Identification of N-benzyltetrahydroisoquinolines as novel anti-neuroinflammatory agents

A series of simplified berberine analogs was designed, synthesized, and evaluated for anti-inflammatory activity. SAR studies identified N-benzyltetrahydroisoquinoline 7d as a potent berberine analog. 7d suppressed LPS-induced inflammatory cytokine levels in both BV2 cells and primary microglia. Taken together, our results suggest that simplified BB analogs have therapeutic potential as a novel class of anti-neuroinflammatory agents.     

Landfill methane oxidation in soil and bio-based cover systems: a review

Mitigation of landfill gases has gained the utmost importance in recent years due to the increase in methane (CH₄) emissions from landfills worldwide. This, in turn, can contribute to global warming and climatic changes. The concept of microbially mediated methane oxidation in landfill covers by using methanotrophic microorganisms has been widely adopted as a method to counter the rise in methane emissions. Traditionally, landfill soil covers were used to achieve methane oxidation, thereby reducing methane emissions. Meanwhile, the continual rise of CH₄ emissions from landfills and the significant need to and importance of developing a better technology has led researchers to explore different methods to enhance microbial methane oxidation by using organic rich materials such as compost in landfill covers. The development and field application of such bio-based systems, explored by various researches worldwide, eventually led to more widely accepted and better performing cover systems capable of reducing CH₄ emissions from landfills. However, the long-term performance of bio-based cover systems were found to be negatively affected by factors such as the material’s ability to self-degrade, causing CH₄ to be generated rather than oxidized as well as the greater potential for forming pore-clogging exopolymeric substances. In order to design an effective cover system for landfills, it is essential to have a thorough understanding of the concepts incorporated into methodologies currently in favor along with their pros and cons. This review summarizes previous laboratory and field-scale studies conducted on various soil and bio-based cover systems, along with the modeling mechanisms adopted for quantifying CH₄ oxidation rates. Finally, several issues and challenges in developing effective and economical soil and bio-based cover systems are presented.     

Estrogen receptor beta and truncated variants enhance the expression of transfected MMP-1 promoter constructs in response to specific mechanical loading

Background

Joint diseases such as osteoarthritis (OA) predominantly afflict post-menopausal women, suggesting a pertinent role for female hormones. Estrogen receptor beta (ER-β) has been detected in connective tissues of the knee joint suggesting that these tissues are responsive to the hormone estrogen. Matrix metalloproteinase-1 (MMP-1) activity contributes to cartilage degradation, a key factor leading to OA development in synovial joints. Two polymorphic forms of MMP-1 exist due to a deletion/insertion of the guanine residue in the promoter, and the 2G allelic variant of MMP-1 exhibits more activity than the 1G allele. Previous studies have demonstrated that the polymorphic forms of the human MMP-1 are influenced by the modulating effects of estrogen receptor isoforms. In addition to hormonal influences, physiological factors such as altered mechanical loading are also contributory features of OA. In the present study, the combined influence of biomechanical and hormonal variables on the activity of MMP-1 isoforms was evaluated. We hypothesized that the combined effects of ER-β and sheer stress will differentially activate the two allelic forms of MMP-1 in a hormone-independent manner.

Methods

HIG-82 synoviocytes were transiently transfected with 1G or 2G alleles (±) ER-β and subjected to either shear or equibiaxial stress. Next, 1G/2G promoter activity was measured to determine the combined influence of physiological stimuli. Truncated ER-β constructs were used to determine the importance of different domains of ER-β on 1G/2G activation.

Results

The 2G allele exhibited a constitutively higher activity than the 1G allele, which was further increased when the transfected cells were subject to shear stress, but not equibiaxial stress. Moreover, the combination of ER-β and shear stress further increased the activity levels of the 1G/2G allelic variants. Additionally, select AF-2 truncated ER-β variants led to increased activity levels for the 2G allele, indicating the AF-1 domain was likely involved in the response to mechanical stimulation.

Conclusions

These results suggest that the 1G/2G alleles of MMP-1 are influenced by specific mechanical stimuli like shear stress, as well as the ER-β receptor. These findings contribute to the potential allelic involvement in connective tissue diseases such as OA in females compared to males.

     

Estimating binding affinities of the nicotinic receptor for low-efficacy ligands using mixtures of agonists and two-dimensional concentration-response relationships

The phenomenon of ligand-induced ion channel gating hinges upon the ability of a receptor channel to bind ligand molecules with conformation-specific affinities. However, our understanding of this fundamental phenomenon is notably limited, not only because the changes in binding site structure and ligand conformation that occur upon gating are largely unknown but, also, because the strength of these ligand-receptor interactions are experimentally elusive. Both high- and low-efficacy ligands pose a number of analytical and experimental challenges that can render the estimation of their conformation-specific binding affinities impossible. In this paper, we present a novel assay that overcomes some of the hurdles presented by weak agonists of the muscle nicotinic receptor and allows the estimation of their closed-state affinities. The method, which we have termed the "activation-competition" assay, consists of a single-channel concentration-response assay performed in the presence of a binary mixture of ligands of widely different efficacies. By plotting the channel response (i.e., the open probability) as a function of the concentration of each agonist in the mixture, interpreting the observed response in the framework of a plausible kinetic scheme, and fitting the open probability surface with the corresponding function, the affinities of the closed receptor for the two agonists can be simultaneously extracted as free parameters. Here, we applied this methodology to estimate the closed-state affinity of the muscle nicotinic receptor for choline (a very weak agonist) using acetylcholine (ACh) as the partner in the mixture. We estimated the dissociation equilibrium constant of choline (K(D)) from the wild type's closed state to be 4.1 +/- 0.5 mM (and that of ACh to be 106 +/- 6 microM). We also discuss the use of accurate estimates of affinities for low-efficacy agonists as a tool to discriminate between binding and gating effects of mutations, and in the context of the rational design of therapeutic drugs.     

Indel‐seq: a fast‐forward genetics approach for identification of trait‐associated putative candidate genomic regions and its application in pigeonpea (Cajanus cajan)

Identification of candidate genomic regions associated with target traits using conventional mapping methods is challenging and time‐consuming. In recent years, a number of single nucleotide polymorphism (SNP)‐based mapping approaches have been developed and used for identification of candidate/putative genomic regions. However, in the majority of these studies, insertion–deletion (Indel) were largely ignored. For efficient use of Indels in mapping target traits, we propose Indel‐seq approach, which is a combination of whole‐genome resequencing (WGRS) and bulked segregant analysis (BSA) and relies on the Indel frequencies in extreme bulks. Deployment of Indel‐seq approach for identification of candidate genomic regions associated with fusarium wilt (FW) and sterility mosaic disease (SMD) resistance in pigeonpea has identified 16 Indels affecting 26 putative candidate genes. Of these 26 affected putative candidate genes, 24 genes showed effect in the upstream/downstream of the genic region and two genes showed effect in the genes. Validation of these 16 candidate Indels in other FW‐ and SMD‐resistant and FW‐ and SMD‐susceptible genotypes revealed a significant association of five Indels (three for FW and two for SMD resistance). Comparative analysis of Indel‐seq with other genetic mapping approaches highlighted the importance of the approach in identification of significant genomic regions associated with target traits. Therefore, the Indel‐seq approach can be used for quick and precise identification of candidate genomic regions for any target traits in any crop species.     

Microspheres based on mannosylated lysine-co-sodium alginate for macrophage-specific delivery of isoniazid

The present investigation reports coupling of ?- and α-amino groups of lysine (LS) with mannose (m-LS) and sodium alginate (SA), respectively, to reduce its toxicity. Prepared conjugate, m-LS-co-SA, was characterized through infra-red spectroscopy and differential scanning calorimetry. Cell viability studies were undertaken to assess the safety profile of the prepared conjugate. Microspheres, based on the conjugate, were prepared using spray drying technique and studied for targeting of isoniazid to alveolar macrophages (AMs). Pharmacokinetic studies of the optimized formulation batch were performed in Charles Foster rats.Infra-red spectral data of the synthesized conjugate were in agreement to the presumptive sequence of the conjugation process. Dispersibility, thermal stability and safety of the conjugate were conducive to its biomedical application. Microspheres, formulated from the conjugate, were of uniform size and offered satisfactory drug loading efficiency and in vitro release characteristics. X-ray diffraction studies established that drug was entrapped within the microspheres rather than being adsorbed on to the surface. Pharmacokinetic studies revealed that the conjugate could be a potential vehicle towards both active targeting of isoniazid to AMs and controlling its release rate.     

Investigating Pervaporation for In Situ Acetone Removal as Process Intensification Tool in ω-Transaminase Catalyzed Chiral Amine Synthesis

Hydrophobic pervaporation (PV), allowing for the separation of an organic component from an aqueous stream, was investigated for in situ acetone removal from a transamination reaction. A poly(dimethylsiloxane) membrane was applied in a coupled enzymatic process at 5 L scale. Among the four components, there was no loss of donor and product amines through PV which was highly desirable. However, in addition to removal of acetone, there was also an unwanted loss of acetophenone (substrate ketone) because of PV. The coupled enzyme-PV process resulted in 13% more product formation compared to the control process (where no PV was applied) after 9 h. Results from a qualitative simulation study (based on partial vapor pressures and a vapor–liquid equilibrium of the feed solution) indicated that PV might have an advantage over direct distillation strategy for selective removal of acetone from the reaction medium. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2731, 2019