Differences in variation of human immunodeficiency virus type 1 sequences from Henan and Shanghai regions of China

Illegally paid blood donation was a risk factor for HIV acquisition exclusively in Henan and Hubei Provinces of China, and not in Shanghai. Nucleotide sequences in the gag and env genes of HIV-1 were compared between isolates from Henan and Shanghai regions of China to test whether an expected higher degree of a common source of infections from this unique blood donation transmission risk would be evident as decreased variation among Henan isolates in an exploratory cross-sectional analysis. Among 38 isolates studied, 23 of 23 (100%) from Henan and 8 of 15 (54%) from Shanghai were subtype B. In addition, fewer sequence differences were found in gp41 of subtype B isolates from Henan than from Shanghai isolates. Further studies with additional controls are therefore warranted to confirm the role of the degree of a common source of infections in differences in HIV variation across populations. Fundation items: The Vanderbilt-Meharry Center for AIDS Research (P30 AI 54999); R.T.D (R01 AI 29193); Start Fund of Ministry of Education of China (for Hong-zhou LU, 2004BA719A10).     

A cancer protein microarray platform using antibody fragments and its clinical applications

Antibody microarrays have shown great potential for measurement of either a spectrum of target proteins in proteomics or disease-associated antigens in molecular diagnostics. Despite its importance, the applications of antibody microarrays are still limited by a variety of fundamental problems. Among them, cross-reactivity significantly limits the multiplexing ability in parallel sandwich immunoassays. As a result, it is very important to design new capture probes in order to incorporate a universal label into the assay configuration. In this report, an antibody fragments (F(ab')2) microarray platform for serum tumor markers was developed. Each antigen was detected at different concentrations to assemble its calibration curve, and combinations of different markers were tested to examine the specificity of simultaneous detection based on the F(ab')2 microarrays. Diagnostics of serum samples with this cancer antibody microarray platform and immunoradiometric assays (IRMA) were also performed. Wide range calibration curves (0-1280 U mL(-1)) were obtained for each tumor marker. Comparative studies demonstrated that such F(ab')2 microarrays exhibited both moderately improved sensitivity and better specificity than full-sized monoclonal antibody microarrays. It is also demonstrated that this microarray platform is quantitative, highly specific and reasonably sensitive. More importantly, clinical applications of our F(ab')2 microarray platform for upwards of 100 patient serum samples clearly show its potential in cancer diagnostics.     

Analysis of Protein Three-Dimension Structure Using Amino Acids Depths

The issue of amino acid depth in proteins gives important insights to our understanding of protein’s three-dimensional structure. There has already been much research done in mathematical and statistical sciences regarding the general definitions, properties and algorithms describing the particle depth of spatially extended systems. We constructed a method of calculating the amino acids depths and applied it to a set of 527 protein structures. We propose the introduction of amino acid depth tendency factors for three-dimensional structures of proteins. The depth tendency factors relate not only to the hydrophobicity indices but also to the electrostatic charge. We found a relationship between the protein size and the number of residues using the distance between the deepest residue and surface residues. We made a prediction regarding the number of residues on the surface of a protein, the deepest amino acid, and the average depth, all of which are fitted well to a linear functional relationship with the length of the protein. Finally, we have predicted the depths of multiple peptides in protein’s three-dimension structure. Electronic supplementary material The online version of this article () contains supplementary material, which is available to authorized users.     

Structure and function of the human calcium-sensing receptor: insights from natural and engineered mutations and allosteric modulators

The human extracellular Ca(2+)-sensing receptor (CaR), a member of the G protein-coupled receptor family 3, plays a key role in the regulation of extracellular calcium homeostasis. It is one of just a few G protein-coupled receptors with a large number of naturally occurring mutations identified in patients. In contrast to the small sizes of its agonists, this large dimeric receptor consists of domains with topologically distinctive orthosteric and allosteric sites. Information derived from studies of naturally occurring mutations, engineered mutations, allosteric modulators and crystal structures of the agonist-binding domain of homologous type 1 metabotropic glutamate receptor and G protein-coupled rhodopsin offers new insights into the structure and function of the CaR.     

Glucosides from the roots of Capparis tenera

Two new lignan glucosides, compounds 2 and 3, two new 1H-indole-alkaloid glucosides, 5 and 6, as well as two new phenolic glucosides, 7 and 10, were isolated from the roots of Capparis tenera, together with five known compounds. Their structures were characterized by chemical and spectroscopic methods. Most of these isolates were obtained for the first time from Capparidaceae. The antioxidant and anti-inflammatory activities of the new compounds were investigated.     

Immunohistochemical localization of estrogen receptors ERα and ERβ in the spiny mouse (<Emphasis Type="Italic">Acomys cahirinus</Emphasis>) ovary during postnatal development

This study was designed to determine the expression pattern of estrogen receptor (ER) subtypes in the Acomys cahirinus ovarian cells during its postnatal development. Immunohistochemical studies revealed the presence of ERα and ERβ in germinal epithelium cells and interstitial tissue. Both these ER subtypes were also seen in granulosa cells and oocytes of growing follicles, however, the level of ERβ expression was higher in comparison with ERα. In contrast to ERβ, ERα protein was also present in theca cells. The expression of ERs increased with animals’ age, but it decreased during follicular maturation. Moreover, the immunolocalization of ER subtypes in luteal cells showed that not ERβ, but ERα expression is up-regulated throughout corpus luteum development. These immunohistochemical studies demonstrate, for the first time, that ERα is also expressed in the mouse granulosa cells and it may be a mediator of estrogen action in granulosa cells proliferation and differentiation.     

The sensitivity of the dinoflagellate Crypthecodinium cohnii to transient hydrodynamic forces and cell-bubble interactions

The increased interest in the benefits of omega-3 fatty acids for human health has resulted in the commercial development of the dinoflagellate Crypthecodinium cohnii for production of docosahexaenoic acid (DHA). The growing market demand for DHA requires highly efficient, very large scale cultures of DHA. While the effects of hydrodynamic forces on dinoflagellates have been investigated for several decades, the majority of the work focused on the negative effects of oceanic turbulence on the population growth of environmentally important dinoflagellates. In contrast, significantly less is known on the effect of hydrodynamic forces encountered by algae in bioprocesses. Unlike other studies conducted on algae, this study employed a microfluidic, flow contraction device to evaluate the effect of transient hydrodynamic forces on C. cohnii cells. It was found that C. cohnii cells can sustain the energy dissipation rate of 5.8 x 10(7) W/m3 without lysis. However, an obvious sublethal effect, the loss of flagella, was observed at a lower level of 1.6 x 10(7) W/m3. Finally the cell-bubble interaction and the effect of bubble rupture were also explored to simulate the conditions of sparged bioreactors.     

Determining antibody stability: creation of solid-liquid interfacial effects within a high shear environment

The purpose of this study was to assess the stability of protein formulations using a device designed to generate defined, quantifiable levels of shear in the presence of a solid-liquid interface. The device, based on a rotating disk, produced shear strain rates of up to 3.4 x 10(4) s(-1) (at 250 rps) and was designed to exclude air-liquid interfaces and enable temperature to be controlled. Computational fluid dynamics (CFD) was used to study the fluid flow patterns within the device and to determine the shear strain rate (s(-1)) at a range of disk speeds. The device was then used to study the effect on a monoclonal IgG4 of high levels of shear at the solid-liquid interface. Monomeric antibody concentration and aggregation of the protein in solution were monitored by gel permeation HPLC and turbidity at 350 nm. High shear strain rates were found to cause significant levels of protein aggregation and precipitation with reduction of protein monomer following first-order kinetics. Monomer reduction rate was determined for a range of disk speeds and found to have a nonlinear relationship with shear strain rate, indicating the importance of identifying and minimizing such environments during processing.     

Highly efficient genome editing in Xanthomonas oryzae pv. oryzae through repurposing the endogenous type I-C CRISPR-Cas system

Efficient and modular genome editing technologies that manipulate the genome of bacterial pathogens will facilitate the study of pathogenesis mechanisms. However, such methods are yet to be established for Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of rice bacterial blight. We identified a single type I-C CRISPR-Cas system in the Xoo genome and leveraged this endogenous defence system for high-efficiency genome editing in Xoo. Specifically, we developed plasmid components carrying a mini-CRISPR array, donor DNA, and a phage-derived recombination system to enable the efficient and programmable genome editing of precise deletions, insertions, base substitutions, and gene replacements. Furthermore, the type I-C CRISPR-Cas system of Xoo cleaves target DNA unidirectionally, and this can be harnessed to generate large genomic deletions up to 212 kb efficiently. Therefore, the genome-editing strategy we have developed can serve as an excellent tool for functional genomics of Xoo, and should also be applicable to other CRISPR-harbouring bacterial plant pathogens.     

Endocytic protein Pal1 regulates appressorium formation and is required for full virulence of Magnaporthe oryzae

Endocytosis plays key roles during infection of plant-pathogenic fungi, but its regulatory mechanisms are still largely unknown. Here, we identified a putative endocytosis-related gene, PAL1, which was highly expressed in appressorium of Magnaporthe oryzae, and was found to be important for appressorium formation and maturation. Deletion of PAL1 significantly reduced the virulence of M. oryzae due to defects in appressorial penetration and invasive growth in host cells. The Pal1 protein interacted and colocalized with the endocytosis protein Sla1, suggesting it is involved in endocytosis. The Δpal1 mutant was significantly reduced in appressorium formation, which was recovered by adding exogenous cAMP and 3-isobutyl-1-methylxanthine (IBMX). Moreover, the phosphorylation level of Pmk1 in Δpal1 was also reduced, suggesting Pal1 functions upstream of both the cAMP and Pmk1 signalling pathways. As a consequence, the utilization of glycogen and lipid, appressorial autophagy, actin ring formation, localization of septin proteins, as well as turgor accumulation were all affected in the Δpal1 mutant. Taken together, Pal1 regulates cAMP and the Pmk1 signalling pathway for appressorium formation and maturation to facilitate infection of M. oryzae.