Mutational dissection of a hole hopping route in a lytic polysaccharide monooxygenase (LPMO).

Oxidoreductases have evolved tyrosine/tryptophan pathways that channel highly oxidizing holes away from the active site to avoid damage. Here we dissect such a pathway in a bacterial LPMO, member of a widespread family of C-H bond activating enzymes with outstanding industrial potential. We show that a strictly conserved tryptophan is critical for radical formation and hole transference and that holes traverse the protein to reach a tyrosine-histidine pair in the protein's surface. Real-time monitoring of radical formation reveals a clear correlation between the efficiency of hole transference and enzyme performance under oxidative stress. Residues involved in this pathway vary considerably between natural LPMOs, which could reflect adaptation to different ecological niches. Importantly, we show that enzyme activity is increased in a variant with slower radical transference, providing experimental evidence for a previously postulated trade-off between activity and redox robustness.

The role of BST4 in the pyrenoid of Chlamydomonas reinhardtii.

In many eukaryotic algae, CO2 fixation by Rubisco is enhanced by a CO2-concentrating mechanism, which utilizes a Rubisco-rich organelle called the pyrenoid. The pyrenoid is traversed by a network of thylakoid-membranes called pyrenoid tubules, proposed to deliver CO2. In the model alga Chlamydomonas reinhardtii (Chlamydomonas), the pyrenoid tubules have been proposed to be tethered to the Rubisco matrix by a bestrophin-like transmembrane protein, BST4. Here, we show that BST4 forms a complex that localizes to the pyrenoid tubules. A Chlamydomonas mutant impaired in the accumulation of BST4 (bst4) formed normal pyrenoid tubules and heterologous expression of BST4 in Arabidopsis thaliana did not lead to the incorporation of thylakoids into a reconstituted Rubisco condensate. Chlamydomonas bst4 mutant did not show impaired growth at air level CO2. By quantifying the non-photochemical quenching (NPQ) of chlorophyll fluorescence, we show that bst4 displays a transiently lower thylakoid lumenal pH during dark to light transition compared to control strains. When acclimated to high light, bst4 had sustained higher NPQ and elevated levels of light-induced H2O2 production. We conclude that BST4 is not a tethering protein, but rather is an ion channel involved in lumenal pH regulation possibly by mediating bicarbonate transport across the pyrenoid tubules.

Oxysterols Protect Epithelial Cells Against Pore-Forming Toxins.

Many species of bacteria produce toxins such as cholesterol-dependent cytolysins that form pores in cell membranes. Membrane pores facilitate infection by releasing nutrients, delivering virulence factors, and causing lytic cell damage - cytolysis. Oxysterols are oxidized forms of cholesterol that regulate cellular cholesterol and alter immune responses to bacteria. Whether oxysterols also influence the protection of cells against pore-forming toxins is unresolved. Here we tested the hypothesis that oxysterols stimulate the intrinsic protection of epithelial cells against damage caused by cholesterol-dependent cytolysins. We treated epithelial cells with oxysterols and then challenged them with the cholesterol-dependent cytolysin, pyolysin. Treating HeLa cells with 27-hydroxycholesterol, 25-hydroxycholesterol, 7α-hydroxycholesterol, or 7ß-hydroxycholesterol reduced pyolysin-induced leakage of lactate dehydrogenase and reduced pyolysin-induced cytolysis. Specifically, treatment with 10 ng/ml 27-hydroxycholesterol for 24 h reduced pyolysin-induced lactate dehydrogenase leakage by 88%, and reduced cytolysis from 74% to 1%. Treating HeLa cells with 27-hydroxycholesterol also reduced pyolysin-induced leakage of potassium ions, prevented mitogen-activated protein kinase cell stress responses, and limited alterations in the cytoskeleton. Furthermore, 27-hydroxycholesterol reduced pyolysin-induced damage in lung and liver epithelial cells, and protected against the cytolysins streptolysin O and Staphylococcus aureus α-hemolysin. Although oxysterols regulate cellular cholesterol by activating liver X receptors, cytoprotection did not depend on liver X receptors or changes in total cellular cholesterol. However, oxysterol cytoprotection was partially dependent on acyl-CoA:cholesterol acyltransferase (ACAT) reducing accessible cholesterol in cell membranes. Collectively, these findings imply that oxysterols stimulate the intrinsic protection of epithelial cells against pore-forming toxins and may help protect tissues against pathogenic bacteria.

Corrigendum to design data for the 3D printer modification to print gels and pastes and the corresponding firmware.

[This corrects the article DOI: 10.1016/j.dib.2021.106974.].

Dual stimuli-sensitive carrageenan-based formulation for additive manufacturing.

The design and development of controlled release systems of molecules of interest (nutrients, flavors, and drugs) have attracted significant attention over several years. Herein, we report a formulation of dual temperature and electro responsive κ- and ι-carrageenan based hydrogel for efficient food material and drug delivery. The microstructure and the thermal behavior of the hydrogel were characterized. The in-vitro drug release from the hydrogel was also studied. Using this carrageenan-based formulation and folic acid as the drug model, a high drug loading, and a sustained release because of either electric field or temperature were observed. In principle, the proposed formulation does not rely on 3D printing to perform its function; however, it adds to the feedstocks for 3D printing in the food and pharmaceutical industries. For the future, this could allow potentially more complex smart structures to be created from this material, further tuning release behavior.

Design data for the 3D printer modification to print gels and pastes and the corresponding firmware.

In order to deposit gel and paste-like materials, a commercially available HICTOP Prusa i3 plastic 3D printer was modified. The modification included replacing the existing plastic microextruder with a customised 3D printed syringe pump which could hold a syringe containing the printing material. The arrangement also allowed the temperature in the syringe to be controlled. Since the hardware of the printer was changed significantly, a new firmware was loaded on the 3D printer which was customised to enable it to perform its new function. The present data consists of the 3D image files of the syringe pump assembly and instructions on how to assemble the components. It also provides a copy of the modified firmware with a list of the changes made to it. This data will allow the readers to modify a similar type of 3D printer to print pastes and gels. This can be achieved by recreating the entire syringe pump assembly by 3D printing the given 3D image file data. With some changes, these designs can also be adapted to a variety of different printers. Similarly, the given firmware can also be loaded onto a similar type of printer. The list and explanation of the changes made to the firmware also allow such changes to be made to the respective firmwares of a variety of different printers.

A recombineering pipeline to clone large and complex genes in Chlamydomonas.

The ability to clone genes has greatly advanced cell and molecular biology research, enabling researchers to generate fluorescent protein fusions for localization and confirm genetic causation by mutant complementation. Most gene cloning is polymerase chain reaction (PCR)�or DNA synthesis-dependent, which can become costly and technically challenging as genes increase in size, particularly if they contain complex regions. This has been a long-standing challenge for the Chlamydomonas reinhardtii research community, as this alga has a high percentage of genes containing complex sequence structures. Here we overcame these challenges by developing a recombineering pipeline for the rapid parallel cloning of genes from a Chlamydomonas bacterial artificial chromosome collection. To generate fluorescent protein fusions for localization, we applied the pipeline at both batch and high-throughput scales to 203 genes related to the Chlamydomonas CO2 concentrating mechanism (CCM), with an overall cloning success rate of 77%. Cloning success was independent of gene size and complexity, with cloned genes as large as 23 kb. Localization of a subset of CCM targets confirmed previous mass spectrometry data, identified new pyrenoid components, and enabled complementation of mutants. We provide vectors and detailed protocols to facilitate easy adoption of this technology, which we envision will open up new possibilities in algal and plant research.

Dynamic thylakoid stacking and state transitions work synergistically to avoid acceptor-side limitation of photosystem I.

TAP38/STN7-dependent (de)phosphorylation of light-harvesting complex II (LHCII) regulates the relative excitation rates of photosystems I and II (PSI, PSII) (state transitions) and the size of the thylakoid grana stacks (dynamic thylakoid stacking). Yet, it remains unclear how changing grana size benefits photosynthesis and whether these two regulatory mechanisms function independently. Here, by comparing Arabidopsis wild-type, stn7 and tap38 plants with the psal mutant, which undergoes dynamic thylakoid stacking but lacks state transitions, we explain their distinct roles. Under low light, smaller grana increase the rate of PSI reduction and photosynthesis by reducing the diffusion distance for plastoquinol; however, this beneficial effect is only apparent when PSI/PSII excitation balance is maintained by state transitions or far-red light. Under high light, the larger grana slow plastoquinol diffusion and lower the equilibrium constant between plastocyanin and PSI, maximizing photosynthesis by avoiding PSI photoinhibition. Loss of state transitions in low light or maintenance of smaller grana in high light also both bring about a decrease in cyclic electron transfer and over-reduction of the PSI acceptor side. These results demonstrate that state transitions and dynamic thylakoid stacking work synergistically to regulate photosynthesis in variable light.

Assembly of the algal CO2-fixing organelle, the pyrenoid, is guided by a Rubisco-binding motif.

Approximately one-third of the Earth's photosynthetic CO2 assimilation occurs in a pyrenoid, an organelle containing the CO2-fixing enzyme Rubisco. How constituent proteins are recruited to the pyrenoid and how the organelle's subcompartments-membrane tubules, a surrounding phase-separated Rubisco matrix, and a peripheral starch sheath-are held together is unknown. Using the model alga Chlamydomonas reinhardtii, we found that pyrenoid proteins share a sequence motif. We show that the motif is necessary and sufficient to target proteins to the pyrenoid and that the motif binds to Rubisco, suggesting a mechanism for targeting. The presence of the Rubisco-binding motif on proteins that localize to the tubules and on proteins that localize to the matrix-starch sheath interface suggests that the motif holds the pyrenoid's three subcompartments together. Our findings advance our understanding of pyrenoid biogenesis and illustrate how a single protein motif can underlie the architecture of a complex multilayered phase-separated organelle.

How to Formulate for Structure and Texture via Medium of Additive Manufacturing-A Review.

Additive manufacturing, which is also known as 3D printing, is an emerging and growing technology. It is providing significant innovations and improvements in many areas such as engineering, production, medicine, and more. 3D food printing is an area of great promise to provide an indulgence or entertaining experience, personalized food product, or specific nutritional needs. This paper reviews the additive manufacturing methods and materials in detail as well as their advantages and disadvantages. After a full discussion of 3D food printing, the reports on edible printed materials are briefly presented and discussed. In the end, the current and future outlook of additive manufacturing in the food industry is shown.

The Neuronal Encoding of Oral Fat by the Coefficient of Sliding Friction in the Cerebral Cortex and Amygdala.

Fat in the diet contributes to the pleasant mouthfeel of many foods, but overconsumption may contribute to obesity. Here we analyze what properties of fat in the mouth are sensed, by analyzing the responses of neurons in the macaque insular taste cortex, and two areas to which it projects the orbitofrontal cortex where the pleasantness of fat is represented, and the amygdala. We discovered that the firing rate responses of these fat-responsive neurons are correlated with the coefficient of sliding friction (CSF) and not with viscosity which reflects food thickness. Other, not fat-sensitive, neurons encoded viscosity and not the CSF. Neuronal population analyses confirmed that fat-responsive neurons conveyed information about the CSF but not about viscosity. Conversely the viscosity-sensitive neuronal population conveyed information about viscosity but not about the CSF. This new understanding of the representation of oral fat in the cerebral cortex and amygdala opens the way for the systematic development of foods with the pleasant mouthfeel of fat, together with ideal nutritional content and has great potential to contribute to healthy eating and a healthy body weight.

Interaction of Mannitol and Sucrose with Gellan Gum in Freeze-Dried Gel Systems.

The effect of sucrose and mannitol addition to low-acyl (LA) gellan gum gels at both the molecular and macroscopic levels prior to, and after freeze-drying has been investigated. It has been shown that the gel network order as well as the mechanical properties are changed with the solute content, especially in the case of sucrose. The freeze-dried gel structure, containing either mannitol or sucrose, was studied, reporting for the first time the interaction of mannitol with the gellan gum gel. The generated freeze-dried gel network was evaluated in terms of porosity, pore size and wall thickness distributions. The solute physical state was correlated the water activity trend as a function of the solute content. Since mannitol is crystalline, the water activity decreases, in contrast with the amorphous sucrose. The rehydration mechanism was investigated and associated with the solute release from the structure. Specifically, the material properties (surface and bulk) as well as the role of the dissolution medium over time were assessed. It was found that the rehydration for both the gellan/sucrose and gellan/mannitol systems was highly influenced by the additive content, as an increase in water uptake was measured up to 10 wt%. A further increase in solute led to a considerable drop in the rehydration rate and extent due to the change in the freeze-dried structure, with smaller pores and with higher wall thickness values.

What has become of critique? Reassembling sociology after Latour.

This paper offers a defence of sociology through an engagement with Actor Network Theory (ANT) and particularly the critique of 'critical' and politically engaged social science developed by Bruno Latour. It argues that ANT identifies some weaknesses in more conventional sociology and social theory, and suggests that 'critical' and 'public' orientated sociologists can learn from the analytical precision and ethnographic sensibilities that characterize ANT as a framework of analysis and a research programme. It argues, however, that Latour et al. have too hastily dispensed with 'critique' in favour of a value neutral descriptive sociology, and that the symmetrical and horizontalist approach adopted in ANT is particularly ill-suited to the development of scientific knowledge about social structures. It argues that a more straightforwardly realist sociology would share many of the strengths of ANT whilst being better able to interrogate, empirically and normatively, the centres of contemporary social power.

Lipid Based Nanosystems for Curcumin: Past, Present and Future.

Curcumin is one of the principle bioactive compounds used in the ayurvedic medicine system that has the history of over 5000 years for human use. Curcumin an "Indian Gold" is used to treat simple ailments like the common cold to severe life threatening diseases like cancer, and HIV. Though its contribution is immense for the health protection and disease prevention, its clinical use is limited due to its susceptible nature to alkaline pH, high temperature, presence of oxygen and light. Hence it becomes extremely difficult to maintain its bioactivity during processing, storage and consumption. Recent advancements in the application of nanotechnology to curcumin offer an opportunity to enhance its stability, bioactivity and to overcome its pharmacokinetic mismatch. This in turn helps to bridge the gaps that exist between its bench top research data to its clinical findings. Among the various types of nano/micro delivery systems, lipid based delivery systems are well studied and are the best suited delivery systems to enhance the stability and pharmacokinetic profile of curcumin both for pharma and the food application. In the current review, effort will be made to recapitulate the work done in the past to use lipid based delivery systems (liposomes, solid lipid nanoparticles, and emulsions) to enhance the application of curcumin for health promotion and disease prevention. Further, future prospects for the utilization of these lipid-based delivery systems will be discussed in detail.

The application of tribology in assessing texture perception of oral liquid medicines.

The palatability of medicines is likely to have a significant impact on patient adherence and consequently, on the safety and efficacy of a medicinal product. Palatability encompasses properties of medicines not limited to taste including swallowability (e.g. size, shape, texture). However, there has been limited work undertaken to measure the texture of medicines and how this may affect palatability and subsequent adherence. Tribology offers an understanding of oral processes and can allow physical properties of materials to be linked to "mouthfeel". This paper describes a preliminary application of tribology to oral liquid medicines and demonstrates that this technique is useful in the development of future oral liquid medicines.

Size dependence of shape and stiffness of single sessile oil nanodroplets as measured by atomic force microscopy.

This article presents results and guidelines on the quantitative analysis of size, shape, and stiffness of single sessile oil droplets in air and in water. Atomic force microscopy (AFM) facilitates the analysis of micro- and nanoscale droplets which are of growing importance for agrochemicals, cosmetics, or foodstuffs containing emulsions with nanoscale compartments or droplets. Measurement of droplet shape and stiffness provides information on the contact angle with the support surface as well as the interfacial tension of the liquid-liquid interface. In this study, micro- and nanoscale droplets were imaged both in amplitude modulation (AM) and force mapping modes. The effects of the AM mode set point ratio on the measured droplet shape are discussed, and a modified spherical cap model is suggested to extract the droplet-substrate contact angle. This model was applied to a population of different sized oil droplets imaged in water and led to the finding that the contact angle with the solid support varies with the droplet size. Force mapping was undertaken to measure the droplet stiffness as a function of the droplet size. Smaller droplets were found to be stiffer, in reasonable agreement with the Attard-Miklavcic model [Langmuir 2001, 17, 8217-8223] which describes the deformation of a sessile droplet in the nonwetting regime, i.e., by partial wrapping of the droplet around the probe surface. The model limitations are discussed in terms of the diverging droplet stiffness predicted for droplet radii similar to the probe radius as well as the error propagation associated with the droplet shape function.

Cockroach counts and house dust allergen concentrations after professional cockroach control and cleaning.

BACKGROUND: It is known that cockroach allergen exposure is both frequent in inner-city homes and associated with asthma severity in children living in those homes. However, there have been few studies of interventions to reduce exposures in this setting. OBJECTIVE: To evaluate the effect of short-term professional cockroach control and intensive cleaning on allergen concentrations. METHODS: Families of children from a school-based asthma treatment program who had skin test results positive to cockroach allergen were enrolled if the home had cockroaches. Forty-nine homes were randomly assigned to receive professional cleaning with bait traps containing insecticide, professional cleaning with bait traps without insecticide, or no cleaning or bait traps. In all homes, dust was collected repeatedly to evaluate cockroach allergen Bla g 2 in the kitchen and bedroom, and cockroaches were trapped and counted repeatedly for more than 11 weeks. RESULTS: Median cockroach counts were reduced in the homes treated with insecticide bait traps but not in other groups. There were significant reductions in allergen concentration in the kitchen in homes that received professional cleaning and had higher initial cockroach counts (54), regardless of whether bait traps had insecticide or not. CONCLUSIONS: We conclude that intensive cleaning can produce significant reductions in cockroach allergen in homes with heavy initial cockroach infestations.