Probing binding mechanism of interleukin-6 and olokizumab: in silico design of potential lead antibodies for autoimmune and inflammatory diseases

Computer-aided antibody engineering has been successful in the design of new biologics for disease diagnosis and therapeutic interventions. Interleukin-6 (IL-6), a well-recognized drug target for various autoimmune and inflammatory diseases such as rheumatoid arthritis, multiple sclerosis, and psoriasis, was investigated in silico to design potential lead antibodies. Here, crystal structure of IL-6 along with monoclonal antibody olokizumab was explored to predict antigen–antibody (Ag???Ab)-interacting residues using DiscoTope, Paratome, and PyMOL. Tyr56, Tyr103 in heavy chain and Gly30, Ile31 in light chain of olokizumab were mutated with residues Ser, Thr, Tyr, Trp, and Phe. A set of 899 mutant macromolecules were designed, and binding affinity of these macromolecules to IL-6 was evaluated through Ag???Ab docking (ZDOCK, ClusPro, and Rosetta server), binding free-energy calculations using Molecular Mechanics/Poisson Boltzman Surface Area (MM/PBSA) method, and interaction energy estimation. In comparison to olokizumab, eight newly designed theoretical antibodies demonstrated better result in all assessments. Therefore, these newly designed macromolecules were proposed as potential lead antibodies to serve as a therapeutics option for IL-6-mediated diseases.     

The cytoplasmic tail of CD4 targets chimeric molecules to a degradative pathway.

Many different cell surface receptors undergo endocytosis via coated pits. Once having entered the cell, the receptors are sorted into diverse pathways. Which path a given receptor will follow is determined by signals inherent in the receptor's structure. The nature of these structural features is not yet known. In this study, we have taken the approach of constructing chimeric molecules to localize the domain of the T-cell surface molecule CD4 which is responsible for targeting it for degradation. Chimeric molecules bearing the cytoplasmic domain of CD4 and the extracellular domain of either the low-density lipoprotein receptor or a major histocompatibility complex (MHC) class I molecule were both internalized in response to phorbol 12-myristate 13-acetate and were subsequently degraded, indicating that the cytoplasmic tail of CD4 contains all the information required for both processes. The ability to modulate the level of MHC class I molecules on the cell surface offers an approach to investigating quantitative aspects of antigen presentation, the initial possibilities of which are explored herein.     

Circadian rhythm of airways caliber and its autonomic modulation

ABSTRACT

The autonomic nervous system (ANS) is one of the effector pathways for circadian variation of many physiological parameters. Autonomic tone and airways caliber have been reported to exhibit circadian variation in separate studies. A simultaneous investigation of heart rate variability (HRV) and airway caliber might ascertain how airway caliber is modulated by autonomic tone. This study was planned to identify the variations in airway caliber and autonomic function tone during a 24-hour span. A total of 56 healthy male subjects with almost similar daily routines were studied. Time domain, frequency domain and nonlinear analysis of R-R interval from 5 min electrocardiogram (ECG) was done seven times during the daytime wake span at 3-hour intervals starting at 05:00 h in the morning until 23:00 h in the night. Simultaneously peak expiratory flow rate (PEFR) was determined using a mini Wright’s peak flow meter. Rhythmometric analysis was done for PEFR and HRV parameters. Significant circadian variation in low frequency (LF) and high frequency (HF) variance was identified in this group of healthy subjects. The circadian rhythm of LF variance was characterized by a gradual increase and corresponding reciprocal change in HF variance from morning until night. The LF/HF ratio and SD2/SD1 ratio reflecting sympatho-vagal balance showed low to high values from morning to evening. The acrophase of the PEFR temporal pattern is similar to that of LF power and almost opposite in phase to that of HF power. PEFR is positively correlated with LF power. The circadian rhythm of airway caliber co-varies with cardiac autonomic tone. It appears that the temporal pattern of cardiac autonomic tone precedes in time that of airways caliber, thereby suggesting the latter operates under the modulatory effect of the 24-hour pattern in sympatho-vagal balance.     

The Cytoprotective Effects of E-α-(4-Methoxyphenyl)-2’,3,4,4'-Tetramethoxychalcone (E-α-p-OMe-C6H4-TMC)—A Novel and Non-Cytotoxic HO-1 Inducer

Cell protection against different noxious stimuli like oxidative stress or chemical toxins plays a central role in the treatment of many diseases. The inducible heme oxygenase isoform, heme oxygenase-1 (HO-1), is known to protect cells against a variety of harmful conditions including apoptosis. Because a number of medium strong electrophiles from a series of α-X-substituted 2’,3,4,4’-tetramethoxychalcones (α-X-TMCs, X = H, F, Cl, Br, I, CN, Me, p-NO₂-C₆H₄, Ph, p-OMe-C₆H₄, NO₂, CF₃, COOEt, COOH) had proven to activate Nrf2 resulting in HO-1 induction and inhibit NF-κB downstream target genes, their protective effect against staurosporine induced apoptosis and reactive oxygen species (ROS) production was investigated. RAW264.7 macrophages treated with 19 different chalcones (15 α-X-TMCs, chalcone, 2’-hydroxychalcone, calythropsin and 2’-hydroxy-3,4,4’-trimethoxychalcone) prior to staurosporine treatment were analyzed for apoptosis and ROS production, as well as HO-1 protein expression and enzyme activity. Additionally, Nrf2 and NF-κB activity was assessed. We found that amongst all tested chalcones only E-α-(4-methoxyphenyl)-2’,3,4,4''-tetramethoxychalcone (E-α-p-OMe-C₆H₄-TMC) demonstrated a distinct, statistically significant antiapoptotic effect in a dose dependent manner, showing no toxic effects, while its double bond isomer Z-α-p-OMe-C₆H₄-TMC displayed no significant activity. Also, E-α-p-OMe-C₆H₄-TMC induced HO-1 protein expression and increased HO-1 activity, whilst inhibition of HO-1 by SnPP-IX abolished its antiapoptotic effect. The only weakly electrophilic chalcone E-α-p-OMe-C₆H₄-TMC reduced the staurosporine triggered formation of ROS, while inducing the translocation of Nrf2 into the nucleus. Furthermore, staurosporine induced NF-κB activity was attenuated following E-α-p-OMe-C₆H₄-TMC treatment. Overall, E-α-p-OMe-C₆H₄-TMC demonstrated its effective cytoprotective potential via a non-toxic induction of HO-1 in RAW264.7 macrophages. The observed cytoprotective effect may partly be related to both, the activation of the Nrf2- and inhibition of the NF-κB pathway.     

Can Newts Cope with the Heat? Disparate Thermoregulatory Strategies of Two Sympatric Species in Water

Many ectotherms effectively reduce their exposure to low or high environmental temperatures using behavioral thermoregulation. In terrestrial ectotherms, thermoregulatory strategies range from accurate thermoregulation to thermoconformity according to the costs and limits of thermoregulation, while in aquatic taxa the quantification of behavioral thermoregulation have received limited attention. We examined thermoregulation in two sympatric newt species, Ichthyosaura alpestris and Lissotriton vulgaris, exposed to elevated water temperatures under semi-natural conditions. According to a recent theory, we predicted that species for which elevated water temperatures pose a lower thermal quality habitat, would thermoregulate more effectively than species in thermally benign conditions. In the laboratory thermal gradient, L. vulgaris maintained higher body temperatures than I. alpestris. Semi-natural thermal conditions provided better thermal quality of habitat for L. vulgaris than for I. alpestris. Thermoregulatory indices indicated that I. alpestris actively thermoregulated its body temperature, whereas L. vulgaris remained passive to the thermal heterogeneity of aquatic environment. In the face of elevated water temperatures, sympatric newt species employed disparate thermoregulatory strategies according to the species-specific quality of the thermal habitat. Both strategies reduced newt exposure to suboptimal water temperatures with the same accuracy but with or without the costs of thermoregulation. The quantification of behavioral thermoregulation proves to be an important conceptual and methodological tool for thermal ecology studies not only in terrestrial but also in aquatic ectotherms.     

Modelling of the disulphide-swapped isomer of human insulin-like growth factor-1: implications for receptor binding.

Insulin-like growth factor-1 (IGF-1) is a serum protein which unexpectedly folds to yield two stable tertiary structures with different disulphide connectivities; native IGF-1 [18-61,6-48,47-52] and IGF-1 swap [18-61,6-47, 48-52]. Here we demonstrate in detail the biological properties of recombinant human native IGF-1 and IGF-1 swap secreted from Saccharomyces cerevisiae. IGF-1 swap had a approximately 30 fold loss in affinity for the IGF-1 receptor overexpressed on BHK cells compared with native IGF-1.The parallel increase in dose required to induce negative cooperativity together with the parallel loss in mitogenicity in NIH 3T3 cells implies that disruption of the IGF-1 receptor binding interaction rather than restriction of a post-binding conformational change is responsible for the reduction in biological activity of IGF-1 swap. Interestingly, the affinity of IGF-1 swap for the insulin receptor was approximately 200 fold lower than that of native IGF-1 indicating that the binding surface complementary to the insulin receptor (or the ability to attain it) is disturbed to a greater extent than that to the IGF-1 receptor. A 1.0 ns high-temperature molecular dynamics study of the local energy landscape of IGF-1 swap resulted in uncoiling of the first A-region alpha-helix and a rearrangement in the relative orientation of the A- and B-regions. The model of IGF-1 swap is structurally homologous to the NMR structure of insulin swap and CD spectra consistent with the model are presented. However, in the model of IGF-1 swap the C-region has filled the space where the first A-region alpha-helix has uncoiled and this may be hindering interaction of Val44 with the second insulin receptor binding pocket.     

Host location in Oomyzus gallerucae (Hymenoptera: Eulophidae), an egg parasitoid of the elm leaf beetle Xanthogaleruca luteola (Coleoptera: Chrysomelidae)

Eggs of the elm leaf beetle Xanthogaleruca luteola are often heavily attacked by the chalcidoid wasp Oomyzus gallerucae. We studied the chemical signals mediating interactions between the egg parasitoid, its host, and the plant Ulmus campestris. Olfactometer bioassays with O. gallerucae showed that volatiles of the host-plant complex attract the parasitoid. In order to determine the source of attractive volatiles within this host-plant-complex, we tested separately the effect of odours of eggs, gravid elm leaf beetle females, faeces of the beetles and elm twigs (with undamaged leaves and leaves damaged either mechanically or by feeding of the beetles). Odours of faeces of the elm leaf beetle were attractive, whereas neither volatiles from eggs nor from gravid females acted as attractants. Volatiles from undamaged or damaged plants did not elicit a positive reaction in O. gallerucae, whereas volatiles from feeding-damaged plants onto which host eggs had been deposited were attractive. This latter result suggests that it is not feeding but deposition of host eggs onto elm leaves that induces the production of plant volatiles attractive to the egg parasitoid. Investigations of the search patterns of O. gallerucae within the habitat by laboratory bioassays revealed that the egg parasitoid encounters host eggs by chance. Contact kairomones from faeces were demonstrated to be important in microhabitat acceptance, while contact kairomones isolated from the host eggs are relevant for host recognition. Received: 12 February 1997 / Accepted: 29 April 1997     

The impact of phenotypic heterogeneity of tumour cells on treatment and relapse dynamics

Intratumour heterogeneity is increasingly recognized as a frequent problem for cancer treatment as it allows for the evolution of resistance against treatment. While cancer genotyping becomes more and more established and allows to determine the genetic heterogeneity, less is known about the phenotypic heterogeneity among cancer cells. We investigate how phenotypic differences can impact the efficiency of therapy options that select on this diversity, compared to therapy options that are independent of the phenotype. We employ the ecological concept of trait distributions and characterize the cancer cell population as a collection of subpopulations that differ in their growth rate. We show in a deterministic model that growth rate-dependent treatment types alter the trait distribution of the cell population, resulting in a delayed relapse compared to a growth rate-independent treatment. Whether the cancer cell population goes extinct or relapse occurs is determined by stochastic dynamics, which we investigate using a stochastic model. Again, we find that relapse is delayed for the growth rate-dependent treatment type, albeit an increased relapse probability, suggesting that slowly growing subpopulations are shielded from extinction. Sequential application of growth rate-dependent and growth rate-independent treatment types can largely increase treatment efficiency and delay relapse. Interestingly, even longer intervals between decisions to change the treatment type may achieve close-to-optimal efficiencies and relapse times. Monitoring patients at regular check-ups may thus provide the temporally resolved guidance to tailor treatments to the changing cancer cell trait distribution and allow clinicians to cope with this dynamic heterogeneity.     

Malathion biodegradation by a psychrotolerant bacteria Ochrobactrum sp. M1D and metabolic pathway analysis

An organophosphorus pesticide malathion biodegradation was investigated by using the bacteria Ochrobactrum sp. M1D isolated from a soil sample of peach orchards in Palampur, District Kangra, Himachal Pradesh (India). The bacterium was able to utilize malathion as the sole source of carbon and energy. The isolated bacterium was found psychrotolerant and could degrade 100% of 100 mg l⁻¹ malathion in minimal salt medium at 20°C, pH 7·0 within 12 days with no major significant metabolites left at the end of the study. Through GCMS analysis, methyl phosphate, diethyl maleate, and diethyl 2-mercaptosuccinate were detected and identified as the major pathway metabolites. Based on the GCMS profile, three probable degradation pathways were interpreted. The present study is the first report of malathion biodegradation at both the psychrophilic and mesophilic conditions by any psychrotolerant strain and also through multiple degradation pathways. In the future, the strain can be explored to bio-remediate the malathion contaminated soil in the cold climatic region and to utilize the enzymatic systems for advanced biotechnology applications.