One-Step Purification and N-Terminal Functionalization of Bioactive Proteins via Atypically Split Inteins.

Site-specific installation of non-natural functionality onto proteins has enabled countless applications in biotechnology, chemical biology, and biomaterials science. Though the N-terminus is an attractive derivatization location, prior methodologies targeting this site have suffered from low selectivity, a limited selection of potential chemical modifications, and/or challenges associated with divergent protein purification/modification steps. In this work, we harness the atypically split VidaL intein to simultaneously N-functionalize and purify homogeneous protein populations in a single step. Our method─referred to as VidaL-tagged expression and protein ligation (VEPL)─enables modular and scalable production of N-terminally modified proteins with native bioactivity. Demonstrating its flexibility and ease of use, we employ VEPL to combinatorially install 4 distinct (multi)functional handles (e.g., biotin, alkyne, fluorophores) to the N-terminus of 4 proteins that span three different classes: fluorescent (Enhanced Green Fluorescent Protein, mCherry), enzymatic (ß-lactamase), and growth factor (epidermal growth factor). Moving forward, we anticipate that VEPL's ability to rapidly generate and isolate N-modified proteins will prove useful across the growing fields of applied chemical biology.

Method to derive the infrared complex refractive indices n(λ) and k(λ) for organic solids from KBr pellet absorption measurements.

Obtaining the complex refractive index vectors n(ν~) and k(ν~) allows calculation of the (infrared) reflectance spectrum that is obtained from a solid in any of its many morphological forms. We report an adaptation to the KBr pellet technique using two gravimetric dilutions to derive quantitative n(ν~)/k(ν~) for dozens of powders with greater repeatability. The optical constants of bisphenol A and sucrose are compared to those derived by other methods, particularly for powdered materials. The variability of the k values for bisphenol A was examined by 10 individual measurements, showing an average coefficient of variation for k peak heights of 5.6%. Though no established standards exist, the pellet-derived k peak values of bisphenol A differ by 11% and 31% from their single-angle- and ellipsometry-derived values, respectively. These values provide an initial estimate of the precision and accuracy of complex refractive indices that can be derived using this method. Limitations and advantages of the method are discussed, the salient advantage being a more rapid method to derive n/k for those species that do not readily form crystals or specular pellets.

Chemical and Biological Engineering Strategies to Make and Modify Next-Generation Hydrogel Biomaterials.

There is a growing interest in the development of technologies to probe and direct in vitro cellular function for fundamental organoid and stem cell biology, functional tissue and metabolic engineering, and biotherapeutic formulation. Recapitulating many critical aspects of the native cellular niche, hydrogel biomaterials have proven to be a defining platform technology in this space, catapulting biological investigation from traditional two-dimensional (2D) culture into the 3D world. Seeking to better emulate the dynamic heterogeneity characteristic of all living tissues, global efforts over the last several years have centered around upgrading hydrogel design from relatively simple and static architectures into stimuli-responsive and spatiotemporally evolvable niches. Towards this end, advances from traditionally disparate fields including bioorthogonal click chemistry, chemoenzymatic synthesis, and DNA nanotechnology have been co-opted and integrated to construct 4D-tunable systems that undergo preprogrammed functional changes in response to user-defined inputs. In this Review, we highlight how advances in synthetic, semisynthetic, and bio-based chemistries have played a critical role in the triggered creation and customization of next-generation hydrogel biomaterials. We also chart how these advances stand to energize the translational pipeline of hydrogels from bench to market and close with an outlook on outstanding opportunities and challenges that lay ahead.

Solid phase characterization and transformation of illite mineral with gas-phase ammonia treatment.

In situ remediation applications of ammonia (NH3) gas have potential for sequestration of subsurface contamination. Ammonia gas injections initially increase the pore water pH leading to mineral dissolution followed by formation of secondary precipitates as the pH is neutralized. However, there is a lack of understanding of fundamental alteration processes due to NH3 treatment. In these batch studies, phyllosilicate minerals (illite and montmorillonite) were exposed to NH3 gas with subsequent aeration to simulate in situ remediation. Following treatments, solids were characterized using a variety of techniques, including X-ray diffraction, N2 adsorption-desorption analysis for surface area, Fourier transform infrared (FTIR) spectroscopy, nuclear magnetic resonance (NMR), and microscopy methods to investigate physicochemical transformations. Results indicate that, at high pH, the clays are altered as observed by differences in morphology and particle size via microscopy. However, the two clays interact differently with NH3. While montmorillonite interlayers collapsed due to intercalation, illite layers were unaffected as confirmed by FTIR analysis. Further, structural changes in silicate ([SiO4]n-) and aluminol (Al-OH) groups were identified by NMR and FTIR. This research showed that mineral alteration processes occur during and after NH3 gas treatment which may be used to remove radionuclides from the aqueous phase through sorption, co-precipitation, and coating with secondary phyllosilicate alteration products.

Age and aerobic training status effects on plasma and skeletal muscle tPA and PAI-1.

INTRODUCTION: Reductions in fibrinolytic potential occur with both aging and physical inactivity and are associated with an increased cardiovascular disease risk. Plasmin, the enzyme responsible for the enzymatic degradation of fibrin clots, is activated by tissue plasminogen activator (tPA), while plasminogen activator inhibitor-1 (PAI-1) inhibits its activation. Currently, fibrinolysis research focuses almost exclusively on changes within the plasma. However, tPA and PAI-1 are expressed by human skeletal muscle (SM). Currently, no studies have focused on changes in SM fibrinolytic activity with regard to aging and aerobic fitness. PURPOSE: The purpose of this study was to cross-sectionally evaluate effects of age and aerobic fitness on tPA and PAI-1 expressions and activity in SM. METHODS: Twenty-six male subjects were categorized into the following groups: (1) young aerobically trained (n = 8); (2) older aerobically trained (n = 6); (3) young aerobically untrained (n = 7); and (4) older aerobically untrained (n = 5). Muscle biopsies were obtained from each subject. SM tPA activity was assessed using gel zymography and SM tPA and PAI-1 expressions were assessed using RT-PCR. RESULTS: Trained subjects had higher SM tPA activity compared to untrained (25.3 ± 2.4 × 10(3) vs. 21.5 ± 5.6 × 10(3) pixels, respectively; p = 0.03) with no effect observed for age. VO2 max and SM tPA activity were also significantly correlated (r = 0.42; p < 0.04). SM tPA expression was higher in older participants, but no effect of fitness level was observed. No differences were observed for PAI-1 expression in SM. CONCLUSIONS: Higher levels of aerobic fitness are associated with increased fibrinolytic activity in SM.

Comparison of oxidative capacity among leg muscles in humans using gated 31P 2-D chemical shift imaging.

In many small animals there are distinct differences in fiber-type composition among limb muscles, and these differences typically correspond to marked disparities in the oxidative capacities. However, whether there are similar differences in the oxidative capacity among leg muscles in humans is less clear. The purpose of this study was to compare the rate of phosphocreatine (PCr) recovery, a functional in vivo marker of oxidative capacity, in the lateral and medial gastrocnemius, soleus, and the anterior compartment of the leg (primarily the tibialis anterior) of humans. Subjects performed plantar flexion and dorsiflexion gated exercise protocols consisting of 70 sets of three rapid dynamic contractions (<2.86 s) at 20 s intervals (total: 23.3 min). Starting after the sixth set of contractions, (31)P 2-D CSI (8 x 8 matrix, 14-16 cm FOV, 3 cm slice, TR 2.86 s) were acquired via a linear transmit/receive surface coil using a GE 3T Excite System. The CSI data were zero-filled (32 x 32) and a single FID was produced for each time point in the lateral and medial gastrocnemius, soleus, and anterior compartment. The time constant for PCr recovery was calculated from tau = -Deltat/ln[D/(D + Q)], where Q is the percentage change in PCr due to contraction during the steady-state portion of the protocol, D the additional drop in PCr from rest, and Deltat is the interval between contractions. The tau of PCr recovery was longer (p < 0.05) in the anterior compartment (32 +/- 3 s) than in the lateral (23 +/- 2 s) and medial gastrocnemius muscles (24 +/- 3 s) and the soleus (22 +/- 3 s) muscles. These findings suggest that the oxidative capacity is lower in the anterior compartment than in the triceps surae muscles and is consistent with the notion that fiber-type phenotypes vary among the leg muscles of humans.