The secondary pocket of cryptochrome 2 is important for the regulation of its stability and localization.

Human clock-gene variations contribute to the phenotypic differences observed in various behavioral and physiological processes, such as diurnal preference, sleep, metabolism, mood regulation, addiction, and fertility. However, little is known about the possible effects of identified variations at the molecular level. In this study, we performed a functional characterization at the cellular level of rare cryptochrome 2 (CRY2) missense variations that were identified from the Ensembl database. Our structural studies revealed that three variations (p.Pro123Leu, p.Asp406His, and p.Ser410Ile) are located at the rim of the secondary pocket of CRY2. We show that these variants were unable to repress CLOCK (circadian locomotor output cycles kaput)/BMAL1 (brain and muscle ARNT-like-1)-driven transcription in a cell-based reporter assay and had reduced affinity to CLOCK-BMAL1. Furthermore, our biochemical studies indicated that the variants were less stable than the WT CRY2, which could be rescued in the presence of period 2 (PER2), another core clock protein. Finally, we found that these variants were unable to properly localize to the nucleus and thereby were unable to rescue the circadian rhythm in a Cry1-/-Cry2-/- double KO mouse embryonic fibroblast cell line. Collectively, our data suggest that the rim of the secondary pocket of CRY2 plays a significant role in its nuclear localization independently of PER2 and in the intact circadian rhythm at the cellular level.

The Arg-293 of Cryptochrome1 is responsible for the allosteric regulation of CLOCK-CRY1 binding in circadian rhythm.

Mammalian circadian clocks are driven by transcription/translation feedback loops composed of positive transcriptional activators (BMAL1 and CLOCK) and negative repressors (CRYPTOCHROMEs (CRYs) and PERIODs (PERs)). CRYs, in complex with PERs, bind to the BMAL1/CLOCK complex and repress E-box-driven transcription of clock-associated genes. There are two individual CRYs, with CRY1 exhibiting higher affinity to the BMAL1/CLOCK complex than CRY2. It is known that this differential binding is regulated by a dynamic serine-rich loop adjacent to the secondary pocket of both CRYs, but the underlying features controlling loop dynamics are not known. Here we report that allosteric regulation of the serine-rich loop is mediated by Arg-293 of CRY1, identified as a rare CRY1 SNP in the Ensembl and 1000 Genomes databases. The p.Arg293His CRY1 variant caused a shortened circadian period in a Cry1-/-Cry2-/- double knockout mouse embryonic fibroblast cell line. Moreover, the variant displayed reduced repressor activity on BMAL1/CLOCK driven transcription, which is explained by reduced affinity to BMAL1/CLOCK in the absence of PER2 compared with CRY1. Molecular dynamics simulations revealed that the p.Arg293His CRY1 variant altered a communication pathway between Arg-293 and the serine loop by reducing its dynamicity. Collectively, this study provides direct evidence that allosterism in CRY1 is critical for the regulation of circadian rhythm.

Transcriptome analysis of the circadian clock gene BMAL1 deletion with opposite carcinogenic effects.

We have previously reported that the deletion of BMAL1 gene has opposite effects in respect to its contribution to the pathways that are effective in the multistage carcinogenesis process. BMAL1 deletion sensitized nearly normal breast epithelial (MCF10A) and invasive breast cancer cells (MDA-MB-231) to cisplatin- and doxorubicin-induced apoptosis, while this deletion also aggravated the invasive potential of MDA-MB-231 cells. However, the mechanistic relationship of the seemingly opposite contribution of BMAL1 deletion to carcinogenesis process is not known at genome-wide level. In this study, an RNA-seq approach was taken to uncover the differentially expressed genes (DEGs) and pathways after treating BMAL1 knockout (KO) or wild-type (WT) MDA-MB-231 cells with cisplatin and doxorubicin to initiate apoptosis. Gene set enrichment analysis with the DEGs demonstrated that enrichment in multiple genes/pathways contributes to sensitization to cisplatin- or doxorubicin-induced apoptosis in BMAL1-dependent manner. Additionally, our DEG analysis suggested that non-coding transcript RNA (such as lncRNA and processed pseudogenes) may have role in cisplatin- or doxorubicin-induced apoptosis. Protein-protein interaction network obtained from common DEGs in cisplatin and doxorubicin treatments revealed that GSK3ß, NACC1, and EGFR are the principal genes regulating the response of the KO cells. Moreover, the analysis of DEGs among untreated BMAL1 KO and WT cells revealed that epithelial-mesenchymal transition genes are up-regulated in KO cells. As a negative control, we have also analyzed the DEGs following treatment with an endoplasmic reticulum (ER) stress-inducing agent, tunicamycin, which was affected by BMAL1 deletion minimally. Collectively, the present study suggests that BMAL1 regulates many genes/pathways of which the alteration in BMAL1 KO cells may shed light on pleotropic phenotype observed.

Sulfobetaine Methacrylate Polymers of Unconventional Polyzwitterion Architecture and Their Antifouling Properties.

Combining high hydrophilicity with charge neutrality, polyzwitterions are intensely explored for their high biocompatibility and low-fouling properties. Recent reports indicated that in addition to charge neutrality, the zwitterion's segmental dipole orientation is an important factor for interacting with the environment. Accordingly, a series of polysulfobetaines with a novel architecture was designed, in which the cationic and anionic groups of the zwitterionic moiety are placed at equal distances from the backbone. They were investigated by in vitro biofouling assays, covering proteins of different charges and model marine organisms. All polyzwitterion coatings reduced the fouling effectively compared to model polymer surfaces of poly(butyl methacrylate), with a nearly equally good performance as the reference polybetaine poly(3-(N-(2-(methacryloyloxy)ethyl)-N,N-dimethylammonio)propanesulfonate). The specific fouling resistance depended on the detailed chemical structure of the polyzwitterions. Still, while clearly affecting the performance, the precise dipole orientation of the sulfobetaine group in the polyzwitterions seems overall to be only of secondary importance for their antifouling behavior.

Feasibility of anaerobic membrane bioreactors (AnMBR) for onsite sanitation and resource recovery (nutrients, energy and water) in urban slums.

Slums are challenging locations for sanitation technologies. High population densities, a lack of water and electricity infrastructure, and space constraints combine to ensure that many traditional waste treatment technologies fail when implemented in this context. This paper proposes the use of anaerobic membrane bioreactors (AnMBRs) for slum sanitation. AnMBRs allow for localized water reuse, high quality treatment, and energy production at the point of treatment. A water, energy, nutrient, and mass balance was conducted on a theoretical AnMBR directly coupled to a public toilet. The combined system would be capable of recycling its water for use in toilet flushing and would be capable of providing enough energy to power both the toilet and AnMBR operation. The addition of food waste to the feed would help to ensure process stability and energy production by the AnMBR. Ammonia accumulation within the system would have to be managed through struvite precipitation, ion exchange, oxidation, plant uptake or other means. Generated biogas can be converted into heat and/or electricity using small scale gas generators. AnMBR technology has high potential for success in slum settings, if considerations for maintenance and supplies are made as part of the design and system delivery.

Single-Material Solvent-Driven Polydimethylsiloxane Sponge Bending Actuators.

Soft robots mimic the agility of living organisms without rigid joints and muscles. Continuum bending (CB) is one type of motion living organisms can display. CB can be achieved using pneumatic, electroactive, or thermal actuators prepared by casting an active layer on a passive layer. The corresponding input actuates only the active layer in the assembly resulting in the bending of the structure. These two different layers must be laminated well during manufacturing. However, the formed bilayer can still delaminate later, and the detachment hampers the actuator's reversible, long-time use. An approach to creating a single material bending actuator was previously reported, for which spatial gradient swelling was used. This authentic approach allows a single material to be manufactured as a bending actuator, allowing easy access to such actuators without lamination. In this study, we show spatial porosity differences in the sponges of polydimethylsiloxane (PDMS) (a common material in soft robotics) can be used to create the required anisotropy for bending. The spongy polymers are manufactured through table sugar templates and actuated by (organic) solvent absorption/desorption. This enables some versatility in the mechanical properties, shape, actuation force, and actuation speed. The one-material system's straightforward production and seamless nature are advantageous for reversible and repetitive bending. This simple method can further be developed in hydrogels and polymers for soft robotics and functional materials.

Dynamic regulation of the serine loop by distant mutations reveals allostery in cryptochrome1.

Cryptochromes (CRYs) are essential components of the molecular clock that generates circadian rhythm. They inhibit BMAL1/CLOCK-driven transcription at the molecular level. There are two CRYs that have differential functions in the circadian clock in mammals. It is not precisely known how they achieve such differential functions. In this study, we performed molecular dynamic simulations on eight CRY mutants that have been experimentally shown to exhibit reduced repressor activities. Our results revealed that mutations in CRY1 affect the dynamic behavior of the serine loop and the availability of the secondary pocket, but not in CRY2. Further analysis of these CRY1 mutants indicated that the differential flexibility of the serine loop leads to changes in the volume of the secondary pocket. We also investigated the weak interactions between the amino acids in the serine loop and those in close proximity. Our findings highlighted the crucial roles of S44 and S45 in the dynamic behavior of the serine loop, specifically through their interactions with E382 in CRY1. Considering the clinical implications of altered CRY1 function, our study opens up new possibilities for the development of drugs that target the allosteric regulation of CRY1.Communicated by Ramaswamy H. Sarma.

Single nucleotide polymorphisms (SNPs) in circadian genes: Impact on gene function and phenotype.

Circadian rhythm is an endogenous timing system that allows an organism to anticipate and adapt to daily changes and regulate various physiological variables such as the sleep-wake cycle. This rhythm is governed by a molecular circadian clock mechanism, generated by a transcriptional and translational feedback loop (TTFL) mechanism. In mammals, TTFL is determined by the interaction of four main clock proteins: BMAL1, CLOCK, Cryptochromes (CRY), and Periods (PER). BMAL1 and CLOCK form dimers and initiate the transcription of clock-controlled genes (CCG) by binding an E-box element with the promotor genes. Among CCGs, PERs and CRYs accumulate in the cytosol and translocate into the nucleus, where they interact with the BMAL1/CLOCK dimer and inhibit its activity. Several epidemiological and genetic studies have revealed that circadian rhythm disruption causes various types of disease. In this chapter, we summarize the effect of core clock gene SNPs on circadian rhythm and diseases in humans.

Control and study of bio-inspired quadrupedal gaits on an underactuated miniature robot.

This paper presents a linear quadratic Gaussian (LQG) controller for controlling the gait of a miniature, foldable quadruped robot with individually actuated and controlled legs (MinIAQ-III). The controller is implemented on a palm-size robot made by folding an acetate sheet. MinIAQ-III has four DC motors for actuation and four rotary sensors for feedback. It is one of the few untethered robots on a miniature scale capable of working with different gaits with the help of its individually-actuated legs and the developed controller. The presented LQG controller controls each leg's positions and rotational speeds by measuring the positions and estimating the rotational speeds, respectively. With the precise gait control on the robot, we demonstrate different gaits inspired by quadrupeds in nature and compare the simulation and experiment results for some of the gaits. An extensive simulation environment developed for robot dynamics helps us to predict the locomotion behavior of the robot in various environments. The match between the simulation and the experiment results shows that the proposed LQG controller can successfully control the miniature robot's gaits. We also conduct a case study that shows the potential to use the simulation to achieve different robot behavior. In a case study, we present our robot performing a prancing similar to horses. We use the simulation environment to find the required motor configuration phases and physical parameters, which can make our robot prance. After finding the parameters in simulation, we replicate the configuration in our robot and observe the robot making the same moves as the simulation.

TW68, cryptochromes stabilizer, regulates fasting blood glucose levels in diabetic ob/ob and high fat-diet-induced obese mice.

Cryptochromes (CRYs), transcriptional repressors of the circadian clock in mammals, inhibit cAMP production when glucagon activates G-protein coupled receptors. Therefore, molecules that modulate CRYs have the potential to regulate gluconeogenesis. In this study, we discovered a new molecule called TW68 that interacts with the primary pockets of mammalian CRY1/2, leading to reduced ubiquitination levels and increased stability. In cell-based circadian rhythm assays using U2OS Bmal1-dLuc cells, TW68 extended the period length of the circadian rhythm. Additionally, TW68 decreased the transcriptional levels of two genes, Phosphoenolpyruvate carboxykinase 1 (PCK1) and Glucose-6-phosphatase (G6PC), which play crucial roles in glucose biosynthesis during glucagon-induced gluconeogenesis in HepG2 cells. Oral administration of TW68 in mice showed good tolerance, a good pharmacokinetic profile, and remarkable bioavailability. Finally, when administered to fasting diabetic animals from ob/ob and HFD-fed obese mice, TW68 reduced blood glucose levels by enhancing CRY stabilization and subsequently decreasing the transcriptional levels of Pck1 and G6pc. These findings collectively demonstrate the antidiabetic efficacy of TW68 in vivo, suggesting its therapeutic potential for controlling fasting glucose levels in the treatment of type 2 diabetes mellitus.

Modeling of simultaneous growth and storage kinetics variation under unsteady feast conditions for aerobic heterotrophic biomass.

The heterotrophic biomass has the capacity of utilizing substrate predominantly for growth or storage processes under steady-state conditions. In this study, the short-term variations in growth and storage kinetics of activated sludge under disturbed feeding conditions were analyzed using a multi-component biodegradation model. The variations in growth and storage kinetics were investigated with the aid of multi-response modeling and identifiability analysis. It was found that the heterotrophic biomass is able to increase its direct growth activity together with reducing the substrate storage capability under the availability of external substrate. Reducing the sludge age (SRT) from 10 to 2 days increased the maximum specific growth rate, µ (OHO,Max) from 3.9 to 7.0 day(-1), but did not considerably affected the maximum storage rate, k (Stor,OHO). The alteration of sludge age also elevated the half-saturation constant for growth (K (S,OHO)) from 5 to 25 mg COD/L. The increase in primary growth metabolism together with reduced storage rate was validated by model for two different sludge ages in the availability of external substrate. Aside from having a lower storage capability, the biomass had fast adaptation ability to direct growth process at low SRTs. The alteration of feed conditions was found to have different impacts on storage and growth kinetics. These results are significant and advance the field of activated sludge modeling under dynamic conditions by incorporation of short-term effects. Appropriate modifications including short-term effects in model structure may also reduce dynamic model recalibration efforts in the future.

Discovery of a small molecule that selectively destabilizes Cryptochrome 1 and enhances life span in p53 knockout mice.

Cryptochromes are negative transcriptional regulators of the circadian clock in mammals. It is not clear how reducing the level of endogenous CRY1 in mammals will affect circadian rhythm and the relation of such a decrease with apoptosis. Here, we discovered a molecule (M47) that destabilizes Cryptochrome 1 (CRY1) both in vitro and in vivo. The M47 selectively enhanced the degradation rate of CRY1 by increasing its ubiquitination and resulted in increasing the circadian period length of U2OS Bmal1-dLuc cells. In addition, subcellular fractionation studies from mice liver indicated that M47 increased degradation of the CRY1 in the nucleus. Furthermore, M47-mediated CRY1 reduction enhanced oxaliplatin-induced apoptosis in Ras-transformed p53 null fibroblast cells. Systemic repetitive administration of M47 increased the median lifespan of p53-/- mice by ~25%. Collectively our data suggest that M47 is a promising molecule to treat forms of cancer depending on the p53 mutation.

Gait and locomotion analysis of a soft-hybrid multi-legged modular miniature robot.

The locomotion performance of the current legged miniature robots remains inferior compared to even the most simple insects. The inferiority has led researchers to utilize biological principles and control in their designs, often resulting in improved performance and robot capabilities. Additionally, optimizing the locomotion patterns compatible with the robot's limitations (such as the gaits achievable by the robot) improves the performance significantly and results in a robot operating with its maximum capabilities. This paper studies the locomotion characteristics of running/walkingn-legged modular miniature robots with soft or rigid module connections. The locomotion study is done using the presented dynamic model, and the results are verified using a legged modular miniature robot with soft and rigid backbones (SMoLBot). The optimum foot contact sequences for ann-legged robot with different compliance values between the modules are derived using the locomotion analyses and the dynamic and kinematic formulations. Our investigations determine unique optimum foot contact sequences for multi-legged robots with different body compliances and module numbers. Locomotion analyses of a multi-legged robot with different backbones operating with optimum gaits show two main motion characteristics; the rigid robots minimize the number of leg-ground contacts to increase velocity, whereas soft-backbone robots use a lift-jump-fall motion sequence to maximize the translational speeds. These two behaviors are similar between different soft-backbone and rigid-backbone robots; however, the optimal foot contact sequences are different and unpredictable.

In silico identification of widely used and well-tolerated drugs as potential SARS-CoV-2 3C-like protease and viral RNA-dependent RNA polymerase inhibitors for direct use in clinical trials.

Despite strict measures taken by many countries, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to be an issue of global concern. Currently, there are no clinically proven pharmacotherapies for coronavirus disease 2019, despite promising initial results obtained from drugs such as azithromycin and hydroxychloroquine. Therefore, the repurposing of clinically approved drugs for use against SARS-CoV-2 has become a viable strategy. Here, we searched for drugs that target SARS-CoV-2 3C-like protease (3CLpro) and viral RNA-dependent RNA polymerase (RdRp) by in silico screening of the U.S. Food and Drug Administration approved drug library. Well-tolerated and widely used drugs were selected for molecular dynamics (MD) simulations to evaluate drug-protein interactions and their persistence under physiological conditions. Tetracycline, dihydroergotamine, ergotamine, dutasteride, nelfinavir, and paliperidone formed stable interactions with 3CLpro based on MD simulation results. Similar analysis with RdRp showed that eltrombopag, tipranavir, ergotamine, and conivaptan bound to the enzyme with high binding free energies. Docking results suggest that ergotamine, dihydroergotamine, bromocriptine, dutasteride, conivaptan, paliperidone, and tipranavir can bind to both enzymes with high affinity. As these drugs are well tolerated, cost-effective, and widely used, our study suggests that they could potentially to be used in clinical trials for the treatment of SARS-CoV-2-infected patients.Communicated by Ramaswamy H. Sarma.

Spoilage assessment of chicken breast fillets by means of fourier transform infrared spectroscopy and multispectral image analysis.

The objective of this research was the evaluation of Fourier transforms infrared spectroscopy (FT-IR) and multispectral image analysis (MSI) as efficient spectroscopic methods in tandem with multivariate data analysis and machine learning for the assessment of spoilage on the surface of chicken breast fillets. For this purpose, two independent storage experiments of chicken breast fillets (n â€‹= â€‹215) were conducted at 0, 5, 10, and 15 â€‹°C for up to 480 â€‹h. During storage, samples were analyzed microbiologically for the enumeration of Total Viable Counts (TVC) and Pseudomonas spp. In addition, FT-IR and MSI spectral data were collected at the same time intervals as for microbiological analyses. Multivariate data analysis was performed using two software platforms (a commercial and a publicly available developed platform) comprising several machine learning algorithms for the estimation of the TVC and Pseudomonas spp. population of the surface of the samples. The performance of the developed models was evaluated by intra batch and independent batch testing. Partial Least Squares- Regression (PLS-R) models from the commercial software predicted TVC with root mean square error (RMSE) values of 1.359 and 1.029 log CFU/cm2 for MSI and FT-IR analysis, respectively. Moreover, RMSE values for Pseudomonas spp. model were 1.574 log CFU/cm2 for MSI data and 1.078 log CFU/cm2 for FT-IR data. From the implementation of the in-house sorfML platform, artificial neural networks (nnet) and least-angle regression (lars) were the most accurate models with the best performance in terms of RMSE values. Nnet models developed on MSI data demonstrated the lowest RMSE values (0.717 log CFU/cm2) for intra-batch testing, while lars outperformed nnet on independent batch testing with RMSE of 1.252 log CFU/cm2. Furthermore, lars models excelled with the FT-IR data with RMSE of 0.904 and 0.851 log CFU/cm2 in intra-batch and independent batch testing, respectively. These findings suggested that FT-IR analysis is more efficient than MSI to predict the microbiological quality on the surface of chicken breast fillets.

Performance of AnMBR in Treatment of Post-consumer Food Waste: Effect of Hydraulic Retention Time and Organic Loading Rate on Biogas Production and Membrane Fouling.

Anaerobic digestion of food waste (FW) is typically limited to large reactors due to high hydraulic retention times (HRTs). Technologies such as anaerobic membrane reactors (AnMBRs) can perform anaerobic digestion at lower HRTs while maintaining high chemical oxygen demand (COD) removal efficiencies. This study evaluated the effect of HRT and organic loading rate (OLR) on the stability and performance of a side-stream AnMBR in treating diluted fresh food waste (FW). The reactor was fed with synthetic FW at an influent concentration of 8.24 (± 0.12) g COD/L. The OLR was increased by reducing the HRT from 20 to 1 d. The AnMBR obtained an overall removal efficiency of >97 and >98% of the influent COD and total suspended solids (TSS), respectively, throughout the course of operation. The biological process was able to convert 76% of the influent COD into biogas with 70% methane content, while the cake layer formed on the membrane gave an additional COD removal of 7%. Total ammoniacal nitrogen (TAN) and total nitrogen (TN) concentrations were found to be higher in the bioreactor than in the influent, and average overall removal efficiencies of 17.3 (± 5) and 61.5 (± 3)% of TAN and TN, respectively, were observed with respect to the bioreactor concentrations after 2 weeks. Total phosphorus (TP) had an average removal efficiency of 40.39 (± 5)% with respect to the influent. Membrane fouling was observed when the HRT was decreased from 7 to 5 d and was alleviated through backwashing. This study suggests that the side-stream AnMBR can be used to successfully reduce the typical HRT of wet anaerobic food waste (solids content 7%) digesters from 20 days to 1 day, while maintaining a high COD removal efficiency and biogas production.

Parallelized microfluidic diatom accumulation assay to test fouling-release coatings.

Assessing the efficiency of the next generation of protective marine coatings is highly relevant for their optimization. In this paper, a parallelized microfluidic testing device is presented to quantify the accumulation of a model organism (Navicula perminuta) under constant laminar flow. Using automated microscopy in conjunction with image analysis, the adhesion densities on the tested surfaces could be determined after exposure to a flow of suspended algae for 90 min. The optimized protocol for the assay is presented, and the reproducibility of the densities of attached diatoms was verified on four identical surfaces (self-assembled dodecanethiol monolayers). A set of well-characterized self-assembled monolayers with different chemical terminations was used to validate the performance of the assay and its capability to discriminate diatom accumulation on different surface chemistries under dynamic conditions. The observed trends are in good agreement with previously published results obtained in single channel accumulation and detachment assays. To demonstrate the practical relevance of the dynamic experiment, diatom attachment on four technically relevant silicone coatings with different fouling-release properties could clearly be distinguished.

Bioaugmentation of the anaerobic digestion of food waste by dungs of herbivore, carnivore, and omnivore zoo animals.

The potential improvement of biomethanation of food waste (FW) by adding dung of herbivore (giraffe, llama, koala), carnivore (tiger), and omnivore (sloth bear) animals to anaerobic sludge (AnS) was investigated. Adding 30% giraffe, sloth bear or koala dung to the AnS inoculum yielded, respectively, a 11.17 (±4.51), 10.10 (±1.23), and 1.41 (±0.56)% higher biomethane production, as compared to the control (FW with solely AnS). The highest biomethane production of 564.00 (±3.88) ml CH4/gVSadded obtained with 30% giraffe dung and 70% AnS was attributed to a higher solubilization of proteins (6.96 ± 2.76%) and recalcitrant carbohydrates (344.85 ± 54.31 mg/L as compared to zero). The biomethanation process could have been stimulated by the microorganisms or enzymes newly introduced, and/or the trace elements (Ni, Zn, and Co) present in the giraffe dung. These results indicate that bioaugmentation with zoo animals dung is worthy of further investigation as a strategy for improving the biomethane recovery from organic wastes.