A quantitative and qualitative RNA expression profiling assay for cell culture with single cell resolution

Cells are often characterized by their gene expression profile. However, commonly used methods to detect mRNA require cell pooling and could therefore mask differences in gene expression within heterogeneous cell populations. q²PISH allows for the analysis of both qualitative and quantitative (q²) gene expression on cultured cells for quality control measures with single cell resolution. q²PISH was optimized for the subsequent use of two alkaline phosphatase substrates in combination with a cell nucleus count to allow for accurate quantification of gene expression per cell and simultaneously qualitative assessment of potential culture population drift or heterogeneity. As proof of principle the assay was applied to cell lines derived from different areas of the bovine intervertebral disc, showing significant difference in the expression of Col1a1, Col2a1, Acan and Sox9. Furthermore, the assay served to explore a potential impact on cultured cells when substituting a critical media component, fetal bovine serum (FBS), suggesting no significant difference in gene expression for the biomarkers analyzed. As a tool, q²PISH serves as an accurate quality control with single cell resolution for cultured cells.     

Cardiovascular effects of cadmium on intravenous and intracerebroventricular administration in rats

Cardiovascular responses to the intravenous (i.v.) and the intracerebroventricular (i.c.v.) administration of cadmium acetate were evaluated in rats anaesthetized with urethane. Cadmium acetate (1 mg/kg i.v.) caused an initial fall followed by a persistent rise in blood pressure. Cadmium acetate (1 microgram i.c.v.) produced a more marked hypertensive effect. In the spinal-transected rat, the effect of intravenous cadmium was reduced but the effect of intraventricularly administered cadmium was completely abolished. It is, therefore, suggested that both central and peripheral mechanisms are involved in the pressor response to cadmium exposure.     

Response of methylenetetrahydrofolate levels to methotrexate in Krebs ascites cells.

Levels of methylenetetrahydrofolate in Krebs ascites cells subsequent to transplantation and the effects of methotrexate on these levels have been measured. To directly measure methylenetetrahydrofolate in tissue extracts, the cofactor was incorporated into a covalent ternary complex with thymidylate synthase and 3H-labelled fluorodeoxyuridine monophosphate. A 3-4-day lag preceded rapid growth of the tumour cells, and this same kinetic behaviour was observed for methylenetetrahydrofolate levels in the tumour cells. Liver and kidney tissue from the same animals also showed an increase in methylenetetrahydrofolate over the same time period. The impact of methotrexate on methylenetetrahydrofolate in the tumour cells depended upon concentration and the post-transplantation time at which treatment was initiated. Levels of methylenetetrahydrofolate in the tumour cells were most sensitive to the drug at the beginning of the rapid growth phase and were more sensitive to a given level of methotrexate in the presence of phospholipids. A slight but significant increase in methylenetetrahydrofolate occurred in some cases in response to the presence of methotrexate.     

Inheritance of dwarfness in pigeon pea (Cajanus cajan (L.) millsp.)

Summary A mutant pigeon pea, showing dwarf and bushy growth, very late maturity, poor yield and abnormal flowers was isolated from the tall variety, Brazil P/2. It is proposed that the mutant be called dwarf.A single recessive gene appears to be responsible for dwarfness and seems to have pleiotropic effect on maturation. The symbol proposed for the dwarfness gene is d.

---

Zusammenfassung Aus einer hochwüchsigen Straucherbse (Cajanus cajan (L.) Millsp.) der Sorte Brazil P/2 wurde eine Mutante mit verzwergtem und buschigem Wuchstyp, sehr später Reife, geringer Ertragsfähigkeit und abweichender Blütenform isoliert, für die der Name dwarf vorgeschlagen wird.Nur ein rezessives Gen scheint für den Zwergwuchs verantwortlich zu sein, das einen pleiotropen Effekt auf die Reife ausübt. Für dieses Gen wird das Symbol d vorgeschlagen.

     

Increased Expression of Phosphorylated Polo-Like Kinase 1 and Histone in Bypass Vein Graft and Coronary Arteries following Angioplasty

Interventional procedures, including percutaneous transluminal coronary angioplasty (PTCA) and coronary artery bypass surgery (CABG) to re-vascularize occluded coronary arteries, injure the vascular wall and cause endothelial denudation and medial vascular smooth muscle cell (VSMCs) metaplasia. Proliferation of the phenotypically altered SMCs is the key event in the pathogenesis of intimal hyperplasia (IH). Several kinases and phosphatases regulate cell cycle in SMC proliferation. It is our hypothesis that increased expression and activity of polo-like kinase-1 (PLK1) in SMCs, following PTCA and CABG, contributes to greater SMC proliferation in the injured than uninjured blood vessels. Using immunofluorescence (IF), we assessed the expression of PLK1 and phosphorylated-PLK1 (pPLK1) in post-PTCA coronary arteries, and superficial epigastric vein grafts (SEV) and compared it with those in the corresponding uninjured vessels. We also compared the expressions of mitotic marker phospho-histone, synthetic-SMC marker, contractile SMC marker, IFN-γ and phosphorylated STAT-3 in the post-PTCA arteries, SEV-grafts, and the uninjured vessels. Immunostaining demonstrated an increase in the number of cells expressing PLK1 and pPLK1 in the neointima of post PTCA-coronary arteries and SEV-grafts compared to their uninjured counterparts. VSMCs in the neointima showed an increased expression of phospho-histone, synthetic and contractile SMC markers, IFN-γ and phosphorylated STAT-3. However, VSMCs of uninjured coronaries and SEV had no significant expression of the aforementioned proteins. These data suggest that PLK1 might play a critical role in VSMC mitosis in hyperplastic intima of the injured vessels. Thus, novel therapies to inhibit PLK1 could be developed to inhibit the mitogenesis of VSMCs and control neointimal hyperplasia.     

Large‐scale coral reef rehabilitation after blast fishing in Indonesia

The severely degraded condition of many coral reefs worldwide calls for active interventions to rehabilitate their physical and biological structure and function, in addition to effective management of fisheries and no‐take reserves. Rehabilitation efforts to stabilize reef substratum sufficiently to support coral growth have been limited in size. We documented a large coral reef rehabilitation in Indonesia aiming to restore ecosystem functions by increasing live coral cover on a reef severely damaged by blast fishing and coral mining. The project deployed small, modular, open structures to stabilize rubble and to support transplanted coral fragments. Between 2013 to 2015, approximately 11,000 structures covering 7,000 m² were deployed over 2 ha of a reef at a cost of US$174,000. Live coral cover on the structures increased from less than 10% initially to greater than 60% depending on depth, deployment date and location, and disturbances. The mean live coral cover in the rehabilitation area in October 2017 was higher than reported for reefs in many other areas in the Coral Triangle, including marine protected areas, but lower than in the no‐take reference reef. At least 42 coral species were observed growing on the structures. Surprisingly, during the massive coral bleaching in other regions during the 2014–2016 El Niño–Southern Oscillation event, bleaching in the rehabilitation area was less than 5% cover despite warm water (≥30°C). This project demonstrates that coral rehabilitation is achievable over large scales where coral reefs have been severely damaged and are under continuous anthropogenic disturbances in warming waters.     

Synthesis and antitumor activity of fluorocyclopentenyl-pyrimidines

Synthesis of fluorocyclopentenyl pyrimidine nucleosides 6-9 was enantiopurely accomplished employing oxidative rearrangement, RCM reaction and electrophilic fluorination starting from d-ribose. Cytosine analog 8 was found to exhibit significant anticancer activity in various human tumor cell lines.     

Analysis of codon usage patterns and predicted highly expressed genes for six phytopathogenic Xanthomonas genomes shows a high degree of conservation

Members of the genus Xanthomonas are significant phytopathogens, which cause diseases in several economically important crops including rice, canola, tomato, citrus, etc. We have analyzed the genomes of six recently sequenced Xanthomonas strains for their synonymous codon usage patterns for all of protein coding genes and specific genes associated with pathogenesis, and determined the predicted highly expressed (PHX) genes by the use of the codon adaptation index (CAI). Our results show considerable heterogeneity among the genes of these moderately G+C rich genomes. Most of the genes were moderate to highly biased in their codon usage. However, unlike ribosomal protein genes, which were governed by translational selection, those genes associated with pathogenesis (GAP) were affected by mutational pressure and were predicted to have moderate to low expression levels. Only two out of 339 GAP genes were in the PHX category. PHX genes present in clusters of orthologous groups of proteins (COGs) were identified. Genes in the plasmids present in two strains showed moderate to low expression level and only a couple of genes featured in the PHX list. Common genes present in the top-20 PHX gene-list were identified and their possible functions are discussed. Correspondence analysis showed that genes are highly confined to a core in the plot.     

Temporal Evolution of MRI Characteristics in Dogs with Collagenase-Induced Intracerebral Hemorrhage

Intracerebral hemorrhage (ICH) is one of the most lethal types of stroke. Neuroimaging techniques, particularly MRI, have improved the diagnostic accuracy of ICH. The MRI characteristics of the evolving stages of ICH in humans—but not those in dogs—have been described. In this study, we document the temporal MRI characteristics in a canine model of collagenase-induced ICH. Specifically, ICH was induced in 5 healthy beagles by injecting 500 U of bacterial collagenase from Clostridium histolyticum, which was delivered into the parietal lobe over 5 min by using a microinfusion pump. T1- and T2-weighted, fluid-attenuated inversion recovery, gradient-echo (GRE), and diffusion-weighted (DWI) imaging and measurement of the apparent diffusion coefficient (ADC) were performed serially at 6 different time points (before and 12 h, 3 d, 5 d, 10 d and 24 d after hemorrhage) by using a 3-T MR system. The temporal changes of T1 signal intensity (SI) corresponded well with the reported human data. The temporal changes of T2 and GRE sequences, with the exception of T2 and GRE hyperintensities at the early subacute stage, also matched. ADC measurements were high at the early subacute stage, and DWI-SI positively correlated with T2- and GRE-SI from the early subacute stage onward. In conclusion, MRI is an ideal method for characterizing the temporal evolution of parenchymal alterations after ICH in dogs. These data might be useful for differentiating clinical stages of ICH in dogs.Abbreviations: ADC, apparent diffusion coefficient; DWI, diffusion-weighted imaging; FLAIR, fluid attenuation inversion recovery; GRE, gradient echo; ICH, intracerebral hemorrhage; ROI, region of interest; SIR, signal intensity ratio; WI, weighted imagingIntracerebral hemorrhage (ICH) occurs in 15% to 20% of all stroke patients.¹⁰ In comparison with ischemic stroke, patients with ICH are at higher risk of death and long-term functional disability.¹⁰ Most survivors remain disabled, owing to the hematoma within the brain parenchyma, which can cause severe neurologic deficits.Because of the rapid progression of brain damage during the first hours after ICH, quick recognition and diagnosis are key. Clinical signs are helpful for early diagnosis but are insufficient for the differentiation of ICH from other stroke subtypes. Brain imaging techniques, including CT and MRI, have helped improve the accuracy of diagnosis, which is necessary for the appropriate treatment of acute cerebrovascular accidents.⁵ Previously, MRI was considered unsuitable for detecting early-stage hemorrhage. However, MRI recently was shown to have high sensitivity for detecting hyperacute ICH, superior even to CT.²¹ MRI is now considered the ideal imaging modality for characterizing the temporal and spatial evolution of parenchymal changes after ICH.³⁰The MRI characteristics of ICH vary with the duration of the hematoma, the type of MRI sequence, and various biologic factors.¹⁷ The several forms of hemoglobin (oxyhemoglobin, deoxyhemoglobin, and methemoglobin), which have different magnetic properties, are observed at different times points during hemorrhage, depending on whether they contain unpaired electrons.²⁸ The MR signal intensity (SI) of hemorrhage has been reported for various animal models, from in vitro studies, and during clinical observations.^2,6,12,24 Reflecting the breakdown products of hemoglobin, the MRI features of 5 distinctive stages of ICH have been reported in humans: hyperacute (within 24 h of hemorrhage; intracellular oxyhemoglobin; long T1 and T2 values; iso- to hypointense on T1-weighted images [WI], hyperintense on T2WI); acute (1 to 3 d; intracellular deoxyhemoglobin; long T1, short T2 values; hypointense on T1WI, hypointense on T2WI); early subacute (3 to 7 d; intracellular methemoglobin with intact erythrocyte; short T1 and T2 values; hyperintense on T1WI, hypointense on T2WI); late subacute (7 to 14 d; extracellular methemoglobin with erythrocyte lysis; short T1, long T2 values; hyperintense on T1WI and T2WI); and chronic (after 14 d; ferritin and hemosiderin; long T1, short T2 values; hypointense on T1WI and T2WI).⁴So far, very few clinical studies have assessed the time-dependent evaluation of MRI patterns beyond 24 h in dogs.²³ The clinical setting presents several difficulties to the study of ICH. Because ICH patients are often critically ill, requiring physiologic support, most patients with ICH are unsuitable for MRI due to their medical instability.¹⁵ In dogs, it is frequently impossible to ascertain the precise interval between hemorrhage and MRI scanning. An animal model offers several advantages for studying ICH: histologic analysis in survivors of ICH, the initial testing of novel interventions, homogeneous experimental groups, and a predictable onset of ICH.²⁷An experimental animal model used in human medicine should exhibit certain characteristics, such as ease of standardization and reproducibility, and representation of the principal mechanisms associated with the particular condition in humans.⁸ Small-animal (for example, mice, rats, gerbils) and large-animal (for example, cats, dogs, pigs, sheep, monkeys) models that demonstrate these characteristics have been developed. Although small animals are often more cost-effective and allow for relatively simpler genetic manipulation and management, the use of large-animal models is important in preclinical studies of ICH because these animals have gyrencephalic brains with well-developed white matter that are structurally and functionally similar to human brains.¹⁶ Notably, numerous experimental treatment strategies have been evaluated successfully in rodent models and in vitro, but the vast majority of such modalities subsequently have failed in clinical trials.¹⁸ For all of these reasons, the Stroke Therapy Academic Industry Roundtable strongly recommends the use of appropriate large-animal models of stroke.¹⁹ Among large animals, dogs are readily and economically available, easy to care for, and have predictable intercurrent diseases.Presently, MRI of the brain is extremely useful in confirming stroke, determining the extent of the lesion, and distinguishing between ischemic and hemorrhagic stroke. However, MRI findings of canine ICH are largely based on results from human studies, given the paucity of relevant canine studies. A previous study demonstrated that the time course of ICH stages is much faster in rats than in humans.³ Therefore, we hypothesized that the temporal MRI characteristics of evolving stages of canine ICH differ from those of ICH in humans.Advanced MRI techniques may enable clarification of mechanisms that mediate injury after ICH. Diffusion-weighted imaging (DWI) has already proven useful in the diagnosis and investigation of the natural history of ischemic stroke.⁷ However, the usefulness of those techniques to assess mechanisms of neuronal injury after ICH remains a topic of debate, both in humans and in dogs. Therefore, the purpose of the current study was to evaluate the utility of advanced MRI techniques, including DWI, in the diagnosis of canine ICH, by monitoring the temporal changes in MR images relative to the stage of hematoma in affected dogs.