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Abstract Background Our companion paper discussed the yield benefits achieved by integrating deacetylation, mechanical refining, and washing with low acid and low temperature pretreatment. To evaluate the impact of the modified process on the economic feasibility, a techno-economic analysis (TEA) was performed based on the experimental data presented in the companion paper. Results The cost benefits of dilute acid pretreatment technology combined with the process alternatives of deacetylation, mechanical refining, and pretreated solids washing were evaluated using cost benefit analysis within a conceptual modeling framework. Control cases were pretreated at much lower acid loadings and temperatures than used those in the NREL 2011 design case, resulting in much lower annual ethanol production. Therefore, the minimum ethanol selling prices (MESP) of the control cases were $0.41-$0.77 higher than the $2.15/gallon MESP of the design case. This increment is highly dependent on the carbohydrate content in the corn stover. However, if pretreatment was employed with either deacetylation or mechanical refining, the MESPs were reduced by $0.23-$0.30/gallon. Combing both steps could lower the MESP further by $0.44 ~ $0.54. Washing of the pretreated solids could also greatly improve the final ethanol yields. However, the large capital cost of the solid–liquid separation unit negatively influences the process economics. Finally, sensitivity analysis was performed to study the effect of the cost of the pretreatment reactor and the energy input for mechanical refining. A 50% cost reduction in the pretreatment reactor cost reduced the MESP of the entire conversion process by $0.11-$0.14/gallon, while a 10-fold increase in energy input for mechanical refining will increase the MESP by $0.07/gallon. Conclusion Deacetylation and mechanical refining process options combined with low acid, low severity pretreatments show improvements in ethanol yields and calculated MESP for cellulosic ethanol production.

Abstract Background Dilute acid pretreatment is a promising pretreatment technology for the biochemical production of ethanol from lignocellulosic biomass. During dilute acid pretreatment, xylan depolymerizes to form soluble xylose monomers and oligomers. Because the xylan found in nature is highly acetylated, the formation of xylose monomers requires two steps: 1) cleavage of the xylosidic bonds, and 2) cleavage of covalently bonded acetyl ester groups. Results In this study, we show that the latter may be the rate limiting step for xylose monomer formation. Furthermore, acetyl groups are also found to be a cause of biomass recalcitrance and hydrolyzate toxicity. While the removal of acetyl groups from native corn stover by alkaline de-esterification prior to pretreatment improves overall process yields, the exact impact is highly dependent on the corn stover variety in use. Xylose monomer yields in pretreatment generally increases by greater than 10%. Compared to pretreated corn stover controls, the deacetylated corn stover feedstock is approximately 20% more digestible after pretreatment. Finally, by lowering hydrolyzate toxicity, xylose utilization and ethanol yields are further improved during fermentation by roughly 10% and 7%, respectively. In this study, several varieties of corn stover lots were investigated to test the robustness of the deacetylation-pretreatment-saccharification-fermentation process. Conclusions Deacetylation shows significant improvement on glucose and xylose yields during pretreatment and enzymatic hydrolysis, but it also reduces hydrolyzate toxicity during fermentation, thereby improving ethanol yields and titer. The magnitude of effect is dependent on the selected corn stover variety, with several varieties achieving improvements of greater than 10% xylose yield in pretreatment, 20% glucose yield in low solids enzymatic hydrolysis and 7% overall ethanol yield.

Molnupiravir (EIDD-2801) (MLN) is an oral antiviral drug for COVID-19 treatment, being integrated into viral RNA through RNA-dependent RNA polymerase (RdRp). Upon ingestion, MLN is transformed into two active metabolites: β-d-N 4 -hydroxycytidine (NHC) (EIDD-1931) in the host plasma, and EIDD-1931-triphosphate (MTP) within the host cells. However, recent studies provide increasing evidence of MLN's interactions with off-target proteins beyond the viral genome, suggesting that the complete mechanisms of action of MLN remain unclear. The aim of this study was therefore to investigate the molecular interactions of MLN in the form of NHC and MTP with the non-RNA structural components of avian influenza (hemagglutinin, neuraminidase) and SARS-CoV-2 (spike glycoprotein, Mpro, and RdRp) viruses and to elucidate whether these two metabolites possess the ability to form stable complexes with these major viral components. Molecular docking of NHC and MTP was performed using AutoDock 4.2.6 and the obtained protein-drug complexes were submitted to 200-ns molecular dynamics simulations in triplicate with subsequent free energy calculations using GROMACS. Docking scores, molecular dynamics and MM/GBSA results showed that MTP was tightly bound within the active site of SARS-CoV-2 RdRp and remained highly stable throughout the 200-ns simulations. Besides, it was also shown that NHC and MTP formed moderately-to-highly stable molecular complexes with off-target receptors hemagglutinin, neuraminidase and Mpro, but rather weak interactions with spike glycoprotein. Our computational findings suggest that NHC and MTP may directly inhibit these receptors, and propose that additional studies on the off-target effects of MLN, i.e. real-time protein binding assays, should be performed.Communicated by Ramaswamy H. Sarma.

Expensive and time-consuming approaches of immunoelectron microscopy of biopsy tissues continues to serve as the gold-standard for diagnostic pathology. The recent development of the new approach of expansion microscopy (ExM) capable of fourfold lateral expansion of biological specimens for their morphological examination at approximately 70 nm lateral resolution using ordinary diffraction limited optical microscopy, is a major advancement in cellular imaging. Here we report (1) an optimized fixation protocol for retention of cellular morphology while obtaining optimal expansion, (2) an ExM procedure for up to eightfold lateral and over 500-fold volumetric expansion, (3) demonstrate that ExM is anisotropic or differential between tissues, cellular organelles and domains within organelles themselves, and (4) apply image analysis and machine learning (ML) approaches to precisely assess differentially expanded cellular structures. We refer to this enhanced ExM approach combined with ML as differential expansion microscopy (DiExM), applicable to profiling biological specimens at the nanometer scale. DiExM holds great promise for the precise, rapid and inexpensive diagnosis of disease from pathological specimen slides.

The cell plasma membrane is a highly dynamic organelle governing a wide range of cellular activities including ion transport, secretion, cell division, growth, and development. The fundamental process involved in the addition of new membranes to pre-existing plasma membranes, however, is unclear. Here, we report, using biophysical, morphological, biochemical, and molecular dynamic simulations, the selective incorporation of proteins and lipids from the cytosol into the cell plasma membrane dictated by membrane stretch and composition. Stretching of the cell membrane as a consequence of volume increase following incubation in a hypotonic solution and results in the incorporation of cytosolic proteins and lipids into the existing plasma membrane. Molecular dynamic simulations further confirm that increased membrane stretch results in the rapid insertion of lipids into the existing plasma membrane. Similarly, depletion of cholesterol from the cell plasma membrane selectively alters the incorporation of lipids into the membrane.

Ions greatly influence protein structure-function and are critical to health and disease. A 10, 000-fold higher calcium in the sarcoplasmic reticulum (SR) of muscle suggests elevated calcium levels near active calcium channels at the SR membrane and the impact of localized high calcium on the structure-function of the motor protein myosin. In the current study, combined quantum dot (QD)-based nanothermometry and circular dichroism (CD) spectroscopy enabled detection of previously unknown enthalpy changes and associated structural remodeling of myosin, impacting its function following exposure to elevated calcium. Cadmium telluride QDs adhere to myosin, function as thermal sensors, and reveal that exposure of myosin to calcium is exothermic, resulting in lowering of enthalpy, a decrease in alpha helical content measured using CD spectroscopy, and the consequent increase in motor efficiency. Isolated muscle fibers subjected to elevated levels of calcium further demonstrate fiber lengthening and decreased motility of actin filaments on myosin-functionalized substrates. Our results, in addition to providing new insights into our understanding of muscle structure-function, establish a novel approach to understand the enthalpy of protein-ion interactions and the accompanying structural changes that may occur within the protein molecule.

Despite recent advances in thermometry, determination of temperature at the nanometer scale in single molecules to live cells remains a challenge that holds great promise in disease detection among others. In the present study, we use a new approach to nanometer scale thermometry with a spatial and thermal resolution of 80 nm and 1 mK respectively, by directly associating 2 nm cadmium telluride quantum dots (CdTe QDs) to the subject under study. The 2 nm CdTe QDs physically adhered to bovine cardiac and rabbit skeletal muscle myosin, enabling the determination of heat released when ATP is hydrolyzed by both myosin motors. Greater heat loss reflects less work performed by the motor, hence decreased efficiency. Surprisingly, we found rabbit skeletal myosin to be more efficient than bovine cardiac. We have further extended this approach to demonstrate the gain in efficiency of Drosophila melanogaster skeletal muscle overexpressing the PGC-1α homologue spargel, a known mediator of improved exercise performance in humans. Our results establish a novel approach to determine muscle efficiency with promise for early diagnosis and treatment of various metabolic disorders including cancer.

Objectives: Smokers with posttraumatic stress disorder (PTSD) have increased difficulty achieving and maintaining abstinence. Contingency management approaches to smoking cessation interventions have demonstrated short-term efficacy but are limited by high rates of relapse. The goal of this pilot study was to evaluate the usability and feasibility of a smartphone-based smoking cessation application (Stay Quit Coach) designed to prevent relapse among individuals with PTSD., Methods: Smokers (N = 11) were randomized to (1) QUIT4EVER, an intervention combining mobile contingency management smoking cessation counseling and medications, and Stay Quit Coach or (2) a contact control condition that was identical to QUIT4EVER except Stay Quit Coach was not included. The primary outcome was prolonged smoking abstinence., Results: Among those queried during the follow-up periods, average Stay Quit Coach helpfulness ratings were high and ranged from 7.25 to 10 on a 10-point Likert scale (with higher scores corresponding to greater helpfulness). The Stay Quit Coach was rated by participants as being most effective at helping to quit smoking, helping to remain quit, and providing support and relevant information about quitting. Among the three quitters in the QUIT4EVER group, all reported abstinence at 3 and 6 months; however, abstinence was only bioverified for one quitter at 6 months. Among the four quitters in the contact control condition group, three reported abstinence at 3 and 6 months, but abstinence was not confirmed by bioverification., Conclusions: Smokers with PTSD express interest in and helpfulness of Stay Quit Coach for remaining abstinent after a quit attempt. Combined use of mobile contingency management and Stay Quit Coach is a feasible and acceptable adjunctive smoking cessation treatment for reducing smoking among smokers with PTSD. Adequately powered clinical trials are needed to demonstrate the long-term efficacy of this combined approach to smoking cessation. This study [Use of Technological Advances to Prevent Smoking Relapse among Smokers with PTSD (QUIT4EVER)] was registered on www.clinicaltrials.gov . clinicaltrials.gov identifier: NCT01990079.

Objective: To determine whether botulinum toxin type B (BTX-B) is effective in controlling upper-limb spasticity., Design: A single-site, double-blind, placebo-controlled, randomized trial and open-label study., Setting: Outpatient., Participants: Subjects with an Ashworth Scale score of 2 or more at the elbow, wrist, and fingers., Interventions: Subjects were injected with 10000 U of BTX-B or placebo at the elbow, wrist, and finger flexors. Main outcome measures Measures recorded at weeks 0, 2, 4, 8, 12, and 16, with a 12-week open-label study. Ashworth Scale score, a global assessment of change (GAC), adverse events and mouse neutralization antibody testing., Results: BTX-B did not decrease muscle tone in the elbow, wrist, or finger flexors at 10000 U over the 16-week period. A decrease in Ashworth Scale score for the BTX-B patient group was present at the wrist at week 2 of the double-blind study (P=.003) but was not statistically significant at other visits. In the open-label study, improvement was noted at week 4 for the elbow (P=.039), wrist (P=.002), finger (P=.001), and thumb flexors (P=.002). In the double-blind study, the Physician GAC did not reach significance. Dry mouth was reported by 8 of 9 BTX-B subjects in the double-blind study. Mouse neutralization antibodies were negative., Conclusions: Our study does not show a significant decrease in tone from 10000 U of BTX-B. Dry mouth was common.

CRH is known as the main stimulator of ACTH release. In representatives of all nonmammalian vertebrates, CRH has also been shown to induce TSH secretion, acting directly at the level of the pituitary. We have investigated which cell types and receptors are involved in CRH-induced TSH release in the chicken (Gallus gallus). Because a lack of CRH type 1 receptors (CRH-R1) on the chicken thyrotropes has been previously reported, two hypotheses were tested using in situ hybridization and perifusion studies: 1) TSH secretion might be induced in a paracrine way involving melanocortins from the corticotropes; and 2) thyrotropes might express another type of CRH-R. For the latter, we have cloned a partial cDNA encoding the chicken CRH-R2. Neither alpha-melanotropin (alpha-MSH) nor its powerful analog Nle4,d-Phe7-MSH could mimic the in vitro TSH-releasing effect of ovine CRH. The nonselective melanocortin receptor blocker SHU91199 did not influence CRH- or TRH-induced TSH secretion. On the other hand, we have found that thyrotropes express CRH-R2 mRNA. The involvement of this CRH receptor in the response of thyrotropes to CRH was further confirmed by the fact that TSH release was stimulated by human urocortin III, a CRH-R2-specific agonist, whereas the TSH response to CRH was completely blocked by the CRH-R blocker astressin and the CRH-R2-specific antagonist antisauvagine-30. We conclude that CRH-induced TSH secretion is mediated by CRH-R2 expressed on thyrotropes.

Objective: To determine if botulinum toxin type B (MyoBloc) decreases spasticity., Design: Investigator-initiated trial, open-label, single-treatment session., Setting: Single site., Participants: Ten patients with stable upper-limb spasticity and an Ashworth Scale score of 2 or higher at the elbow, wrist, and fingers., Interventions: Total dose of 10,000U of botulinum toxin type B injected into 5 major muscles., Main Outcome Measures: The Ashworth Scale, goniometry, and functional assessments were performed at injection and weeks 4, 8, and 12. The principal investigator (PI) global assessment of change (GAC) and the patient GAC were done at weeks 4, 8, and 12 postinjection. The safety of the procedure was measured by adverse events and vital signs., Results: Improvements in Ashworth Scale scores were observed at weeks 4, 8, and 12 postinjection. At week 4, the mean changes (in Ashworth score) were elbow, -1.0 (P=.016); wrist, -1.7 (P=.004); finger, -1.35 (P=.02); at week 8: elbow, -.83 (P=.016); wrist, -1.00 (P=.016); finger, -.94 (P=.08); and at week 12: elbow, -.61 (P=.07); wrist, -1.00 (P=.016); and finger, -.89 (P=.10). The PI GAC improved at all visits. Nine of the 10 subjects reported dry mouth at week 4, with resolution by week 12. No changes were seen on the functional measures., Conclusions: Botulinum toxin type B may be useful in treatment of spasticity., (Copyright 2003 by the American Congress of Rehabilitation Medicine and the American Academy of Physical Medicine and Rehabilitation)

The feeding behaviour of male monosex, female monosex, and mixed groups of Oreochromis niloticus was studied under conditions of self-feeding. Feeding activity was observed almost exclusively during the light period. The food intake pattern was similar whatever the sex ratio, and voluntary food intake (VFI) appeared lower in the male monosex groups than in the others. Male monosex groups displayed higher specific growth rates (SGR) and a lower food conversion ratio than female monosex and mixed groups. The SGR of males was higher in the monosex than in the mixed groups, whereas females of mixed and monosex groups displayed no significant difference in SGR. The efficiency of food utilisation was also analysed: nutrient retention ratios were higher in male monosex than in female monosex and mixed groups. Males displayed a distinctly higher metabolic capacity. Differences in sex-related hormones (11 ketotestosterone = 11-KT, 17beta-Oestradiol = 17beta-E2) and a metabolic hormone (triiodothyronine = T3) were observed between males and females. The hypothesis of an involvement of these hormones in the higher metabolic capacity of males is discussed. The observed differences in feeding behaviour between the different groups also suggest an effect of social interactions on the efficiency of food conversion and thus on the differential growth of males and females.

The full endocrine system is developed during the embryonic stages of the domestic fowl and probably in all avian species. This involves the genesis and differentiation of endocrine cells and progressive acquisition of interactions between endocrine tissues leading to integrated series of control systems. The embryonic endocrine organs produce hormones with a variety of physiological roles (including homeostatic, homeorhetic, and anticipatory). The present chapter provides an overview of the recent advances in the endocrinology of the avian embryo. Particular attention is focused on the general phenomenon of endocrine development and on the ontogeny of the hypothalamo-pituitary (growth hormone)-somatomedin-growth axis, the hypothalamo-pituitary-thyroid axis, and the hormones of calcium homeostasis.

Plasma concentrations of thyroxine (T4) and triiodothyronine (T3) were profoundly depressed both in chick embryos and growing chickens after methimazole (MMI) treatment. There was no response of T4 and T3 levels to TRH or TSH injections in the MMI group, either in embryos or growing chickens. Peroxidase activity measured in the thyroid gland was significantly higher in embryos and growing chickens treated with MMI. However, neither TRH nor TSH affected this activity 2 hr after injection in either control or the MMI-treated group. Hepatic 5'-monodeiodinase activity was significantly stimulated in the MMI-treated groups of embryos and growing chickens but only when additional sulphydryl groups (DTT) were provided. In embryos, monodeiodination activity 2 hr after TSH injection was not significantly different from control values for either DTT-stimulated or unstimulated conditions within the control and MMI-infused groups. However, in both control and MMI-treated embryos monodeiodination activity significantly increased 2 hr after TRH injection. In the growing chickens, monodeiodination activity 2 hr after TRH or TSH injection was not significantly different from control values in either stimulated or unstimulated conditions of each group.