrenia can be treated effectively with different neuroleptic treatments. (2) Flupenthixol especially improves the ability to cope with stress, the ability to achieve something and feeling more relaxed. (3) Subjective quality of life significantly increased with no difference between the groups.

Ribosomes and secretory granules in human mast cells: close associations demonstrated by staining with a chelating agent.

The distribution of ribosomes was investigated in mature human mast cells with a chelation-based staining protocol known to bleach DNA-rich structures, leaving RNA-rich structures unbleached. With this method, electron-dense ribosomes were adjacent to, attached to, and within secretory granules, which were also bleached with the chelation method that we used. The finding of these ribosome-secretory granule relationships suggests that secretory granules in mature human mast cells may participate in RNA metabolism.

The vesiculo-vacuolar organelle (VVO). A new endothelial cell permeability organelle.

A newly defined endothelial cell permeability structure, termed the vesiculo-vacuolar organelle (VVO), has been identified in the microvasculature that accompanies tumors, in venules associated with allergic inflammation, and in the endothelia of normal venules. This organelle provides the major route of extravasation of macromolecules at sites of increased vascular permeability induced by vascular permeability factor/vascular endothelial growth factor (VPF/VEGF), serotonin, and histamine in animal models. Continuity of these large sessile structures between the vascular lumen and the extracellular space has been demonstrated in kinetic studies with ultrastructural electron-dense tracers, by direct observation of tilted electron micrographs, and by ultrathin serial sections with three-dimensional computer reconstructions. Ultrastructural enzyme-affinity cytochemical and immunocytochemical studies have identified histamine and VPF/VEGF bound to VVOs in vivo in animal models in which these mediators of permeability are released from mast cells and tumor cells, respectively. The high-affinity receptor for VPF/VEGF, VEGFR-2, was localized to VVOs and their substructural components by pre-embedding ultrastructural immunonanogold and immunoperoxidase techniques. Similar methods were used to localize caveolin and vesicle-associated membrane protein (VAMP) to VVOs and caveolae, indicating a possible commonality of formation and function of VVOs to caveolae.

Glomeruloid microvascular proliferation follows adenoviral vascular permeability factor/vascular endothelial growth factor-164 gene delivery.

Glomeruloid bodies are a defining histological feature of glioblastoma multiforme and some other tumors and vascular malformations. Little is known about their pathogenesis. We injected a nonreplicating adenoviral vector engineered to express vascular permeability factor/vascular endothelial growth factor-164 (VPF/VEGF(164)) into the ears of athymic mice. This vector infected local cells that strongly expressed VPF/VEGF(164) mRNA for 10 to 14 days, after which expression gradually declined. Locally expressed VPF/VEGF(164) induced an early increase in microvascular permeability, leading within 24 hours to edema and deposition of extravascular fibrin; in addition, many pre-existing microvessels enlarged to form thin-walled, pericyte-poor, "mother" vessels. Glomeruloid body precursors were first detected at 3 days as focal accumulations of rapidly proliferating cells in the endothelial lining of mother vessels, immediately adjacent to cells expressing VPF/VEGF(164). Initially, glomeruloid bodies were comprised of endothelial cells but subsequently pericytes and macrophages also participated. As they enlarged by endothelial cell and pericyte proliferation, glomeruloid bodies severely compromised mother vessel lumens and blood flow. Subsequently, as VPF/VEGF(164) expression declined, glomeruloid bodies devolved throughout a period of weeks by apoptosis and reorganization into normal-appearing microvessels. These results provide the first animal model for inducing glomeruloid bodies and indicate that VPF/VEGF(164) is sufficient for their induction and necessary for their maintenance.

Ultrastructural localization of vesicle-associated membrane protein(s) to specialized membrane structures in human pericytes, vascular smooth muscle cells, endothelial cells, neutrophils, and eosinophils.

Vesicle-associated membrane proteins (VAMPs) are important to the trafficking of vesicles between membrane-bound intracytoplasmic organelles, in the facilitation of neurosecretion, and in constitutive and regulated secretion in non-neuronal cells. We used a pre-embedding ultrastructural immunonanogold method to localize VAMPs to subcellular sites in human cells of five lineages known to have cytoplasmic vesicles that may function in vesicular transport. We found VAMPs localized to caveolae in pericytes, vascular smooth muscle cells, and endothelial cells of venules, to the vesiculo-vacuolar organelle, recently defined in venular endothelial cells, to the vesicle-rich intergranular cytoplasm and secretory granule membranes of neutrophils, and to perigranular cytoplasmic secretory vesicles and secretory granule membranes in eosinophils. These specific localizations in five human vascular and granulocyte lineages support the notion that VAMPs have vesicle-associated functions in these cells.

Ultrastructural immunolocalization of basic fibroblast growth factor to lipid bodies and secretory granules in human mast cells.

Isolated human lung mast cells were used to identify subcellular sites of basic fibroblast growth factor using a postembedding immunogold method. The factor was present in quantity in secretory granules and cytoplasmic lipid bodies. Cisterns of smooth endoplasmic reticulum and ribosome clusters, closely associated with lipid bodies, contained the factor as did the nuclear matrix. Factor-positive lipid bodies were adjacent to nuclear pores and often indented perinuclear cisternae. Altered secretory granules with reduced density, characteristic of secretion by piecemeal degranulation in mast cells, showed reduced gold label for basic fibroblast growth factor; small, electron-lucent (80-100 nm) transport vesicles near altered granules were labelled for the factor. Since these mature mast cells do not display extensive arrays of classical secretory organelles, such as rough endoplasmic reticulum and Golgi structures, these new subcellular localizations for basic fibroblast growth factor suggest several possible alternative release routes for a cytokine devoid of a signal sequence characteristic of regulated secretory proteins.

Frequencies of alleles and genotypes of the PRNP gene in Polish Red, Czech Pied and Czech Black-and-White cattle.

Frequencies of alleles and genotypes of the PRNP gene in 65 Polish Red cows kept under a conservation programme and 52 randomly chosen cows of two Czech breeds: Czech Pied (42) and Black-and-White (10) were studied. All cows were at the age of 4 years or older. It was found that the frequency of allele 5 (5 copies of the octapeptide repeat) ranged from 0.11 (Polish Red) to 0.00 (Czech Black-and-White) whereas the frequency of allele 6 (6 copies of the octapeptide repeat) ranged from 1.00 (Czech Black and White) to 0.89 (Polish Red). The highest frequency was found for the homozygous genotype 6/6 (1.00 in Czech Black-and-White) and the lowest frequency was detected for the heterozygous genotype 6/5 (0.143). In the studied cows the genotype 5/5 was not found. The higher frequency of allele 5 in the native breeds and its lower frequency or lack of this allele in the populations intensively selected for high milk production may suggest that alleles of the PRNP gene may be associated with milk production.

Ultrastructural cytochemical, immunocytochemical and in situ hybridization methods with polyuridine probes detect mRNA in human mast cell granules.

Mature human mast cells are classical secretory cells that are filled with secretory-storage granules but are poorly endowed with visible free or membrane-bound cytoplasmic ribosomes. We recently reported close associations of ribosomes and various components essential to RNA metabolism in and close to human mast cell granules using multiple ultrastructural imaging methods. In view of these findings and an increased awareness of RNA sorting and localization to specific subcellular sites and organelles, we used human mast cells purified from non-tumour portions of lung samples resected at surgery for carcinoma and ultrastructural methods to investigate this further. Poly(U) probes were used to detect direct en grid binding, and radiolabelled as well as non-radiolabelled poly(U) probes were used in in situ hybridization protocols to detect poly(A)-positive pre-mRNA and mRNA in nuclear, cytoplasmic and granular compartments of mature human mast cells. Negative controls verified specificity of label; expected nuclear and cytoplasmic locations of poly(A)-positive RNA served as positive controls for each sample. These findings lend support to the hypothesis that site-specific synthesis in secretory-storage granules may occur in secretory cells.

Ultrastructural autoradiographic analysis of RNA in isolated human lung mast cells during secretion and recovery from secretion.

BACKGROUND: Previous work has implicated isolated, control human lung mast cell granules in RNA metabolism using multiple methods of high-magnification imaging based on different mechanistic principles. These methods have demonstrated ribosomes, RNA, U1snRNP and uridine in, around and attached to secretory granules. METHODS: Here, we have extended these studies using ultrastructural autoradiography of radiolabeled uridine incorporation in degranulating and recovering mast cells. RESULTS: We found that control cells incorporated uridine into granules, with values that decreased dramatically in conjunction with stimulated histamine secretion and granule extrusion, and that granule stores of tritiated uridine increased together with the reconstitution of secretory granules in recovering mast cells. CONCLUSION: These findings support a possible new role for secretory granules in RNA metabolism in mast cell biology.