Class 5 transmembrane semaphorins control selective Mammalian retinal lamination and function

In the vertebrate retina, neurites from distinct neuronal cell types are constrained within the plexiform layers, allowing for establishment of retinal lamination. However, the mechanisms by which retinal neurites are segregated within the inner or outer plexiform layers are not known. We find that the transmembrane semaphorins Sema5A and Sema5B constrain neurites from multiple retinal neuron subtypes within the inner plexiform layer (IPL). In Sema5A?/?; Sema5B?/? mice, retinal ganglion cells (RGCs) and amacrine and bipolar cells exhibit severe defects leading to neurite mistargeting into the outer portions of the retina. These targeting abnormalities are more prominent in the outer (OFF) layers of the IPL and result in functional defects in select RGC response properties. Sema5A and Sema5B inhibit retinal neurite outgrowth through PlexinA1 and PlexinA3 receptors both in vitro and in vivo. These findings define a set of ligands and receptors required for the establishment of inner retinal lamination and function.     

Signal amplification of microarray-based immunoassay by optimization of nanoliposome formulations

The use of microarray-based immunoassay is often limited by its sensitivity. To increase the sensitivities of such an immunoassay, liposome encapsulation was explored. Two different liposome formations and several preparation methods were examined to optimize encapsulation and signal-enhancing efficacy for enzyme-linked immunosorbent assay (ELISA) and antibody array. The signal amplification by liposome encapsulation was demonstrated through a detection for foodborne pathogenic Listeria. In plate-trapped antigen (PTA) ELISA, horseradish peroxidase (HRP)-loaded liposome increased signal 9-fold more than the control. Limits of detection (LODs) of HRP-encapsulated liposome were 6.4×10(5) and 5.5×10(6)CFU/ml in sandwich ELISA and antibody array, respectively. Furthermore, when chromogenic 4-chloro-1-naphthol (4-CN) substrate was used for signal development in the antibody array, the signal could be detected with the naked eye. These results suggest that the liposome encapsulation technique can have great potential for signal amplification and, therefore, for increasing assay sensitivity for various formats of immunoassay, especially microarray-based format.     

Mapping of Genes for Cooking and Eating Qualities in Thai Jasmine Rice (KDML105)

Thai jasmine rice, KDML 105, is known as the best quality rice.It is known not only for its aroma but also for its good cookingand eating qualities. Amylose content (AC), gel consistency(GC) and gelatinization temperature (GT) are important traitsdetermining rice quality. A population of recombinant inbredlines (RIL) derived from KDML105 x CT9993 cross was used tostudy the genetic control of AC, GC and GT traits. A total of191 markers were used in the linkage map construction. The 1605.3cM linkage map covering nearly the whole rice genome was usedfor QTL (define QTL) analysis. Four QTLs for AC were detectedon chromosomes 3, 4, 6 and 7. These QTLs accounted for 80% ofphenotypic variation explained (PVE) in AC. The presence ofone major gene as well as several modifiers was responsiblefor the expression of the trait. Two QTLs on chromosome 6 andone on chromosome 7 were detected for GC, which accounts for57% of PVE. A single gene of major effect along with modifiergenes controls GC from this cross. The QTLs in the vicinityof waxy locus were major contributors in the expression of ACand GC. The finding that the position of QTLs for AC and GCwere near each other may reflect tight linkage or pleiotropy.Three QTLs were detected, one on chromosome 2 and two on chromosome6, which accounted for 67% of PVE in GT. Just like AC and GC,one major gene and modifier genes governed the variation inGT resulting from the KDML105 x CT9993 cross. Breeding for cookingand eating qualities will largely rely on the preferences ofthe end users.     

Pre-cut Filter Paper for Detecting Anti-Japanese Encephalitis Virus IgM from Dried Cerebrospinal Fluid Spots

BackgroundThe use of filter paper as a simple, inexpensive tool for storage and transportation of blood, ‘Dried Blood Spots’ or Guthrie cards, for diagnostic assays is well-established. In contrast, there are a paucity of diagnostic evaluations of dried cerebrospinal fluid (CSF) spots. These have potential applications in low-resource settings, such as Laos, where laboratory facilities for central nervous system (CNS) diagnostics are only available in Vientiane. In Laos, a major cause of CNS infection is Japanese encephalitis virus (JEV). We aimed to develop a dried CSF spot protocol and to evaluate its diagnostic performance using the World Health Organisation recommended anti-JEV IgM antibody capture enzyme-linked immunosorbent assay (JEV MAC-ELISA).Conclusions/SignificanceThe novel design of pre-cut filter paper saturated with CSF could provide a useful tool for JEV diagnostics in settings with limited laboratory access. It has the potential to improve national JEV surveillance and inform vaccination policies. The saturation of filter paper has potential use in the wider context of pathogen detection, including dried spots for detecting other analytes in CSF, and other body fluids.     

The Aetiologies and Impact of Fever in Pregnant Inpatients in Vientiane,Laos

Introduction

Laos has the highest maternal mortality ratio in mainland Southeast Asia and a high incidence of infectious diseases. Globally, malaria has been the pathogen most intensively investigated in relation to impact on pregnancy, but there has been relatively little research on the aetiology and impact of other diseases. We therefore aimed to determine the causes and impact of fever in pregnant women admitted to two central hospitals in Vientiane City, Lao PDR (Laos).

Materials and Methods

This hospital-based prospective study was conducted in Mahosot Hospital and the Mother and Child Hospital, Vientiane, between 2006 and 2010, with the aim to recruit 250 consenting pregnant women admitted with tympanic temperature ≥37.5°C. Primary outcome was the cause of fever and secondary outcomes were pregnancy outcomes. Specific investigations (culture, antigen, molecular and serological tests) were performed to investigate causes of fever. After discharge, all pregnant women were asked to return for review and convalescence serum on day 10–14 and were monitored until delivery.

Principle findings

250 pregnant women were recruited to this study between February 2006 and November 2010. Fifty percent were pregnant for the first time. Their median (range) gestational age on admission was 24 (4–43) weeks. The median (range) tympanic admission temperature was 38.5°C (37.5–40.5°C). Fifteen percent of patients stated that they had taken antibiotics before admission. Headache, myalgia, back pain and arthralgia were described by >60% of patients and 149 (60%) were given a laboratory diagnosis. Of those with confirmed diagnoses, 132 (53%) had a single disease and 17 (7%) had apparent mixed diseases. Among those who had a single disease, dengue fever was the most common diagnosis, followed by pyelonephritis, scrub typhus, murine typhus and typhoid. Patients were also diagnosed with tuberculosis, appendicitis, Staphylococcus aureus septicemia, leptospirosis, Japanese encephalitis virus infection and Plasmodium falciparum malaria. Severe consequences, including maternal death, miscarriage, stillbirth, low birth weight and preterm birth, were found among 28 (78%) mothers with dengue fever, rickettsioses and typhoid.

Conclusion

Fevers other than malaria, such as dengue, pyelonephritis, rickettsioses and typhoid are common causes of fever during pregnancy in the Asian tropics. Further investigations of their impact in the community on maternal death, fetal loss, vertical transmission, low birth weight and preterm birth are needed.     

Mucoadhesive property and biocompatibility of methylated N-aryl chitosan derivatives

The methylated N-aryl chitosan derivatives, methylated N-(4-N,N-dimethylaminocinnamyl) chitosan chloride (MDMCMCh) and methylated N-(4-pyridylmethyl) chitosan chloride (MPyMeCh), were synthesized by two steps, the reductive amination and the methylation. The physicochemical properties of chitosan derivatives were determined by ATR-FTIR, NMR, X-ray diffraction (XRD) and thermogravimetric (TG) techniques. The XRD analysis showed that the crystallinity and thermal stability of methylated chitosan derivatives were lower than those of chitosan. The effects of degree of quaternization (DQ), polymer structure and positive charge location on mucoadhesive property and cytotoxicity were investigated by using a mucin particle method and MTT assay compared to N,N,N-trimethylammonium chitosan chloride (TMChC). It was found that the mucoadhesive property and cytotoxicity increased with increasing DQ. At the DQ of 65%, the mucoadhesive property of the MDMCMCh was twofold lower than that of the TMChC. However, this phenomenon did not affect the mucoadhesive property when the DQ was higher than 65%. Surprisingly, the MPyMeCh showed the lowest toxicity even with the high DQ. These could be due to the resonance effect of the positive charge in the pyridine ring and the molecular weight after methylation. Finally, our result revealed that the mucoadhesive property was dependent on the DQ and polymer structure whereas the cytotoxicity was dependent on the combination of the polymer structure, positive charge location and molecular weight after methylation.     

Isolation and characterization of cells with neurogenic potential from adult skeletal muscle

Autologous cell therapies in neurodegenerative diseases and stroke will require an efficient generation of neuroprogenitors or neurons. We have previously shown that presumptive neural progenitors can be obtained from a candidate stem cell population isolated from adult skeletal muscle. Here we describe experimental conditions to isolate and characterize the cells with neurogenic potential from this population. Candidate stem cell population was isolated from adult skeletal muscle and expanded for selection during at least 30 cell divisions. FACS analysis revealed that this population was homogeneous with respect to CD45 (-), CD34 (-), and heterogeneous for CD90 (Thy-1) expression. The population was separated by cell sorting into three sub-populations based on CD90 expression (CD90-, CD90+, and CD90++) and each population expanded rapidly as free-floating spheres. When dissociated and plated in a neuronal differentiation medium, a large number of CD90+ cells acquired morphological characteristics of neuroprogenitors and neurons, and expressed markers of neurons but no markers of glial or muscle cells. In contrast, CD90- and CD90++ cells lacked this ability. Comparison of CD90+ and CD90- populations may be useful for studying the molecular characteristics defining the neuronal potential of stem cells from adult muscle. The selection of CD90+ expressing cells, combined with the growth conditions presented here, allows for rapid generation of a large number of cells which may be useful for autologous cell replacement therapies in the central nervous system.     

Immunocytochemical detection of p16INK4a protein in scraped cervical cells

OBJECTIVE: To develop an immunocytochemical technique for p16INK4a protein detection in scraped cervical cells for cancer screening. STUDY DESIGN: We took duplicate cervical scrapes from each participant, the first for a Pap smear and the second for p16INK4a protein detection. From a 50-microL cell suspension prepared from the scrape rinsing, a 10-microL aliquot was dropped in a 5-mm-diameter circle on a glass slide, air dried and fixed in 0.1% formal saline (1 hour) and in 95% ethanol (10 minutes). Using the immunocytochemical technique, slides from 30 samples of each Pap diagnosis class were stained sequentially with mouse monoclonal anti-p16INK4a (primary antibody), biotinylated goat antimouse IgG (secondary antibody), horse-radish peroxidase-labelled streptavidin and 3,3'-diaminobenzidine and mixed hydrogen peroxide, then counterstained with hematoxylin. A positive sample had to contain > or = 3 immunoreactive cells. Results were confirmed by western blot analysis of lysates from the remaining 40 microL of each cervical cell suspension. RESULTS: Samples were grouped as control (normal cervical cells), mild dysplasia (ASCUS, LSIL) and high abnormality (HSIL, SCC). Using the immunocytochemical technique, > 95% of the positive (SiHa cells) but 0% of the negative controls (human embryonic lung fibroblast cells) showed immunoreactive cells. All slides displayed a clear background without mucus, and positive cells were stained in both the cytoplasm and nucleus. p16INK4a Protein was detected in 17 of 30 (56.67%) ASCUS and 10 of 30 (33.33%) LSIL and increased with the degree of abnormality to 93.33% (28 of 30) and 96.67% (29 of 30) in the HSIL and SCC group, respectively. Normal cervical cells and degenerated malignant cells were nonimmunoreactive. Western blot analysis confirmed similar positive samples in the low-abnormality group, while the whole high-abnormality group was immunoreactive. A sampling error might have caused the 2 HSIL and 1 SCC sample to be negative using our immunocytochemical technique. CONCLUSION: p16INK4a Protein detection in scraped cervical cells using the immunocytochemical technique correlated with western blot analysis and was nontraumatic and precise. It offers a significant diagnostic adjunct to the Pap test for cervical cancer screening.     

Presenilin-dependent receptor processing is required for axon guidance

The Alzheimer's disease-linked gene presenilin is required for intramembrane proteolysis of amyloid-β precursor protein, contributing to the pathogenesis of neurodegeneration that is characterized by loss of neuronal connections, but the role of Presenilin in establishing neuronal connections is less clear. Through a forward genetic screen in mice for recessive genes affecting motor neurons, we identified the Columbus allele, which disrupts motor axon projections from the spinal cord. We mapped this mutation to the Presenilin-1 gene. Motor neurons and commissural interneurons in Columbus mutants lacking Presenilin-1 acquire an inappropriate attraction to Netrin produced by the floor plate because of an accumulation of DCC receptor fragments within the membrane that are insensitive to Slit/Robo silencing. Our findings reveal that Presenilin-dependent DCC receptor processing coordinates the interplay between Netrin/DCC and Slit/Robo signaling. Thus, Presenilin is a key neural circuit builder that gates the spatiotemporal pattern of guidance signaling, thereby ensuring neural projections occur with high fidelity.     

Influence of the interleukin-6 -174 G/C gene polymorphism on exercise training-induced changes in glucose tolerance indexes.

A polymorphism in the IL-6 gene, a G-to-C substitution 176 bp upstream of the ATG translation initiation site, has been associated with diabetes prevalence and insulin resistance. Interventions including exercise training are frequently used to modify cardiovascular disease risk factors. Consequently, this project examined associations between the IL-6 -174 genotype and oral glucose tolerance test outcomes in 50- to 75-yr-old sedentary men and postmenopausal women before and after aerobic exercise training. Among the 87 individuals who started the study, 56 were retested after 6 mo of aerobic exercise training. Subject characteristics at baseline did not differ between the IL-6 genotype groups with the exception of fasting glucose, which was higher (P = 0.02, covariates age, gender, and ethnicity) in the CC genotype group. The training-induced change in glucose area under the curve during the oral glucose tolerance test varied between the IL-6 -174 genotype groups (P = 0.05, covariates age, gender, ethnicity, baseline glucose area under the curve, and percent body fat change) with a significant decrease occurring only in the GG genotype group. Insulin outcomes did not differ among the groups at baseline or after training. Training-induced changes in weight, percent body fat, maximal oxygen consumption, fasting glucose, and an insulin sensitivity index also changed similarly among the genotype groups. In conclusion, fasting glucose and the extent to which glucose tolerance changes with exercise training may be influenced by the IL-6 -174 gene polymorphism.