Development of a powerful synthetic hybrid promoter to improve the cellulase system of Trichoderma reesei for efficient saccharification of corncob residues

Background The filamentous fungus Trichoderma reesei is a widely used workhorse for cellulase production in industry due to its prominent secretion capacity of extracellular cellulolytic enzymes. However, some key components are not always sufficient in this cellulase cocktail, making the conversion of cellulose-based biomass costly on the industrial scale. Development of strong and efficient promoters would enable cellulase cocktail to be optimized for bioconversion of biomass. Results In this study, a synthetic hybrid promoter was constructed and applied to optimize the cellulolytic system of T. reesei for efficient saccharification towards corncob residues. Firstly, a series of 5’ truncated promoters in different lengths were established based on the strong constitutive promoter Pcdna1. The strongest promoter amongst them was Pcdna1-3 (− 640 to − 1 bp upstream of the translation initiation codon ATG), exhibiting a 1.4-fold higher activity than that of the native cdna1 promoter. Meanwhile, the activation region (− 821 to − 622 bp upstream of the translation initiation codon ATG and devoid of the Cre1-binding sites) of the strong inducible promoter Pcbh1 was cloned and identified to be an amplifier in initiating gene expression. Finally, this activation region was fused to the strongest promoter Pcdna1-3, generating the novel synthetic hybrid promoter Pcc. This engineered promoter Pcc drove strong gene expression by displaying 1.6- and 1.8-fold stronger fluorescence intensity than Pcbh1 and Pcdna1 under the inducible condition using egfp as the reporter gene, respectively. Furthermore, Pcc was applied to overexpress the Aspergillus niger β-glucosidase BGLA coding gene bglA and the native endoglucanase EG2 coding gene eg2, achieving 43.5-fold BGL activity and 1.2-fold EG activity increase, respectively. Ultimately, to overcome the defects of the native cellulase system in T. reesei, the bglA and eg2 were co-overexpressed under the control of Pcc promoter. The bglA-eg2 double expression strain QPEB70 exhibited a 178% increase in total cellulase activity, whose cellulase system displayed 2.3- and 2.4-fold higher saccharification efficiency towards acid-pretreated and delignified corncob residues than the parental strain, respectively. Conclusions The synthetic hybrid promoter Pcc was generated and employed to improve the cellulase system of T. reesei by expressing specific components. Therefore, construction of synthetic hybrid promoters would allow particular cellulase genes to be expressed at desired levels, which is a viable strategy to optimize the cellulolytic enzyme system for efficient biomass bioconversion.

Peculiarities of diet, physical activity and body weight of medical university students during COVID-19 restrictive measures

BACKGROUND: Pandemics of new coronavirus infection (COVID-19) introduced additional corrections in the style of life of students. Introduction of restrictive measures, transfer to distance learning produce an ambiguous effect on the vital activity of a human including his physical health and diet. AIM: To evaluate the impact of COVID-19 restrictive measures on nutrition, physical activity and body mass of medical students. MATERIALS AND METHODS: Sociological, analytical, statistical methods were used. According to the results of a questionnaire survey of 167 third-year students of the medical faculty of Smolensk State Medical University, a comparative assessment of nutrition, physical activity and body weight before the introduction of COVID-19 restrictive measures and in 3 4 months after self-isolation is presented. The reliability of differences in the shares of the characteristic was assessed by the value of Fishers angular transformation coefficient (φ). RESULTS: Diets of medical students were unbalanced both before and during COVID-19 restrictive measures. Changes in the diet during the period of self-isolation took place in 54.2% of the respondents. The number of students with 4 meals a day increased 1.6 times, and with 5 meals 12.9 times. The share of respondents with 2 meals a day decreased 6.3 times. Daytime food intake decreased 1.7 times, and 2.8 times more often the respondents did not have a fixed time of food intake. All medical students refused a full breakfast both before and during the period of self-isolation. The predomination of evening meals decreased 2.4 times. Home-made dishes were consumed 1.2 times more often, semi-finished products 2.4 times less often, and consumption of fast food decreased by 4.2%. Physical activity during the period of self-isolation significantly decreased in every fifth student. Every fourth respondent noticed increase in the body weight. CONCLUSION: Restrictive measures did not change the preferences in the choice of food products by medical students and the habits of not eating breakfast, and imbalanced diets. The positive impact of restrictive measures for COVID-19: increase in use of freshly prepared meals, the frequency of meals, and limitation of the possibility to eat in fast food restaurants. The negative effect of self-isolation: frequent infringement of the diet, increased daily amount of fast food consumed, decrease in physical activity, increase in body weight.

Methods of macrophages activation and their modulation for the prospection of new antileishmania drugs: a review

Leishmaniasis are a group of parasitic zoonoses provoked by protozoa from Leishmania genus and belonging to the group of neglected tropical diseases. The search and development for new drugs is necessary not only to investigate the activity against only the parasite, but also to investigate the possible synergistic effect of new drugs with the immune response of the host. In the present review, macrophages are pointed out as potential targets of the investigation of new antileishmanial drugs, and some methodologies in order to assess their activation as response to Leishmania-infected cells are presented. Macrophages are an important role in the cellular immune response, since they are cells from mononuclear phagocytic system, the first line of defense of the host, against parasites from Leishmania genus. Phagocytic capacity, lysosomal activity, increase of nitric oxide and intracellular calcium levels are parameters regarding assessment of macrophages activation which allow them to be more hostile in order to solve the infection and lead the patient to cure. In this context, we bring 19 substances already investigated and that activate macrophages, what makes them promising in the antileishmanial treatment. Therefore, assessment of macrophages activation, are important tools for discovery of immunomodulatory compounds which have potential to act in synergism with host immune response. Such compounds might be promising as monotherapy in the treatment of leishmaniasis, as well as being used as adjuvants in vaccines and/or in combination with conventional drugs.

Macronutrients and essential amino acids on digestive process of the freshwater teleost Matrinxã

The objective of this work was to evaluate the effect of macronutrients and essential amino acids on digestive process of the freshwater teleost Matrinxã (Brycon amazonicus). Juveniles were fed with diets containing starch plus free amino acids or oil plus free amino acids for 15 days. These fish were compared with others fed with diets containing starch or oil without addition of free amino acids. After the experimental span, 12 fish from each treatment were randomly sampled to collect stomach, pyloric cecum, anterior and posterior intestine for assaying digestive enzymes activity. Increase of gastric proteolysis due to dietary amino acids were observed. Amylolytic, proteolytic and lipolytic activities in intestine sections were also positive related to dietary amino acids. However, proteolytic and lipolytic activities in pyloric cecum were not responsive to dietary changes. Moreover, the absence of starch in the diets resulted in decrease of amylolysis, and very low levels of oil did not change the lipolytic activity. In conclusion, activities of amylase, protease and lipase of Matrinxãare selectively responsive to addition of free essential amino acids concerning the gut section.

Methanol Steam Reforming on Bimetallic Catalysts Based on In and Nb Doped Titania or Zirconia: A Support Effect

Methanol steam reforming (MSR) is considered an effective method for hydrogen storage and to generate high-quality hydrogen for fuel cells. In this work, a comprehensive investigation of the methanol steam reforming process using a bimetallic Pt–Rh and Cu–Ni based on different oxide supports is presented. Highly dispersed titania and zirconia doped with indium and niobium ions were synthesized by sol–gel method. The effect of the nature and quantity of the dopant cation (In, Nb) on the catalytic performance of titania supported metal catalysts was investigated. The conclusions obtained show a significant effect of both the metal alloy and the oxide support nature on the activity and selectivity of the methanol steam reforming process. Pt–Rh alloy catalyst shows higher hydrogen yield, but its selectivity in the MSR process is lower than for the catalysts containing the Cu0.8-Ni0.2 alloy. Heterovalent indium doping of titania leads to the catalytic activity increase. It was suggested that this is due to the defects formation in the oxygen TiO2 sublattice. On the contrary, the use of niobium oxide as a dopant decreases the catalyst activity in the methanol steam reforming process but leads to the selectivity increase in the studied process.

A novel Bacillus ligniniphilus catechol 2,3-dioxygenase shows unique substrate preference and metal requirement

Identification of novel enzymes from lignin degrading microorganisms will help to develop biotechnologies for biomass valorization and aromatic hydrocarbons degradation. Bacillus ligniniphilus L1 grows with alkaline lignin as the single carbon source and is a great candidate for ligninolytic enzyme identification. The first dioxygenase from strain L1 was heterologously expressed, purified, and characterized with an optimal temperature and pH of 32.5 °C and 7.4, respectively. It showed the highest activity with 3-ethylcatechol and significant activities with other substrates in the decreasing order of 3-ethylcatechol > 3-methylcatechol > 3-isopropyl catechol > 2, 3-dihydroxybiphenyl > 4-methylcatechol > catechol. It did not show activities against other tested substrates with similar structures. Most reported catechol 2,3-dioxygenases (C23Os) are Fe2+-dependent whereas Bacillus ligniniphilus catechol 2,3-dioxygenase (BLC23O) is more Mn2+- dependent. At 1 mM, Mn2+ led to 230-fold activity increase and Fe2+ led to 22-fold increase. Sequence comparison and phylogenetic analyses suggested that BL23O is different from other Mn-dependent enzymes and uniquely grouped with an uncharacterized vicinal oxygen chelate (VOC) family protein from Paenibacillus apiaries. Gel filtration analysis showed that BLC23O is a monomer under native condition. This is the first report of a C23O from Bacillus ligniniphilus L1 with unique substrate preference, metal-dependency, and monomeric structure.

Effect of Lewis Acids on the Catalyst Activity for Alkene Metathesis, Z-/E- Selectivity and Stability of Tungsten Oxo Alkylidenes

Lewis acids increase the catalytic activity of classical heterogeneous catalysts and molecular d0 tungsten oxo alkylidenes in a variety of olefin metathesis processes. The formation of labile adducts between the metal complex and the Lewis acid has been observed experimentally and suggested to be involved in the catalyst activity increase. In this contribution, DFT (M06) calculations have been performed to determine the role of Lewis acids on catalyst activity, Z-/E- selectivity and stability by comparing three W(E)(CHR)(2,5-dimethylpyrrolide)(O-2,6-dimesithylphenoxide) (E = oxo, imido or oxo-Lewis acid adduct) alkylidenes. Results show that the formation of the alkylidene—Lewis acid adducts influences the reactivity of tungsten oxo alkylidenes due to both steric and electronic effects. The addition of the Lewis acid on the E group increases its bulkiness and this decreases catalyst Z-selectivity. Moreover, the interaction between the oxo ligand and the Lewis acid decreases the donating ability of the former toward the metal. This is important when the oxo group has either a ligand in trans or in the same plane that is competing for the same metal d orbitals. Therefore, the weakening of oxo donating ability facilitates the cycloaddition and cycloreversion steps and it stabilizes the productive trigonal bipyramid metallacyclobutane isomer. The two factors increase the catalytic activity of the complex. The electron donating tuneability by the coordination of the Lewis acid also applies to catalyst deactivation and particularly the key β-hydride elimination step. In this process, the transition states show a ligand in pseudo trans to the oxo. Therefore, the presence of the Lewis acid decreases the Gibbs energy barrier significantly. Overall, the optimization of the E group donating ability in each step of the reaction makes tungsten oxo alkylidenes more reactive and this applies both for the catalytic activity and catalyst deactivation.

A case in which TAS-102 produced disease control without severe adverse events in a patient with recurrent colorectal cancer and dihydropyrimidine dehydrogenase deficiency

Fluoropyrimidine is commonly used to treat unresectable cases of metastatic colorectal cancer or as an adjuvant therapy for colorectal cancer. Dihydropyrimidine dehydrogenase (DPD) is an enzyme encoded by the DPYD gene, which is responsible for the rate-limiting step in pyrimidine catabolism and breaks down >80% of standard doses of 5-fluorouracil (5-FU). Reductions in DPD activity increase the half-life of 5-FU, resulting in excess 5-FU accumulation and toxicity, which can lead to life-threatening side effects. There have been several published case reports about DPD deficiency in colorectal cancer patients from Western countries. However, case reports of DPD deficiency in Japanese colorectal cancer patients are rare because the measurement of DPD activity is not covered by the public medical insurance system in Japan, and DPD activity is not currently measured in daily clinical practice. Furthermore, there have not been any reports about anticancer drug therapy for Japanese patients with DPD deficiency. In this report, we describe a case in which a Japanese patient with colorectal cancer was diagnosed with DPD deficiency. The DPD deficiency arose as a severe adverse effect of mFOLFOX6/CapOX treatment for recurrent colorectal cancer, and the patient was subsequently treated with TAS-102, without experiencing any severe adverse effects. We report this case along with a review of the literature.

Detection of Changes on Parameters Related to Heart Rate Variability after Applying Current Interferential Therapy in Subjects with Non-Specific Low Back Pain

Interferential current therapy (ICT) is an electrotherapeutic intervention that combines the advantages of high permeability from middle frequency currents and efficient tissue stimulation from low frequency currents, delivering the maximum current with high tissue permeability. The aim was to evaluate the effects of ICT on heart rate variability (HRV) and on pain perception in patients with non-specific chronic low back pain (NSCLBP). In the study, 49 patients with NSCLBP were randomly divided into an experimental (EG) and a sham group (SG). All participants received a single intervention, ICT, or simulated intervention. Outcome measures including baseline (sit-down position) and postintervention (prone position) pain, heart rate (HR), time domain parameter (rMSSD), diameters of the Poincaré plot (SD1, SD2), stress score (SS), and sympathetic/parasympathetic (S/PS) ratio were investigated. In both groups, significant statistical differences were found in perceived pain and in all HRV parameters except in HRmax. Between-group comparisons showed statistically significant differences in all variables except for HRmin and HRmean in favor of the experimental group. These changes reported an increase in parasympathetic activity (rMSSD) (p < 0.05) and a decrease in sympathetic activity (increase in SD2 and decrease in SS) (p < 0.001) and perceived pain (p < 0.001), with a greater size effect (η2 = 0.44) in favor of the experimental group. In conclusion, a single session of ICT can shift the autonomic balance towards increase parasympathetic dominance and decrease the sympathetic dominance and intensity of pain perceived by patients with NSCLBP.

A corticothalamic circuit trades off speed for safety during decision-making under motivational conflict

Decisions to act while pursuing goals in the presence of danger must be made quickly but safely. Premature decisions risk injury or death whereas postponing decisions risk goal loss. Here we show how mice resolve these competing demands. Using microstructural behavioral analyses, we identified the spatiotemporal dynamics of approach-avoidance decisions under motivational conflict. Then we used cognitive modelling to show that these dynamics reflect the speeded decision-making mechanisms used by humans and non-human primates, with mice trading off decision speed for safety of choice when danger loomed. Using calcium imaging and functional circuit analyses, we show that this speed-safety trade off occurs because increases in paraventricular thalamus (PVT) activity increase decision caution, thereby increasing approach-avoid decision times in the presence of danger. Our findings demonstrate that a discrete brain circuit involving the PVT and its prefrontal cortical input dynamically adjusts decision caution during motivational conflict, trading off decision speed for decision safety when danger is close. They identify the corticothalamic pathway as central to cognitive control during decision-making under conflict.