Combining Chemical Protein Synthesis and Random Nonstandard Peptides Integrated Discovery for Modulating Biological Processes.

Chemical manipulation of naturally occurring peptides offers a convenient route for generating analogs to screen against different therapeutic targets. However, the limited success of the conventional chemical libraries has urged chemical biologists to adopt alternative methods such as phage and mRNA displays and create libraries of a large number of variants for the screening and selection of novel peptides. Messenger RNA (mRNA) display provides great advantages in terms of the library size and the straightforward recovery of the selected polypeptide sequences. Importantly, the integration of the flexible in vitro translation (FIT) system with the mRNA display provides the basis of the random nonstandard peptides integrated discovery (RaPID) approach for the introduction of diverse nonstandard motifs, such as unnatural side chains and backbone modifications. This platform allows the discovery of functionalized peptides with tight binding against virtually any protein of interest (POI) and therefore shows great potential in the pharmaceutical industry. However, this method has been limited to targets generated by recombinant expression, excluding its applications to uniquely modified proteins, particularly those with post-translational modifications.Chemical protein synthesis allows a wide range of changes to the protein's chemical composition to be performed, including side chain and backbone modifications and access to post-translationally modified proteins, which are often inaccessible or difficult to achieve via recombinant expression methods. Notably, d-proteins can be prepared via chemical synthesis, which has been used in mirror image phase display for the discovery of nonproteolytic d-peptide binders.Combining chemical protein synthesis with the RaPID system allows the production of a library of trillions of cyclic peptides and subsequent selection for novel cyclic peptide binders targeting a uniquely modified protein to assist in studying its unexplored biology and possibly the discovery of new drug candidates.Interestingly, the small post-translational modifier protein ubiquitin (Ub), with its various polymeric forms, regulates directly or indirectly many biochemical processes, e.g., proteasomal degradation, DNA damage repair, cell cycle regulation, etc. In this Account, we discuss combining the RaPID approach against various synthetic Ub chains for selecting effective and specific macrocyclic peptide binders. This offers an advancement in modulating central Ub pathways and provides opportunities in drug discovery areas associated with Ub signaling. We highlight experimental approaches and conceptual adaptations required to design and modulate the activity of Lys48- and Lys63-linked Ub chains by macrocyclic peptides. We also present the applications of these approaches to shed light on related biological activities and ultimately their activity against cancer. Finally, we contemplate future developments still pending in this exciting multidisciplinary field.

Exploration of Potential Anti-inflammatory cum Anti-Rheumatoid-arthritis Phyto-molecule through Integrated Green Approach: Network Pharmacology, Molecular Docking, Molecular dynamics, In-vitro and Ex-vivo study.

Rheumatoid arthritis (RA) and associated inflammatory complications are the most prevalent illnesses and can turn into fatal conditions if left untreated. Allopathic medicine is not satisfactory for curing RA. Scientific literature reports reveal that several phyto-compounds viz. flavonoids, saponins, and terpenoids, can heal joints and organs from auto-inflammatory rheumatoid arthritis and pain. Gene ontology, gene network analysis, molecular clustering, and literature review were used to optimise RA-specific highly expressed genes. In-silico molecular docking was performed to short-out potential phytomolecules (Neohesperidin dihydrochalcone (NHDC)) from 1000 datasets-library against RA and validate using MD simulation running at 100 ns. In-vitro anti-inflammatory assays of NHDC inhibited egg-albumin denaturation, IC50 of 47.739 ± 0.51 µg/ml. The ex-vivo MTT assay with NHDC rendered 67.209% inhibition at 100 µM against fd-FLS-cells. NHDC downregulated pro-inflammatory cytokine IL-17A production by 61.11% and 50% at 300 and 200 µM, respectively. Thus, this Studies recommend that NHDC may be highlighted as a novel multi-target PADI4 and JAK3 inhibitor with better efficacy and minimal toxicity in RA warranted to In-Vivo and clinical investigation. The current findings have uncovered remarkable genes and signalling pathways linked to RA, which could enhance our existing comprehension of the molecular mechanisms that drive its development and progression.

Suspension Bead Loading (SBL): An Economical Protein Delivery Platform to Study URM1's Behavior in Live Cells.

Uniquely modified synthetic proteins are difficult to produce in large quantities, which could limit their use in various in vitro settings and in cellular studies. In this study, we developed a method named "suspension bead loading" (SBL), to deliver protein molecules into suspended living cells using glass beads, which significantly reduces the amount of protein required for effective delivery. We investigated the delivery efficiency of functionally different proteins and evaluated the cytotoxic effect of our method and the chemical and functional integrity of the delivered protein. We utilized SBL to address questions related to ubiquitin-related modifier 1 (URM1). Employing minimal protein quantities, SBL has enabled us to study its behavior within live cells under different redox conditions, including subcellular localization and conjugation patterns. We demonstrate that oxidative stress alters both the localization and conjugation pattern of URM1 in cells, highlighting its possible role in cellular response to such extreme conditions.

Exocyclic and Linker Editing of Lys63-linked Ubiquitin Chains Modulators Specifically Inhibits Non-homologous End-joining Repair.

Despite the great advances in discovering cyclic peptides against protein targets, their reduced aqueous solubility, cell permeability, and activity of the cyclic peptide restrict its utilization in advanced biological research and therapeutic applications. Here we report on a novel approach of structural alternation of the exocyclic and linker parts that led to a new derivative with significantly improved cell activity allowing us to dissect its mode of action in detail. We have identified an effective cyclic peptide (CP7) that induces approximately a 9-fold increase in DNA damage accumulation and a remarkable increase in apoptotic cancer cell death compared to the reported molecule. Notably, treating cells with CP7 leads to a dramatic decrease in the efficiency of non-homologous end joining (NHEJ) repair of DNA double-strand breaks (DSBs), which is accompanied by an increase in homologous recombination (HR) repair. Interestingly, treating BRCA1-deficient cells with CP7 restores HR integrity, which is accompanied by increased resistance to CP7. Additionally, CP7 treatment increases the sensitivity of cancer cells to ionizing radiation. Collectively, our findings demonstrate that CP7 is a selective inhibitor of NHEJ, offering a potential strategy to enhance the effectiveness of radiation therapy.

Neural ODE to model and prognose thermoacoustic instability.

Thermoacoustic instability in a reacting flow field is characterized by high amplitude pressure fluctuations driven by a positive coupling between the unsteady heat release rate and the acoustic field of the combustor. In a turbulent flow, the transition of a thermoacoustic system from a state of chaos to periodic oscillations occurs via a state of intermittency. During the transition to periodic oscillations, the unsteady heat release rate synchronizes with the acoustic pressure fluctuations. Thermoacoustic systems are traditionally modeled by coupling the model for the heat source and the acoustic subsystem, each estimated independently. The response of the unsteady heat source, i.e., the flame, to acoustic fluctuations is characterized by introducing unsteady external forcing. The forced response of the flame need not be the same in the presence of an acoustic field due to their nonlinear coupling. Instead of characterizing individual subsystems, we introduce a neural ordinary differential equation (neural ODE) framework to model the thermoacoustic system as a whole. The neural ODE model for the thermoacoustic system uses time series of the heat release rate and the pressure fluctuations, measured simultaneously without introducing any external perturbations, to model their coupled interaction. Furthermore, we use the parameters of neural ODE to define an anomaly measure that represents the proximity of system dynamics to limit cycle oscillations and thus provide an early warning signal for the onset of thermoacoustic instability.

Structure-Uptake Relationship Study of DABCYL Derivatives Linked to Cyclic Cell-Penetrating Peptides for Live-Cell Delivery of Synthetic Proteins.

Modifying cyclic cell-penetrating deca-arginine (cR10) peptides with 4-(4-dimethylaminophenylazo)benzoic acid (DABCYL) improves the uptake efficiency of synthetic ubiquitin (Ub) cargoes into living cells. To probe the role of the DABCYL moiety, we performed time-lapse microscopy and fluorescence lifetime imaging microscopy (FLIM) of fluorescent DABCYL-R10 to evaluate the impact on cell entry by the formation of nucleation zones. Furthermore, we performed a structure-uptake relationship study with 13 DABCYL derivatives coupled to CPP to examine their effect on the cell-uptake efficiency when conjugated to mono-Ub through disulfide linkages. Our results show that through structure variations of the DABCYL moiety alone we could reach, at nanomolar concentration, an additional threefold increase in the cytosolic delivery of Ub, which will enable studies on various intracellular processes related to Ub signaling.

An opinion on the multiscale nature of Covid-19 type disease spread.

Recognizing the multiscale, interdisciplinary nature of the Covid-19 transmission dynamics, we discuss some recent developments concerning an attempt to construct a disease spread model from the flow physics of infectious droplets and aerosols and the frequency of contact between susceptible individuals with the infectious aerosol cloud. Such an approach begins with the exhalation event-specific, respiratory droplet size distribution (both airborne/aerosolized and ballistic droplets), followed by tracking its evolution in the exhaled air to estimate the probability of infection and the rate constants of the disease spread model. The basic formulations and structure of submodels, experiments involved to validate those submodels, are discussed. Finally, in the context of preventive measures, respiratory droplet-face mask interactions are described.

Role of soft-gel substrates on bouncing-merging transition in drop impact on a liquid film.

Liquid droplets impacting on liquid films is common in many industrial and natural processes. It is crucial to understand the impact of droplets on a liquid film resting on soft deformable substrates in some of the applications including 3D printing of engineering structures, prosthetic implants and tissue engineering. By recognizing the practical relevance of soft-substrates, we present an experimental investigation to assess the role of deformable substrates on bouncing-to-merging transition in droplet impact on the liquid film. First, we prepared polyacrylamide (PAAm) soft-gel substrates with various "softness" (i.e., Young's modulus) by modulating the concentration of a crosslinker, N,N-methylene-bis-acrylamide (BIS). We found that the Young's modulus of PAAm initially increases with the concentration of crosslinker, and subsequently becomes almost constant due to inhomogeneity of crosslinking. Next, through the experiments of droplet impact on the liquid film resting on soft substrates with different Young's moduli, we observe that the early merging and corresponding bouncing-to-merging transitional boundaries remain unaffected by the "softness" since such merging occurs further away from the substrate. However, the late merging, which appears during the retraction process of the deformed droplet, occurs relatively close to the substrate, and hence is found to be significantly affected by its "softness". A scaling analysis is presented to quantify the role of change in Young's modulus of the substrate on late merging, which is supported by the experimental data.

Convolutional neural networks to predict the onset of oscillatory instabilities in turbulent systems.

Many fluid dynamic systems exhibit undesirable oscillatory instabilities due to positive feedback between fluctuations in their different subsystems. Thermoacoustic instability, aeroacoustic instability, and aeroelastic instability are some examples. When the fluid flow in the system is turbulent, the approach to such oscillatory instabilities occurs through a universal route characterized by a dynamical regime known as intermittency. In this paper, we extract the peculiar pattern of phase space attractors during the regime of intermittency by constructing recurrence networks corresponding to the phase space topology. We further train a convolutional neural network to classify the periodic and aperiodic structures in the recurrence networks and define a measure that indicates the proximity of the dynamical state to the onset of oscillatory instability. We show that this measure can predict the onset of oscillatory instabilities in three different fluid dynamic systems governed by different physical phenomena.

Primitive layered gabbros from fast-spreading lower oceanic crust.

Three-quarters of the oceanic crust formed at fast-spreading ridges is composed of plutonic rocks whose mineral assemblages, textures and compositions record the history of melt transport and crystallization between the mantle and the sea floor. Despite the importance of these rocks, sampling them in situ is extremely challenging owing to the overlying dykes and lavas. This means that models for understanding the formation of the lower crust are based largely on geophysical studies and ancient analogues (ophiolites) that did not form at typical mid-ocean ridges. Here we describe cored intervals of primitive, modally layered gabbroic rocks from the lower plutonic crust formed at a fast-spreading ridge, sampled by the Integrated Ocean Drilling Program at the Hess Deep rift. Centimetre-scale, modally layered rocks, some of which have a strong layering-parallel foliation, confirm a long-held belief that such rocks are a key constituent of the lower oceanic crust formed at fast-spreading ridges. Geochemical analysis of these primitive lower plutonic rocks--in combination with previous geochemical data for shallow-level plutonic rocks, sheeted dykes and lavas--provides the most completely constrained estimate of the bulk composition of fast-spreading oceanic crust so far. Simple crystallization models using this bulk crustal composition as the parental melt accurately predict the bulk composition of both the lavas and the plutonic rocks. However, the recovered plutonic rocks show early crystallization of orthopyroxene, which is not predicted by current models of melt extraction from the mantle and mid-ocean-ridge basalt differentiation. The simplest explanation of this observation is that compositionally diverse melts are extracted from the mantle and partly crystallize before mixing to produce the more homogeneous magmas that erupt.

A cellular automata model for expanding turbulent flames.

Cellular automata models based on population dynamics, introduced by Von Neumann in the 1950s, has been successfully used to describe pattern development and front propagation in many applications, such as crystal growth, forest fires, fractal growth in biological media, etc. We, herein, explore the possibility of using a cellular automaton, based on the population dynamics of flamelets, as a low-order model to describe the dynamics of an expanding flame propagating in a turbulent environment. A turbulent flame is constituted by numerous flamelets, each of which interacts with their neighborhood composed of other flamelets, as well as unburned and burnt fluid particles. This local interaction leads to global flame dynamics. The effect of turbulence is simulated by introducing stochasticity in the local interaction and hence in the temporal evolution of the flamefront. Our results show that the model preserves various multifractal characteristics of the expanding turbulent flame and captures several characteristics of expanding turbulent flames observed in experiments. For example, at low turbulence levels, an increase in global burning rate leads to an increase in the turbulence level, while beyond a critical turbulence level, the expanding flame becomes increasingly fragmented, and consequently, the total burning rate decreases with increasing turbulence. Furthermore, at an extremely high turbulence level, the ignition kernel quenches at its nascent state and consequently loses its ability to propagate as an expanding flame.

pH-Regulated anion transport activities of bis(iminourea) derivatives across the cell and vesicle membrane.

Recently, synthetic anion transporters have gained considerable attention because of their ability to disrupt cellular anion homeostasis and promote cell death. Herein, we report the development of bis(iminourea) derivatives as a new class of selective Cl- ion carrier. The bis(iminourea) derivatives were synthesized via a one-pot approach under mild reaction conditions. The presence of iminourea moieties suggests that the bis(iminourea) derivatives can be considered as unique guanidine mimics, indicating that the protonated framework could have much stronger anion recognition properties. The cooperative interactions of H+ and Cl- ions with these iminourea moieties results in the efficient transport of HCl across the lipid bilayer in an acidic environment. Under physiological conditions these compounds weakly transport Cl- ions via an antiport exchange mechanism. This pH-dependent gating/switching behavior (9-fold) within a narrow window could be due to the apparent pKa values (6.2-6.7) of the compounds within the lipid bilayer. The disruption of ionic homeostasis by the potent compounds was found to induce cell death.

Azidophosphonate Chemistry as a Route for a Novel Class of Vesicle-Forming Phosphonolipids.

Membrane forming synthetic lipids constitutes a new class of biomaterials with impressive applications in the field of biological and pharmaceutical sciences. Interestingly, alteration(s) in the headgroup region of the lipids offer a wide chemical space to investigate their specific properties. In this regard, we have utilized ß-azidophosphonate chemistry to gain access to a novel class of triazole-phosphonate (TP) amphiphiles with fascinating physicochemical properties of lipids. TP lipids form stable vesicles that exhibit negative surface potential across a broad pH range. These anionic lipids have high phase-transition temperatures, phospholipase resistance, slow vesicle leakage profiles, and doxorubicin delivery efficacy. We hypothesize that these readily synthesizable phosphonolipids could find several applications as phospholipid substituents.

Bouncing-to-Merging Transition in Drop Impact on Liquid Film: Role of Liquid Viscosity.

When a drop impacts on a liquid surface, it can either bounce back or merge with the surface. The outcome affects many industrial processes, in which merging is preferred in spray coating to generate a uniform layer and bouncing is desired in internal combustion engines to prevent accumulation of the fuel drop on the wall. Thus, a good understanding of how to control the impact outcome is highly demanded to optimize the performance. For a given liquid, a regime diagram of bouncing and merging outcomes can be mapped in the space of Weber number (ratio of impact inertia and surface tension) versus film thickness. In addition, recognizing that the liquid viscosity is a fundamental fluid property that critically affects the impact outcome through viscous dissipation of the impact momentum, here we investigate liquids with a wide range of viscosity from 0.7 to 100 cSt, to assess its effect on the regime diagram. Results show that while the regime diagram maintains its general structure, the merging regime becomes smaller for more viscous liquids and the retraction merging regime disappears when the viscosity is very high. The viscous effects are modeled and subsequently the mathematical relations for the transition boundaries are proposed which agree well with the experiments. The new expressions account for all the liquid properties and impact conditions, thus providing a powerful tool to predict and manipulate the outcome when a drop impacts on a liquid film.

Correction: Nonmonotonic response of drop impacting on liquid film: mechanism and scaling.

Correction for 'Nonmonotonic response of drop impacting on liquid film: mechanism and scaling' by Xiaoyu Tang et al., Soft Matter, 2016, DOI: 10.1039/c6sm00397d.

Nonmonotonic response of drop impacting on liquid film: mechanism and scaling.

Drop impacting on a liquid film with a finite thickness is omnipresent in nature and plays a critical role in numerous industrial processes. The impact can result in either bouncing or merging, which is mainly controlled by the impact inertia of the drop and film thickness. Although it is known that impact with inertia beyond a critical value on a thick film promotes merging through the breakage of the interfacial gas layer, here we demonstrate that for an impact inertia less than that critical value, increasing the film thickness leads to a nonmonotonic transition from merging to bouncing to merging and finally to bouncing again. For the first time, two different merging mechanisms are identified and the scaling laws of the nonmonotonic transitions are developed. These results provide important insights into the role of the film thickness in the impact dynamics, which is critical for optimizing operating conditions for spray or ink-jet systems among others.

Facilitated ignition in turbulence through differential diffusion.

Contrary to the belief that ignition of a combustible mixture by a high-energy kernel is more difficult in turbulence than in quiescence because of the increased dissipation rate of the deposited energy, we experimentally demonstrate that it can actually be facilitated by turbulence for mixtures whose thermal diffusivity sufficiently exceeds its mass diffusivity. In such cases, turbulence breaks the otherwise single spherical flame of positive curvature, and hence positive aerodynamics stretch, into a multitude of wrinkled flamelets subjected to either positive or negative stretch, such that the intensified burning of the latter constitutes local sources to facilitate ignition.

Unlocking cancer vaccine potential: What are the key factors?

Cancer is a global health challenge, with changing demographics and lifestyle factors producing an increasing burden worldwide. Screening advancements are enabling earlier diagnoses, but current cancer immunotherapies only induce remission in a small proportion of patients and come at a high cost. Cancer vaccines may offer a solution to these challenges, but they have been mired by poor results in past decades. Greater understanding of tumor biology, coupled with the success of vaccine technologies during the COVID-19 pandemic, has reinvigorated cancer vaccine development. With the first signs of efficacy being reported, cancer vaccines may be beginning to fulfill their potential. Solid tumors, however, present different hurdles than infectious diseases. Combining insights from previous cancer vaccine clinical development and contemporary knowledge of tumor immunology, we ask: who are the 'right' patients, what are the 'right' targets, and which are the 'right' modalities to maximize the chances of cancer vaccine success?

Evaporation dynamics of a surrogate respiratory droplet in a vortical environment.

HYPOTHESIS: Vortex droplet interaction is crucial for understanding the route of disease transmission through expiratory jet where several such embedded droplets continuously interact with vortical structures of different strengths and sizes. EXPERIMENTS: A train of vortex rings with different vortex strength, quantified with vortex Reynolds number (Re'=0,53,221,297) are made to interact with an isolated levitated droplet, and the evolution dynamics is captured using shadowgraphy, particle image velocimetry (PIV), and backlight imaging technique. NaCl-DI water solution of 0, 1, 10 and 20 wt% concentrations are used as test fluids for the droplet. FINDINGS: The results show the dependence of evaporation characteristics on vortex strength, while the crystallization dynamics was found to be independent of it. A reduction of 12.23% and 14.6% in evaporation time was seen in case of de-ionized (DI) water and 1% wt NaCl solution respectively in presence of vortex ring train at Re'=221. In contrast to this, a minimal reduction in evaporation time (0.6% and 0.9% for DI water and 1% wt NaCl solution, respectively) is observed when Re' is increased from 221 to 297. The mechanisms for evaporation time reduction due to enhancement of convective heat and mass transfer from the droplet and shearing away of vapor layer by vortex ring interaction are discussed in this work.