Streptomycin affinity depends on 13 amino acids forming a loop in homology modelled ribosomal S12 protein (rpsL gene) of Lysinibacillus sphaericus DSLS5 associated with marine sponge (Tedania anhelans)

Streptomycin, an antibiotic used against microbial infections, inhibits the protein synthesis by binding to ribosomal protein S12, encoded by rpsL12 gene, and associated mutations cause streptomycin resistance. A streptomycin resistant, Lysinibacillus sphaericus DSLS5 (MIC >300 µg/mL for streptomycin), was isolated from a marine sponge (Tedania anhelans). The characterisation of rpsL12 gene showed a region having similarity to long terminal repeat sequences of murine lukemia virus which added 13 amino acids for loop formation in RpsL12; in addition, a K56R mutation which corresponds to K43R mutation present in streptomycin-resistant Escherichia coli is also present. The RpsL12 protein was modelled and compared with that of Lysinibacillus boronitolerans, Escherichia coli and Mycobacterium tuberculosis. The modelled proteins docked with streptomycin indicate compound had less affinity. The effect of loop on streptomycin resistance was analysed by constructing three different models of RpsL12 by, (i) removing both loop and mutation, (ii) removing the loop alone while retaining the mutation and (iii) without mutation having loop. The results showed that the presence of loop causes streptomycin resistance (decreases the affinity), and it further enhanced in the presence of mutation at 56th codon. Further study will help in understanding the evolution of streptomycin resistance in organisms.     

Optimization and effect of dairy industrial waste as media components in the production of hyaluronic acid by Streptococcus thermophilus

Hyaluronic acid (HA) production using a dairy industrial waste is a more cost-efficient strategy than using an expensive synthetic medium. In this study, we investigated the production of HA using Streptococcus thermophilus under shake flask conditions using dairy industrial waste as nutritional supplements, namely whey permeate (WP) and whey protein hydrolysate (WPH). Preliminary screening using Plackett–Burman design exhibited WP, WPH, initial pH, and inoculum size as significant factors influencing HA titer. Response surface methodology design of four factors was formulated at three levels for enhanced production of HA. Shake flask HA fermentation by S. thermophilus was performed under global optimized process conditions and the optimal HA titer (342.93?mg?L^?1) corroborates with Box–Behnken design prediction. The molecular weight of HA was elucidated as 9.22–9.46?kDa. The ultralow-molecular weight HA reported in this study has a potential role in drug and gene delivery applications.     

Computational analysis reveals the coupling between bistability and the sign of a feedback loop in a TGF-β1 activation model

Background

Bistable behaviors are prevalent in cell signaling and can be modeled by ordinary differential equations (ODEs) with kinetic parameters. A bistable switch has recently been found to regulate the activation of transforming growth factor-β1 (TGF-β1) in the context of liver fibrosis, and an ordinary differential equation (ODE) model was published showing that the net activation of TGF-β1 depends on the balance between two antagonistic sub-pathways.

Results

Through modeling the effects of perturbations that affect both sub-pathways, we revealed that bistability is coupled with the signs of feedback loops in the model. We extended the model to include calcium and Krüppel-like factor 2 (KLF2), both regulators of Thrombospondin-1 (TSP1) and Plasmin (PLS). Increased levels of extracellular calcium, which alters the TSP1-PLS balance, would cause high levels of TGF-β1, resembling a fibrotic state. KLF2, which suppresses production of TSP1 and plasminogen activator inhibitor-1 (PAI1), would eradicate bistability and preclude the fibrotic steady-state. Finally, the loop PLS???TGF-β1???PAI1 had previously been reported as negative feedback, but the model suggested a stronger indirect effect of PLS down-regulating PAI1 to produce positive (double-negative) feedback in a fibrotic state. Further simulations showed that activation of KLF2 was able to restore negative feedback in the PLS???TGF-β1???PAI1 loop.

Conclusions

Using the TGF-β1 activation model as a case study, we showed that external factors such as calcium or KLF2 can induce or eradicate bistability, accompanied by a switch in the sign of a feedback loop (PLS???TGF-β1???PAI1) in the model. The coupling between bistability and positive/negative feedback suggests an alternative way of characterizing a dynamical system and its biological implications.

     

Cu(I) recognition via cation-pi and methionine interactions in CusF

Methionine-rich motifs have an important role in copper trafficking factors, including the CusF protein. Here we show that CusF uses a new metal recognition site wherein Cu(I) is tetragonally displaced from a Met2His ligand plane toward a conserved tryptophan. Spectroscopic studies demonstrate that both thioether ligation and strong cation-pi interactions with tryptophan stabilize metal binding. This novel active site chemistry affords mechanisms for control of adventitious metal redox and substitution chemistry.     

Assignment of function to histidines 260 and 298 by engineering the E1 component of the Escherichia coli 2-oxoglutarate dehydrogenase complex; substitutions that lead to acceptance of substrates lacking the 5-carboxyl group

The first component (E1o) of the Escherichia coli 2-oxoglutarate dehydrogenase complex (OGDHc) was engineered to accept substrates lacking the 5-carboxylate group by subjecting H260 and H298 to saturation mutagenesis. Apparently, H260 is required for substrate recognition, but H298 could be replaced with hydrophobic residues of similar molecular volume. To interrogate whether the second component would allow synthesis of acyl-coenzyme A derivatives, hybrid complexes consisting of recombinant components of OGDHc (o) and pyruvate dehydrogenase (p) enzymes were constructed, suggesting that a different component is the "gatekeeper" for specificity for these two multienzyme complexes in bacteria, E1p for pyruvate but E2o for 2-oxoglutarate.     

Purpose-driven biomaterials research in liver-tissue engineering

Bottom-up engineering of microscale tissue ("microtissue") constructs to recapitulate partially the complex structure-function relationships of liver parenchyma has been realized through the development of sophisticated biomaterial scaffolds, liver-cell sources, and in vitro culture techniques. With regard to in vivo applications, the long-lived stem/progenitor cell constructs can improve cell engraftment, whereas the short-lived, but highly functional hepatocyte constructs stimulate host liver regeneration. With regard to in vitro applications, microtissue constructs are being adapted or custom-engineered into cell-based assays for testing acute, chronic and idiosyncratic toxicities of drugs or pathogens. Systems-level methods and computational models that represent quantitative relationships between biomaterial scaffolds, cells and microtissue constructs will further enable their rational design for optimal integration into specific biomedical applications.