Absence of far-red emission band in aggregated core antenna complexes

Reported herein is a Stark fluorescence spectroscopy study performed on photosystem II core antenna complexes CP43 and CP47 in their native and aggregated states. The systematic mathematical modeling of the Stark fluorescence spectra with the aid of conventional Liptay formalism revealed that induction of aggregation in both the core antenna complexes via detergent removal results in a single quenched species characterized by a remarkably broad and inhomogenously broadened emission lineshape peaking around 700 nm. The quenched species possesses a fairly large magnitude of charge-transfer character. From the analogy with the results from aggregated peripheral antenna complexes, the quenched species is thought to originate from the enhanced chlorophyll-chlorophyll interaction due to aggregation. However, in contrast, aggregation of both core antenna complexes did not produce a far-red emission band at ～730 nm, which was identified in most of the aggregated peripheral antenna complexes. The 730-nm emission band of the aggregated peripheral antenna complexes was attributed to the enhanced chlorophyll-carotenoid (lutein1) interaction in the terminal emitter locus. Therefore, it is very likely that the no occurrence of the far-red band in the aggregated core antenna complexes is directly related to the absence of lutein1 in their structures. The absence of the far-red band also suggests the possibility that aggregation-induced conformational change of the core antenna complexes does not yield a chlorophyll-carotenoid interaction associated energy dissipation channel.     

Association between gap junction protein-alpha 8 polymorphisms and age-related cataract

GJA8 plays an important role in lens growth and transparency. Therefore, we hypothesized that two single nucleotide polymorphisms (SNPs) in GJA8 might be associated with age-related cataract. We investigated the SNPs rs1495960 and rs9437983 using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and DNA sequencing, in 96 age-related cataract patients, and 208 gender- and age-matched healthy controls. No significant differences between cases and controls were seen in genotype or allele distributions of rs1495960 (P > 0.05). The allele distribution of rs9437983 was different between cases and controls, but no difference was detected in its genotype distribution. Cataract patients had a significantly lower G–G haplotype frequency (4.9% vs. 15.5%, P = 0.0001), and a significantly higher G–A haplotype frequency (45.6% vs. 36.4%, P = 0.030) than controls. Limiting to nuclear cataract cases significantly increased the differences between cases and controls for G–G and G–A haplotypes. These results support that the GJA8 gene may be a novel susceptibility gene for age-related cataracts.     

A versatile computational pipeline for bacterial genome annotation improvement and comparative analysis, with Brucella as a use case

We present a bacterial genome computational analysis pipeline, called GenVar. The pipeline, based on the program GeneWise, is designed to analyze an annotated genome and automatically identify missed gene calls and sequence variants such as genes with disrupted reading frames (split genes) and those with insertions and deletions (indels). For a given genome to be analyzed, GenVar relies on a database containing closely related genomes (such as other species or strains) as well as a few additional reference genomes. GenVar also helps identify gene disruptions probably caused by sequencing errors. We exemplify GenVar's capabilities by presenting results from the analysis of four Brucella genomes. Brucella is an important human pathogen and zoonotic agent. The analysis revealed hundreds of missed gene calls, new split genes and indels, several of which are species specific and hence provide valuable clues to the understanding of the genome basis of Brucella pathogenicity and host specificity.     

A versatile method for the preparation of conjugates of peptides with DNA/PNA/analog by employing chemo-selective click reaction in water

The specific 1,3 dipolar Hüisgen cycloaddition reaction known as ‘click-reaction’ between azide and alkyne groups is employed for the synthesis of peptide–oligonucleotide conjugates. The peptide nucleic acids (PNA)/DNA and peptides may be appended either by azide or alkyne groups. The cycloaddition reaction between the azide and alkyne appended substrates allows the synthesis of the desired conjugates in high purity and yields irrespective of the sequence and functional groups on either of the two substrates. The versatile approach could also be employed to generate the conjugates of peptides with thioacetamido nucleic acid (TANA) analog. The click reaction is catalyzed by Cu (I) in either water or in organic medium. In water, ~3-fold excess of the peptide-alkyne/azide drives the reaction to completion in 2 h with no side products.     

High‐throughput ion exchange purification of positively charged recombinant protein in the presence of negatively charged dextran sulfate

Product quality analyses are critical for developing cell line and bioprocess producing therapeutic proteins with desired critical product quality attributes. To facilitate these analyses, a high‐throughput small‐scale protein purification (SSP) is required to quickly purify many samples in parallel. Here we develop an SSP using ion exchange resins to purify a positively charged recombinant growth factor P1 in the presence of negatively charged dextran sulfate supplemented to improve the cell culture performance. The major challenge in this work is that the strong ionic interaction between P1 and dextran sulfate disrupts interaction between P1 and chromatography resins. To solve this problem, we develop a two‐step SSP using Q Sepharose Fast Flow (QFF) and SP Sepharose XL (SPXL) resins to purify P1. The overall yield of this two‐step SSP is 78%. Moreover, the SSP does not affect the critical product quality attributes. The SSP was critical for developing the cell line and process producing P1. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:516–520, 2014     

A cell-based computational modeling approach for developing site-directed molecular probes

Modeling the local absorption and retention patterns of membrane-permeant small molecules in a cellular context could facilitate development of site-directed chemical agents for bioimaging or therapeutic applications. Here, we present an integrative approach to this problem, combining in silico computational models, in vitro cell based assays and in vivo biodistribution studies. To target small molecule probes to the epithelial cells of the upper airways, a multiscale computational model of the lung was first used as a screening tool, in silico. Following virtual screening, cell monolayers differentiated on microfabricated pore arrays and multilayer cultures of primary human bronchial epithelial cells differentiated in an air-liquid interface were used to test the local absorption and intracellular retention patterns of selected probes, in vitro. Lastly, experiments involving visualization of bioimaging probe distribution in the lungs after local and systemic administration were used to test the relevance of computational models and cell-based assays, in vivo. The results of in vivo experiments were consistent with the results of in silico simulations, indicating that mitochondrial accumulation of membrane permeant, hydrophilic cations can be used to maximize local exposure and retention, specifically in the upper airways after intratracheal administration.     

Norbornene derived doxorubicin copolymers as drug carriers with pH responsive hydrazone linker

The synthesis and complete characterization of both norbornene-derived doxorubicin (mono 1) and polyethylene glycol (mono 2) monomers are clearly described, and their copolymerization by ring-opening metathesis polymerization (ROMP) to get the block copolymer (COPY-DOX) is vividly elaborated. The careful design of these conjugates exhibits properties like well-shielded drug moieties and well-defined nanostructures; additionally, they show solubility in both water and biological medium and also have the important tendency of rendering acid-triggered drug release. The drug release profile suggests the importance of having the hydrazone linker that helps to release the drug exactly at the mild acidic conditions resembling the pH of the cancerous cells. It is also observed that the drug release from micelles of COPY-DOX is significantly accelerated at a mildly acidic pH of 5.5-6, compared to the physiological pH of 7.4, suggesting the pH-responsive feature of the drug delivery system with hydrazone linkages. Confocal laser scanning microscopy (CLSM) measurements indicate that these COPY-DOX micelles are easily internalized by living cells. MTT assays against HeLa and 4T cancer cells showing COPY-DOX micelles have a high anticancer efficacy. All of these results demonstrate that these polymeric micelles that self-assembled from COPY-DOX block copolymers have great scope in the world of medicine, and they also symbolize promising carriers for the pH-triggered intracellular delivery of hydrophobic anticancer drugs.     

Bioprocessing of bacteriophages via rapid drying onto microcrystals

We present an alternative bioprocess for bacteriophages involving room temperature coprecipitation of an aqueous mixture of phage (Siphoviridae) and a crystallizable carrier (glutamine or glycine) in excess of water miscible organic solvent (isopropanol or isobutanol). The resultant suspension of phage-coated microcrystals can be harvested by filtration and the residual solvent removed rapidly by air-drying at a relative humidity of 75%. Albumin or trehalose added at 5% w/w of the crystalline carrier provide for better stabilization of the phage during co-precipitation. Free-flowing dry powders generated from an aqueous solution of phage (～13 log(10) pfu/mL) can be reconstituted in the same aqueous volume to a phage titer of almost 10 log(10) pfu/mL; high enough to permit subsequent formulation steps following bioprocessing. The phage-coated microcrystals remain partially stable at room temperature for at least one month, which compares favorably with phage immobilized into polyester microcarriers or lyophilized with excipient (1-5% polyethylene glycol 6000 or 0.1-0.5 M sucrose). We anticipate that this bioprocessing technique will have application to other phage families as required for the development of phage therapies.     

Sentinel node biopsy should be supplemented by axillary sampling in patients with small breast cancers

Axillary clearance provides important prognostic information but is associated with significant morbidity. Sentinel node biopsy can provide staging .141 patients with node negative early breast cancers-tumour size less than 1.5 cm measured clinically or by imaging had guided axillary sampling (sentinel lymph node biopsy in combination with axillary sampling). Four node axillary sampling improved the detection rate of axillary node metastases by 13.6% as compared to blue dye sentinel node biopsy alone. Positive sampled nodes strongly indicated the likelihood of further metastatic being revealed by axillary dissection (67%). Negative sampled nodes in combination with a positive sentinel node biopsy were associated with a much lower rate of further nodal involvement in the axillary clearance (8%).