On-line recovery of large molecules from mixtures using reciprocating size exclusion chromatography

Blue Dextran, a standard large molecule, was successfully recovered on-line from the aqueous mixture solution with nickel nitrate using a novel reciprocating size exclusion chromatography. After 7 cycles of repeating operations of frontal mode, 70% of Blue Dextran in the feed was isolated as a pure solution. On-line recovery of large molecules from the mixture is an unusual trial, comparing to the routine practice of filtration where small molecule is isolated from the mixture.     

Deep Whole-Genome Sequencing of 100 Southeast Asian Malays

Whole-genome sequencing across multiple samples in a population provides an unprecedented opportunity for comprehensively characterizing the polymorphic variants in the population. Although the 1000 Genomes Project (1KGP) has offered brief insights into the value of population-level sequencing, the low coverage has compromised the ability to confidently detect rare and low-frequency variants. In addition, the composition of populations in the 1KGP is not complete, despite the fact that the study design has been extended to more than 2,500 samples from more than 20 population groups. The Malays are one of the Austronesian groups predominantly present in Southeast Asia and Oceania, and the Singapore Sequencing Malay Project (SSMP) aims to perform deep whole-genome sequencing of 100 healthy Malays. By sequencing at a minimum of 30× coverage, we have illustrated the higher sensitivity at detecting low-frequency and rare variants and the ability to investigate the presence of hotspots of functional mutations. Compared to the low-pass sequencing in the 1KGP, the deeper coverage allows more functional variants to be identified for each person. A comparison of the fidelity of genotype imputation of Malays indicated that a population-specific reference panel, such as the SSMP, outperforms a cosmopolitan panel with larger number of individuals for common SNPs. For lower-frequency (<5%) markers, a larger number of individuals might have to be whole-genome sequenced so that the accuracy currently afforded by the 1KGP can be achieved. The SSMP data are expected to be the benchmark for evaluating the value of deep population-level sequencing versus low-pass sequencing, especially in populations that are poorly represented in population-genetics studies.     

The C. elegans Male Exercises Directional Control during Mating through Cholinergic Regulation of Sex-Shared Command Interneurons

Background

Mating behaviors in simple invertebrate model organisms represent tractable paradigms for understanding the neural bases of sex-specific behaviors, decision-making and sensorimotor integration. However, there are few examples where such neural circuits have been defined at high resolution or interrogated.

Methodology/Principal Findings

Here we exploit the simplicity of the nematode Caenorhabditis elegans to define the neural circuits underlying the male’s decision to initiate mating in response to contact with a mate. Mate contact is sensed by male-specific sensilla of the tail, the rays, which subsequently induce and guide a contact-based search of the hermaphrodite’s surface for the vulva (the vulva search). Atypically, search locomotion has a backward directional bias so its implementation requires overcoming an intrinsic bias for forward movement, set by activity of the sex-shared locomotory system. Using optogenetics, cell-specific ablation- and mutant behavioral analyses, we show that the male makes this shift by manipulating the activity of command cells within this sex-shared locomotory system. The rays control the command interneurons through the male-specific, decision-making interneuron PVY and its auxiliary cell PVX. Unlike many sex-shared pathways, PVY/PVX regulate the command cells via cholinergic, rather than glutamatergic transmission, a feature that likely contributes to response specificity and coordinates directional movement with other cholinergic-dependent motor behaviors of the mating sequence. PVY/PVX preferentially activate the backward, and not forward, command cells because of a bias in synaptic inputs and the distribution of key cholinergic receptors (encoded by the genes acr-18, acr-16 and unc-29) in favor of the backward command cells.

Conclusion/Significance

Our interrogation of male neural circuits reveals that a sex-specific response to the opposite sex is conferred by a male-specific pathway that renders subordinate, sex-shared motor programs responsive to mate cues. Circuit modifications of these types may make prominent contributions to natural variations in behavior that ultimately bring about speciation.     

Homoeologous recombination in the presence of <Emphasis Type="Italic">Ph1</Emphasis> gene in wheat

A crossover (CO) and its cytological signature, the chiasma, are major features of eukaryotic meiosis. The formation of at least one CO/chiasma between homologous chromosome pairs is essential for accurate chromosome segregation at the first meiotic division and genetic recombination. Polyploid organisms with multiple sets of homoeologous chromosomes have evolved additional mechanisms for the regulation of CO/chiasma. In hexaploid wheat (2n = 6× = 42), this is accomplished by pairing homoeologous (Ph) genes, with Ph1 having the strongest effect on suppressing homoeologous recombination and homoeologous COs. In this study, we observed homoeologous COs between chromosome 5M^g of Aegilops geniculata and 5D of wheat in plants where Ph1 was fully active, indicating that chromosome 5M^g harbors a homoeologous recombination promoter factor(s). Further cytogenetic analysis, with different 5M^g/5D recombinants, showed that the homoeologous recombination promoting factor(s) may be located in proximal regions of 5M^g. In addition, we observed a higher frequency of homoeologous COs in the pericentromeric region between chromosome combination of rec5M^g#2S·5M^g#2L and 5D compared to 5M^g#1/5D, which may be caused by a small terminal region of 5DL homology present in chromosome rec5M^g#2. The genetic stocks reported here will be useful for analyzing the mechanism of Ph1 action and the nature of homoeologous COs.     

The Efficacy of Neurofeedback in Patients with Major Depressive Disorder: An Open Labeled Prospective Study

The purpose of this study was to evaluate the effect of neurofeedback on depressive symptoms and electrophysiological disturbances in patients with major depressive disorder. We recruited participants suffering from depression to evaluate efficacy of left prefrontal beta with alpha/theta training. An 8-week, prospective, open-label study was undertaken. Twenty participants were recruited. The treatment protocol was twice or three times a week training of beta at F3 with alpha/theta at Pz for 8 weeks. When every visit, patients were received beta training for 30 min, and then alpha/theta training for 30 min. Baseline, 4 and 8 week scores of; the Hamilton rating scale for Depression (HAM-D), the Hamilton rating scale for Anxiety (HAM-A), the Beck Depression Inventory (BDI)-II, the Beck Anxiety Inventory (BAI), Clinical global impression-severity (CGI-S), and pre- and post-treatment resting state EEGs were compared. Interhemispheric alpha power asymmetry (A score) was computed for homologous sites F3–F4. Pre- and post-training clinical assessments revealed significant improvements in HAM–D, HAM-A, BDI, and CGI-S scores. Cumulative response rates by HAM-D were 35.0 and 75.0 % at 4 and 8 weeks, respectively, corresponding cumulative remission rates by HAM-D were 15.0 and 55.0 %, respectively. No significant differences were found between pre- and post-treatment A score. Neurofeedback treatment could improve depressive symptoms significantly. In addition, anxiety symptoms and clinical illness severity decreased significantly after neurofeedback treatment. Despite its several limitations, such as, small sample size and lack of a control group, this study suggested neurofeedback has significant effects in patients with major depressive disorder.     

The Chronological Characteristics of SOD1 Activity and Inflammatory Response in the Hippocampi of STZ-Induced Type 1 Diabetic Rats

Because it appears that oxidative stress and inflammation are implicated with disease pathogenesis in the diabetic brain, many researchers have used streptozotocin (STZ)-induced diabetic animals to study superoxide production and the effects of superoxide scavengers like Cu,Zn-superoxide dismutase (SOD1). However, many studies have been conducted without considering temporal changes after STZ injection. Interestingly, though SOD activities were not significantly different among the groups, SOD1 and 4-hydroxy-2-nonenal (4-HNE) immunoreactivities were significantly enhanced at 3 weeks after an STZ injection (STZ3w) versus only marginal levels in sham controls, whereas microglial activity was remarkably reduced in injected rats at this time. However, SOD1 immunoreactivity and microglial activities were only at the sham level at STZ4w. The present study provides important information concerning cell damage by ROS generated by STZ. Microglial response was found to be inactivated at STZ3w and neuronal cells (NeuN) showed a non-significant tendency to be reduced in number at STZ4w except in the dentate gyrus. We speculated that the above oxidative stress-related events should be accomplished at STZ3w in the brains of STZ-induced diabetes animal models. Therefore, the aim of the present study was to investigate chronological changes in SOD1 immunoreactivity associated with lipid peroxidation and inflammatory responses in the hippocampi of STZ-induced type I diabetic rats.     

Sustained release of an anti-glaucoma drug: demonstration of efficacy of a liposomal formulation in the rabbit eye

Topical medication remains the first line treatment of glaucoma; however, sustained ocular drug delivery via topical administration is difficult to achieve. Most drugs have poor penetration due to the multiple physiological barriers of the eye and are rapidly cleared if applied topically. Currently, daily topical administration for lowering the intra-ocular pressure (IOP), has many limitations, such as poor patient compliance and ocular allergy from repeated drug administration. Poor compliance leads to suboptimal control of IOP and disease progression with eventual blindness. The delivery of drugs in a sustained manner could provide the patient with a more attractive alternative by providing optimal therapeutic dosing, with minimal local toxicity and inconvenience. To investigate this, we incorporated latanoprost into LUVs (large unilamellar vesicles) derived from the liposome of DPPC (di-palmitoyl-phosphatidyl-choline) by the film hydration technique. Relatively high amounts of drug could be incorporated into this vesicle, and the drug resides predominantly in the bilayer. Vesicle stability monitored by size measurement and DSC (differential scanning calorimetry) analysis showed that formulations with a drug/lipid mole ratio of about 10% have good physical stability during storage and release. This formulation demonstrated sustained release of latanoprost in vitro, and then tested for efficacy in 23 rabbits. Subconjunctival injection and topical eye drop administration of the latanoprost/liposomal formulation were compared with conventional daily administration of latanoprost eye drops. The IOP lowering effect with a single subconjunctival injection was shown to be sustained for up to 50 days, and the extent of IOP lowering was comparable to daily eye drop administration. Toxicity and localized inflammation were not observed in any treatment groups. We believe that this is the first demonstration, in vivo, of sustained delivery to the anterior segment of the eye that is safe and efficacious for 50 days.     

Time-Course of Changes in Phosphorylated CREB in Neuroblasts and BDNF in the Mouse Dentate Gyrus at Early Postnatal Stages

Cyclic AMP (cAMP) response element-binding protein (CREB) is involved in memory, learning, and synaptic transmission. In this study, we observed changes of phosphorylated CREB (pCREB) immunoreactivity and its protein levels as well as brain-derived neurotrophic factor (BDNF) levels in the hippocampal dentate gyrus at postnatal (P) 1, 7, 14, and 21 in mice. In addition, we also investigated pCREB expression in doublecortin (DCX, a marker for neuronal progenitors) immunoreactive neuroblasts at P21. pCREB immunoreaction at P1 was detected in most of cells in the dentate gyrus, thereafter pCREB immunoreactivity was decreased in all the layers of the dentate gyrus with time, however, strong pCREB immunoreactivity was shown in cells confined to the subgranular zone of the dentate gyrus at P21. In this group, many pCREB immunoreactive cells were co-localized with DCX immunoreactive neuroblasts. In addition, pCREB protein levels were decreased with age, showing that their levels were very low at P21, while BDNF protein levels were increased with age. These results suggest that pCREB may play important roles in functional maturity of granule cells in mice.     

Sexual and somatic determinants of the human Y chromosome: studies in a 46,XYp- phenotypic female.

A case of a 46,XYp- phenotypic female provided an opportunity to evaluate both sexual and somatic determinants for the Y chromosome. The patient had multiple stigmata of Turner syndrome, but normal stature. Laparotomy revealed a normal uterus and tubes, with 1.5 cm undifferentiated gonads. Serological tests for H-Y antigen (ostensibly the product of Y-chromosomal testis-determining genes) indicated absence of the H-Y+ phenotype normally associated with the intact Y chromosome. We conclude that genes exist on the short arm of the human Y chromosome which both suppress some of the somatic stigmata of Turner syndrome and determine normal expression of H-Y antigen and testicular differentiation of the primitive gonad. Our data are consistent with the view that H-Y genes comprise a family of testis-determinants, and that loss of a critical moiety is inconsistent with normal development of the male gonad.     

Independent Relationship between Amyloid Precursor Protein (APP) Dimerization and γ-Secretase Processivity

Altered production of β-amyloid (Aβ) from the amyloid precursor protein (APP) is closely associated with Alzheimer’s disease (AD). APP has a number of homo- and hetero-dimerizing domains, and studies have suggested that dimerization of β-secretase derived APP carboxyl terminal fragment (CTFβ, C99) impairs processive cleavage by γ-secretase increasing production of long Aβs (e.g., Aβ1-42, 43). Other studies report that APP CTFβ dimers are not γ-secretase substrates. We revisited this issue due to observations made with an artificial APP mutant referred to as 3xK-APP, which contains three lysine residues at the border of the APP ectodomain and transmembrane domain (TMD). This mutant, which dramatically increases production of long Aβ, was found to form SDS-stable APP dimers, once again suggesting a mechanistic link between dimerization and increased production of long Aβ. To further evaluate how multimerization of substrate affects both initial γ-secretase cleavage and subsequent processivity, we generated recombinant wild type- (WT) and 3xK-C100 substrates, isolated monomeric, dimeric and trimeric forms of these proteins, and evaluated both ε-cleavage site utilization and Aβ production. These show that multimerization significantly impedes γ-secretase cleavage, irrespective of substrate sequence. Further, the monomeric form of the 3xK-C100 mutant increased long Aβ production without altering the initial ε-cleavage utilization. These data confirm and extend previous studies showing that dimeric substrates are not efficient γ-secretase substrates, and demonstrate that primary sequence determinants within APP substrate alter γ-secretase processivity.