Prediction of DNA-binding specificity in zinc finger proteins

Zinc finger proteins interact via their individual fingers to three base pair subsites on the target DNA. The four key residue positions -1, 2, 3 and 6 on the alpha-helix of the zinc fingers have hydrogen bond interactions with the DNA. Mutating these key residues enables generation of a plethora of combinatorial possibilities that can bind to any DNA stretch of interest. Exploiting the binding specificity and affinity of the interaction between the zinc fingers and the respective DNA can help to generate engineered zinc fingers for therapeutic purposes involving genome targeting. Exploring the structure-function relationships of the existing zinc finger-DNA complexes can aid in predicting the probable zinc fingers that could bind to any target DNA. Computational tools ease the prediction of such engineered zinc fingers by effectively utilizing information from the available experimental data. A study of literature reveals many approaches for predicting DNA-binding specificity in zinc finger proteins. However, an alternative approach that looks into the physico-chemical properties of these complexes would do away with the difficulties of designing unbiased zinc fingers with the desired affinity and specificity. We present a physico-chemical approach that exploits the relative strengths of hydrogen bonding between the target DNA and all combinatorially possible zinc fingers to select the most optimum zinc finger protein candidate.     

Nitric oxide and superoxide generation from endothelial NOS: modulation by HSP90

Previously, we have shown that pulmonary arterial endothelial cells (PAECs) isolated from fetal lambs produce significant levels of nitric oxide (NO) but minimal superoxide upon stimulation, whereas PAECs isolated from 4-wk-old lambs produce significant amounts of both NO and superoxide. These data indicated that a certain degree of uncoupling of endothelial NO synthase (eNOS) occurs in PAECs during postnatal development. In this study, we sought to extend these studies by investigating the potential role of heat shock protein 90 (HSP90) in eNOS coupling. Western blot analyses revealed higher HSP90 expression in PAECs isolated from fetal compared with 4-wk-old lambs, whereas the analysis of recombinant human eNOS activation in vitro in the presence of HSP90 indicated that HSP90 significantly augmented NO production while inhibiting superoxide generation from eNOS. To further investigate whether HSP90 could be involved in uncoupling of eNOS in PAECs isolated from 4-wk-old lambs, we utilized an adenovirus to overexpress HSP90. We found that overexpression of HSP90 significantly increased the shear-stimulated association of HSP90 with eNOS and led to significant increases in NO production and reduced NOS-dependent superoxide generation. Conversely, the exposure of PAECs isolated from fetal lambs to the HSP90 inhibitor radicicol led to significant decreases in eNOS-HSP90 interactions, decreased shear-stimulated NO generation, and increased NOS-dependent superoxide production indicative of eNOS uncoupling. Finally, we examined eNOS-HSP90 interactions in our lamb model of pulmonary hypertension associated with increased pulmonary blood flow (shunt). Our data indicate that HSP90-eNOS interactions were decreased in shunt lambs and that this was associated with decreased NO generation and an increase in eNOS-dependent generation of superoxide. Together, our data support a significant role for HSP90 in promoting NO generation and inhibiting superoxide generation by eNOS and indicate that the disruption of this interaction may be involved in the endothelial dysfunction associated with pulmonary hypertension.     

Green synthesis-assisted copper nanoparticles using Aegle marmelos leaves extract: physical,optical, and antimicrobial properties

In the present report, Aegle marmelos leaf powder was used to synthesize copper nanoparticles (CuNPs) using a simple and cost-effective method. A. marmelos leaves have various medicinal uses including for the treatment of diarrhoea, constipation, diabetes, cholera, skin diseases, earache, blood purification, heart problems, and so on. The plant biomolecules induce the reduction of Cu²⁺ ions to CuNPs and also act as a capping and stabilizing agent. The formation of CuNPs was confirmed using photoluminescence (PL) excitation and emission spectra on a Shimadzu RF-5301 PC spectrofluorophotometer and the absorbance spectra of a UV–visible spectrophotometer at different stages during the synthesis process. In addition, other properties of synthesized CuNPs were also investigated using X-ray diffraction, Fourier transform infrared spectroscopy, and transmission electron microscopy techniques. The average size of the synthesized CuNPs was in the range 20–40 nm. Furthermore, the synthesized NPs were also considered for an antimicrobial study against Gram-positive Staphylococcus aureus and Proteus, and Gram-negative Escherichia coli and Salmonella spp. using the agar well diffusion method. The zone of inhibition against the Gram-positive bacteria was greater than the zone of inhibition against the Gram-negative bacteria. These investigation results suggest that synthesized NPs are promising nanomaterials for use as antimicrobial agents.     

Dental Variation in Megabats (Chiroptera: Pteropodidae): Tooth Metrics Correlate with Body Size and Tooth Proportions Reflect Phylogeny

Journal of Mammalian Evolution - Variation in the dentition yields insight into the evolutionary history of Mammalia. However, to date, there has been limited research on the dental variation in...     

The compressive mechanical properties of diabetic and non-diabetic plantar soft tissue

Diabetic subjects are at an increased risk of developing plantar ulcers. Knowledge of the physiologic compressive properties of the plantar soft tissue is critical to understanding the possible mechanisms of ulcer formation and improving treatment options. The purpose of this study was to determine the compressive mechanical properties of the plantar soft tissue in both diabetic and non-diabetic specimens from six relevant locations beneath the foot, namely the hallux (big toe), first, third, and fifth metatarsal heads, lateral midfoot, and calcaneus (heel). Cylindrical specimens (1.905 cm diameter) from these locations were excised and separated from the skin and bone from 4 diabetic and 4 non-diabetic age-matched, elderly, fresh-frozen cadaveric feet. Specimens were then subjected to biomechanically realistic strains of ～50% in compression using triangle wave tests conducted at five frequencies ranging from 1 to 10 Hz to determine tissue modulus, energy loss, and strain rate dependence. Diabetic vs. non-diabetic results across all specimens, locations, and testing frequencies demonstrated altered mechanical properties with significantly increased modulus (1146.7 vs. 593.0 kPa) but no change in energy loss (68.5 vs. 67.9%). All tissue demonstrated strain rate dependence and tissue beneath the calcaneus was found to have decreased modulus and energy loss compared to other areas. The results of this study could be used to generate material properties for all areas of the plantar soft tissue in diabetic or non-diabetic feet, with implications for foot computational modeling efforts and potentially for pressure alleviating footwear that could reduce plantar ulcer incidence.     

Comparing the MRI appearance of the lymph nodes and spleen in wild-type and immuno-deficient mouse strains

The goal of this study was to investigate the normal MRI appearance of lymphoid organs in immuno-competent and immuno-deficient mice commonly used in research. Four mice from each of four different mouse strains (nude, NOG, C57BL/6, CB-17 SCID (SCID)) were imaged weekly for one month. Images were acquired with a 3D balanced steady state free precession (bSSFP) sequence. The volume of the lymph nodes and spleens were measured from MR images. In images of nude and SCID mice, lymph nodes sometimes contained a hyperintense region visible on MRI images. Volumes of the nodes were highly variable in nude mice. Nodes in SCID mice were smaller than in nude or C57Bl/6 mice (p<0.0001). Lymph node volumes changed slightly over time in all strains. The spleens of C57Bl/6 and nude mice were similar in size and appearance. Spleens of SCID and NOG mice were significantly smaller (p<0.0001) and abnormal in appearance. The MRI appearance of the normal lymph nodes and spleen varies considerably in the various mouse strains examined in this study. This is important to recognize in order to avoid the misinterpretation of MRI findings as abnormal when these strains are used in MRI imaging studies.     

Preserving immunogenicity of lethally irradiated viral and bacterial vaccine epitopes using a radio- protective Mn2+-Peptide complex from Deinococcus

Although pathogen inactivation by γ-radiation is an attractive approach for whole-organism vaccine development, radiation doses required to ensure sterility also destroy immunogenic protein epitopes needed to mount protective immune responses. We demonstrate the use of a reconstituted manganous peptide complex from the radiation-resistant bacterium Deinococcus radiodurans to protect protein epitopes from radiation-induced damage and uncouple it from genome damage and organism killing. The Mn(2+) complex preserved antigenic structures in aqueous preparations of bacteriophage lambda, Venezuelan equine encephalitis virus, and Staphylococcus aureus during supralethal irradiation (25-40 kGy). An irradiated vaccine elicited both antibody and Th17 responses, and induced B and T cell-dependent protection against methicillin-resistant S. aureus (MRSA) in mice. Structural integrity of viruses and bacteria are shown to be preserved at radiation doses far above those which abolish infectivity. This approach could expedite vaccine production for emerging and established pathogens for which no protective vaccines exist.     

Structure, fluorescence quenching and antioxidant activity of a carbohydrate polymer from Eugenia jambolana

Natural products provide an excellent source for novel antioxidants. Herein, we have studied the water-extracted carbohydrate polymer (WE) of Eugenia jambolana using chemical, chromatographic, and spectroscopic methods. A 116 kDa arabinogalactan containing p-coumaric and ferulic acids in monomeric and dimeric forms has been isolated. Cellulase generated oligomeric fragments containing ester linked phenolic acids were also characterized. The antioxidant capacity of this carbohydrate polymer is comparable to butylated hydroxy anisole and butylated hydroxy toluene. Interaction of WE with bovine serum albumin (BSA) was studied by fluorescence quenching measurement. Conformational change of BSA at high carbohydrate polymer concentration was indicated.