Small extracellular vesicles' enrichment from biological fluids using an acoustic trap.

Small extracellular vesicles (sEVs), a form of extracellular vesicles, are lipid bilayered structures released by all cells. Large-scale studies on sEVs from clinical samples are necessary, but a major obstacle is the lack of rapid, reproducible, efficient, and low-cost methods to enrich sEVs. Acoustic microfluidics have the advantage of being label-free and biocompatible, which have been reported to successfully enrich sEVs. In this paper, we present a highly efficient acoustic microfluidic trap that can offer low and large volume compatible ways of enriching sEVs from biological fluids by flexible structure design. It uses the idea of pre-loading larger seed particles in the acoustic trap to enable sub-micron particle capturing. The microfluidic chip is actuated using a piezoelectric plate transducer attached to a silicon-glass bonding plate with circular cavities. Each cavity works as a resonant unit, excited at the frequency of both the half wave resonance in the main plane and inverted quarter wave resonance in the depth direction, which has the ability to strongly trap seed particles at the center, thereby improving the subsequent nanoparticle capture efficiency. Mean trapping efficiencies of 35.62% and 64.27% were obtained using 60 nm and 100 nm nanobeads, respectively. By the use of this technology, we have successfully enriched sEVs from cell culture conditioned media and blood plasma at a flow rate of 10 µL min-1. The isolated sEV subpopulations are characterized by NTA and TEM, and their protein cargo is determined by WB. This acoustic trapping chip provides a rapid and robust method to enrich sEVs from biofluids with high reproducibility and sufficient quantities. Therefore, it can serve as a new tool for biological and clinical research such as cancer diagnosis and drug delivery.

Sheathless acoustic based flow cell sorter for enrichment of rare cells.

Cell enrichment is a powerful tool in many kinds of cell research, especially in applications with low abundance cell types. In this work, we developed a microfluidic fluorescence activated cell sorting device that was able to perform on-demand, low loss cell detection, and sorting. The chip utilizes three-dimensional acoustic standing waves to position all cells in the same fluid velocity regime without sheath. When the cells pass through a laser interrogation region, the scattering and fluorescent signals are detected, translated and transported to software. The target cells are then identified by gating on the plots. Short bursts of standing acoustic waves are triggered by order from PC to sort target cells within predefined gating region. For very low abundance and rare labeled lymphocytes mixed with high concentration unlabeled white blood cells (WBCs), (1-100 labeled lymphocytes are diluted in 106 WBCs in 1 ml volume fluid), the device is able to remove more than 98% WBCs and recover labeled lymphocytes with efficiency of 80%. We further demonstrated that this device worked with real clinical samples by successfully isolating fetal nucleated red blood cells (FNRBCs) in the blood samples from pregnant women with male fetus. The obtained cells were sequenced and the expressions of (sex determining region Y) SRY genes were tested to determine fetal cell proportion. In genetic analysis, the proportion of fetal cells in the final picked sample is up to 40.64%. With this ability, the device proposed could be valuable for biomedical applications involving fetal cells, circulating tumor cells, and stem cells.

Interaction between Curcumin and ß-Casein: Multi-Spectroscopic and Molecular Dynamics Simulation Methods.

Effect of temperature and pH on the interaction of curcumin with ß-casein was explored by fluorescence spectroscopy, ultraviolet-visible spectroscopy and molecular dynamics simulation. The spectroscopic results showed that curcumin could bind to ß-casein to form a complex which was driven mainly by electrostatic interaction. The intrinsic fluorescence of ß-casein was quenched by curcumin through static quenching mechanism. The binding constants of curcumin to ß-casein were 6.48 × 104 L/mol (298 K), 6.17 × 104 L/mol (305 K) and 5.73 × 104 L/mol (312 K) at pH 2.0, which was greater than that (3.98 × 104 L/mol at 298 K, 3.90 × 104 L/mol at 305 K and 3.41 × 104 L/mol at 312 K) at pH 7.4. Molecular docking study showed that binding energy of ß-casein-curcumin complex at pH 2.0 (-7.53 kcal/mol) was lower than that at pH 7.4 (-7.01 kcal/mol). The molecular dynamics simulation study showed that the binding energy (-131.07 kJ/mol) of ß-casein-curcumin complex was relatively low at pH 2.0 and 298 K. α-Helix content in ß-casein was decreased and random coil content was increased in the presence of curcumin. These results can promote a deep understanding of interaction between curcumin and ß-casein and provide a reference for improving the bioavailability of curcumin.

Improved Physicochemical Properties of Yogurt Fortified with Fish Oil/γ-Oryzanol by Nanoemulsion Technology.

Fish oil has several dietary benefits, but its application in food formulations is limited because of its poor water-solubility, easy oxidation and strong odor. The purposes of this study were to produce a fish oil/γ-oryzanol nanoemulsion and to evaluate the effect of adding this nanoemulsion on the physicochemical and sensory characteristics of yogurts. Adding fish oil/γ-oryzanol nanoemulsion resulted in a significant reduction in the acidity and syneresis of yogurt. Yogurt with the nanoemulsion had significantly lower peroxide value (0.28 mmol/L after 21 days) and higher retention of eicosapentaenoic acid and docosahexaenoic acid contents (decreased to 95% and 94% of its initial value, respectively) than yogurt with fish oil/γ-oryzanol (peroxide value = 0.65 mmol/L; eicosapentaenoic acid and docosahexaenoic acid contents decreased to 72% and 53% of its initial value, respectively). Fish oil/γ-oryzanol nanoemulsion incorporated into yogurt had closer sensory attributes scores to plain yogurt. This study may have important implications for the application of fish oil/γ-oryzanol nanoemulsion in yogurt.

Epidermal Growth Factor and Intestinal Barrier Function.

Epidermal growth factor (EGF) is a 53-amino acid peptide that plays an important role in regulating cell growth, survival, migration, apoptosis, proliferation, and differentiation. In addition, EGF has been established to be an effective intestinal regulator helping to protect intestinal barrier integrity, which was essential for the absorption of nutrients and health in humans and animals. Several researches have demonstrated that EGF via binding to the EGF receptor and subsequent activation of Ras/MAPK, PI3K/AKT, PLC-γ/PKC, and STATS signal pathways regulates intestinal barrier function. In this review, the relationship between epidermal growth factor and intestinal development and intestinal barrier is described, to provide a better understanding of the effects of EGF on intestine development and health.

Effects of dietary addition of heat-killed Mycobacterium phlei on growth performance, immune status and anti-oxidative capacity in early weaned piglets.

The contradiction between high susceptibility of early weaned piglets to enteric pathogens and rigid restriction of antibiotic use in the diet is still prominent in the livestock production industry. To address this issue, the study was designed to replace dietary antibiotics partly or completely by an immunostimulant, namely heat-killed Mycobacterium phlei (M. phlei). Piglets (n = 192) were randomly assigned to one of the four groups: (1) basal diet (Group A), (2) basal diet + a mixture of antibiotics (80 mg/kg diet, Group B), (3) basal diet + a mixture of antibiotics (same as in Group B, but 40 mg/kg diet) + heat-killed M. phlei (1.5 g/kg diet) (Group C) and (4) basal diet + heat-killed M. phlei (3 g/kg diet) (Group D). All piglets received the respective diets from days 21 to 51 of age and were weaned at the age of 28 d. Compared with the Control (Group A), in all other groups the average daily gain, average daily feed intake, small intestinal villus height:crypt depth ratio and protein levels of occludin and ZO-1 in the jejunal mucosa were increased. A decreased incidence of diarrhoea in conjunction with an increased sIgA concentration in the intestinal mucosa and serum IL-12 and IFN-γ concentrations was found in groups supplemented with heat-killed M. phlei (Groups C and D), but not in Group B. Groups C and D also showed decreased IL-2 concentrations in the intestinal mucosa with lower TLR4 and phosphor-IκB protein levels. The antioxidant capacity was reinforced in Groups C and D, as evidenced by the reduction in malondialdehyde and enhanced activities of antioxidant enzymes in serum. These data indicate that heat-killed M. phlei is a promising alternative to antibiotic use for early weaned piglets via induction of protective immune responses.

Interferon regulatory factor 9 protects against hepatic insulin resistance and steatosis in male mice.

UNLABELLED: Obesity is a calorie-excessive state associated with high risk of diabetes, atherosclerosis, and certain types of tumors. Obesity may induce inflammation and insulin resistance (IR). We found that the expression of interferon (IFN) regulatory factor 9 (IRF9), a major transcription factor mediating IFN responses, was lower in livers of obese mice than in those of their lean counterparts. Furthermore, whole-body IRF9 knockout (KO) mice were more obese and had aggravated IR, hepatic steatosis, and inflammation after chronic high-fat diet feeding. In contrast, adenoviral-mediated hepatic IRF9 overexpression in both diet-induced and genetically (ob/ob) obese mice showed markedly improved hepatic insulin sensitivity and attenuated hepatic steatosis and inflammation. We further employed a yeast two-hybrid screening system to investigate the interactions between IRF9 and its cofactors. Importantly, we identified that IRF9 interacts with peroxisome proliferator-activated receptor alpha (PPAR-α), an important metabolism-associated nuclear receptor, to activate PPAR-α target genes. In addition, liver-specific PPAR-α overexpression rescued insulin sensitivity and ameliorated hepatic steatosis and inflammation in IRF9 KO mice. CONCLUSION: IRF9 attenuates hepatic IR, steatosis, and inflammation through interaction with PPAR-α.

BmAly is an important factor in meiotic progression and spermatid differentiation in Bombyx mori (Lepidoptera: Bombycidae).

The Drosophila melanogaster "always early" gene (Dmaly), which is required for G2/M cell-cycle control and spermatid differentiation, is one of the meiotic arrest genes. To study the Bombyx mori aly gene (Bmaly), the cDNA of Bmaly was cloned and sequenced, and the results showed that the open reading frame of Bmaly is 1,713 bp in length, encoding 570 amino acid residues, in which a domain in an Rb-related pathway was found. Phylogenetic analysis based on the amino acid sequence of conserved regions showed that Aly from different insects gathered together, except for DmAly and Culex quinquefasciatus Aly, which were not clustered to a subgroup according to insect order. The Bmaly gene was inserted into expression vector pGS-21a(+) and then the recombinant protein was expressed in Escherichia coli and used to immunize mice to prepare the antibody against BmAly. Immunofluorescence examination showed that BmAly was distributed in both the cytoplasm and nucleus of BmN cell. The Bmaly gene expression could not be detected in the silk gland, malpighian tubule, fat body, or midgut of the silkworm. Expression levels of the Bmaly gene were detected in the gonadal tissues, where the levels in the testes were 10 times higher than that in the ovaries. Moreover, Bmaly expression was detected by quantitative polymerase chain reaction at different stages of B. mori testis development, at which fifth instar was relatively grossly expressed. The result suggested Bmaly was abundantly expressed in primary spermatocytes and prespermatids. To further explore the function of Bmaly, Bmaly siRNA was injected into third and fourth instar silkworm larvae, which markedly inhibited the development of sperm cells. These results together suggest that Bmaly is a meiotic arrest gene that plays an important role in spermatogenesis.

Fatty acid specificity of T1 lipase and its potential in acylglycerol synthesis.

BACKGROUND: T1 lipase has received considerable attention due to its thermostability. Fatty acid specificity of T1 lipase (crude and purified) was investigated, and its potential in the synthesis of acylglycerols was also evaluated. RESULTS: Fatty acid specificity of T1 lipase (crude and purified) was investigated in the esterification of fatty acids (C6:0 to C18:3), suggesting that crude and purified T1 lipase had the lowest preference for C18:0 [specificity constant (1/α) = 0.08] followed by C18:1 (1/α = 0.12) and showed the highest preference for C8:0 (1/α = 1). A structural model was constructed to briefly explore interactions between the lipase and its substrate. Furthermore, crude T1 lipase-catalysed synthesis of diacylglycerols (DAGs) and monoacylglycerols (MAGs) by esterification of glycerol with C18:1 was studied for evaluating its potential in acylglycerols synthesis. The optimal conditions were glycerol/oleic acid molar ratio 5:1, the lipase concentration 9.7 U g(-1) of substrates, water content 50 g kg(-1) of substrates and temperature 50 °C, which yielded 42.25% DAGs, 26.34% MAGs and 9.18% triacylglycerols at 2 h. CONCLUSION: DAGs and MAGs were synthesised in good yields although C18:1 (a much poorer substrate) was used. Our work demonstrates that T1 lipase, which was discovered to show 1,3-regio-selectivity, is a promising biocatalyst for lipids modification.

Improved lipase performance by covalent immobilization of Candida antarctica lipase B on amino acid modified microcrystalline cellulose as green renewable support.

Development of immobilized lipase with excellent catalytic performance and low cost is the major challenge for large-scale industrial applications. In this study, green renewable microcrystalline cellulose (MCC) that was hydrophobically modified with D-alanine (Ala) or L-lysine (Lys) was used for immobilizing Candida antarctica lipase B (CALB). The improved catalytic properties were investigated by experimental and computational methods. CALB immobilized on MCC-Ala with higher hydrophobicity showed better catalytic activity than CALB@MCC-Lys because the increased flexibility of the lid region of CALB@MCC-Ala favored the formation of open conformation. Additionally, the low root mean square deviation and the high ß-sheet and α-helix contents of CALB@MCC-Ala indicated that the structure became more stable, leading to a significantly enhanced stability (54.80% and 90.90% relative activity at 70 °C and pH 9.0, respectively) and good reusability (48.92% activity after 5 cycles). This study provides a promising avenue to develop immobilized lipase with high catalytic properties for industry applications.

Improved physicochemical properties of bigels produced with ethyl cellulose-based oleogel and moderately deacetylated konjac glucomannan hydrogel.

The ideal physicochemical properties of bigels are important for food applications. Therefore, a new bigel was prepared based on mixed beef tallow and soybean oil oleogel and deacetylated konjac glucomannan (KGM) hydrogel. The effect of the deacetylation degree of KGM on the physicochemical properties and microstructure of bigels was studied. The bigel containing moderate deacetylation degree of KGM had better rheological properties and hardness (319.84 g) than that with low and high deacetylation degrees of KGM. The interactions among the bigel components were analyzed by Fourier transform infrared spectroscopy and molecular dynamics simulation, indicating that the formation of the bigels was dominated by electrostatic interactions. Overall, the bigels containing moderate deacetylation degree of KGM had better physical properties, which may provide a theoretical foundation to develop bigels with low cholesterol, trans and saturated fats levels to replace traditional solid fats in food industry.

Improved physicochemical properties and in vitro digestion of walnut oil microcapsules with soy protein isolate and highly oxidized konjac glucomannan as wall materials.

This study investigated the effect of the oxidation degrees of oxidized konjac glucomannan (OKGM) on the encapsulation efficiency (EE), physicochemical and in vitro digestive properties of soy protein isolate (SPI)-based microcapsules walnut oil using experimental and computational approaches. Microcapsules had the highest EE when the ratio of OKGM and SPI to oil was 2.5:1. With increasing the oxidation degree of OKGM, the EE of microcapsules was increased and the hygroscopicity was decreased. Molecular dynamics simulation results showed that SPI/oil/highly OKGM had relatively low binding energy (-4.03 × 106 kJ/mol) and strong electrostatic interactions, which may contribute to a higher EE and lower hygroscopicity of microcapsules, respectively. The oxidative stability of the oil was markedly improved by SPI and OKGM, and microcapsules prepared with SPI and highly OKGM had the highest in vitro digestion. This study provided theoretical support for broadening the application of microcapsules prepared with SPI and OKGM.

Enhancing the antioxidant properties and compatibility of protein/sodium alginate film by incorporating Zanthoxylum bungeanum essential oil Pickering emulsion.

Zanthoxylum bungeanum essential oil (ZBEO) Pickering emulsion was incorporated into rice protein (RP)/sodium alginate (SA)-based film to enhance the antioxidant activity and compatibility. With increasing ZBEO content from 2 % to 4 %, the average size of ZBEO Pickering emulsion ranged from 124.28 to 165.65 nm. The best mechanical property with a tensile strength of 14.56 MPa and hydrophobicity with a water vapor permeability of 2.11 × 10-12 g⋅cm-1⋅s-1⋅Pa-1 of emulsion film were achieved with 0.8 % ZBEO. In addition, the loss of ZBEO in the emulsion films was reduced by 11-14 %. The DPPH radical scavenging activity of emulsion film with 1.2 % ZBEO was 65.54 % in 95 % ethanol. The results of Fourier transform infrared spectroscopy and molecular dynamics simulation showed that electrostatic interactions played a leading role in film formation. Overall, ZBEO Pickering emulsion is an effective method to enhance the antioxidant activity, mechanical strength and hydrophobicity of RP/SA film.

Interferon regulatory factor 7 deficiency prevents diet-induced obesity and insulin resistance.

Obesity-related inflammation has been implicated in the pathogenesis of insulin resistance and type 2 diabetes. In this study, we addressed the potential role of interferon regulatory factor 7 (IRF7), a master regulator of type I interferon-dependent immune responses, in the regulation of energy metabolism. The expression levels of IRF7 were increased in white adipose tissue, liver tissue, and gastrocnemius muscle of both diet-induced obese mice and ob/ob mice compared with their lean counterparts. After feeding a high-fat diet (HFD) for 24 wk, IRF7 knockout (KO) mice showed less weight gain and adiposity than wild-type controls. KO of IRF7 improved glucose and lipid homeostasis and insulin sensitivity. Additionally, KO of IRF7 ameliorated diet-induced hepatic steatosis. Next, we assessed the inflammatory state of the IRF7 KO mice on the HFD. These mice showed less macrophage infiltration into multiple organs and were protected from local and systemic inflammation. This study demonstrates a role for IRF7 in diet-induced alterations in energy metabolism and insulin sensitivity. Our results also suggest that IRF7 is involved in the etiology of metabolic abnormalities, which suggests a new strategy for treating obesity and type 2 diabetes.

Interaction, bioaccessibility and stability of bovine serum albumin-gamma-oryzanol complex: Spectroscopic and computational approaches.

The study explored the interaction mechanism between bovine serum albumin (BSA) and gamma-oryzanol (GO) by spectroscopic and computational approaches and the potential to enhance bioaccessibility and chemical stability of GO in the complex with BSA. Fluorescence spectroscopy showed that GO was bound to BSA with static quenching at a single binding site, being consistent with molecular docking results. Thermodynamic analysis and molecular dynamics simulation showed that electrostatic forces dominated interactions between BSA and GO. Besides, BSA-GO complex was more stable at pH 7.4 than at pH 2.0, with low root-mean-square deviation (2.57 Å vs 12.37 Å) and low binding energy (-424.23 kJ/mol at 277 K vs -188.55 kJ/mol at 277 K), but complex stability significantly decreased with increasing temperature. The bioaccessibility and stability of GO in the complex were significantly higher than those in water. This study provided theoretical support for developing proteins as delivery system for GO.

Performance of nitrification-denitrification and denitrifying phosphorus removal driven by in-situ generated biogenic manganese oxides in a moving bed biofilm reactor.

Simultaneous removal of NH4+-N, NO3--N, COD, and P by manganese redox cycling in nutrient wastewater was established with two moving bed biofilm reactors (MBBRs) with in-situ generated biogenic manganese oxides (BioMnOx) and non-BioMnOx. In-situ generated BioMnOx preferentially promoted the denitrification, and the average removal of NO3--N, NH4+-N, and TN in the experimental MBBR with BioMnOx increased to 89.00%, 70.64%, and 76.06% compared with the control MBBR with non-BioMnOx. The relevant enzymes activity, extracellular polymeric substance (EPS), electron transport system activity (ETSA), and reactive oxygen species (ROS) were investigated. The element valence and morphology of purified BioMnOx were characterized by X-ray photoelectron spectroscopy (XPS) and transmission electron microscopy (TEM), as well as the effect of BioMnOx on nitrogen and phosphorus removal. The results suggested that BioMnOx could improve nitrogen conversion. Electrochemical characteristic and microbial community were detected. This study provided a new strategy for nutrients removal in BioMnOx-mediated wastewater treatment.

Development of air-assisted atomization device for the delivery of cells in viscous biological ink prepared with sodium alginate.

Skin wounds, especially large-area skin trauma, would bring great pain and even fatal risk to patients. In recent years, local autologous cell transplantation has shown great potential for wound healing and re-epithelialization. However, when the cell suspension prepared with normal saline is delivered to the wound, due to its low viscosity, it is easy to form big drops in the deposition and lose them from the wound bed, resulting in cell loss and uneven coverage. Here, we developed a novel air-assisted atomization device (AAAD). Under proper atomization parameters, 1% (w/v) sodium alginate (SA) solution carrier could be sprayed uniformly. Compared with normal saline, the run-off of the SA on the surface of porcine skin was greatly reduced. In theory, the spray height of AAAD could be set to achieve the adjustment of a large spray area of 1-12 cm2. In the measurement of droplet velocity and HaCaT cell viability, the spray height of AAAD would affect the droplet settling velocity and then the cell delivery survival rate (CSR). Compared with the spray height of 50 mm, the CSR of 100 mm was significantly higher and could reach 91.09% ± 1.82% (92.82% ± 2.15% in control). For bio-ink prepared with 1% (w/v) SA, the viability remained the same during the 72-h incubation. Overall, the novel AAAD uniformly atomized bio-ink with high viscosity and maintained the viability and proliferation rate during the delivery of living cells. Therefore, AAAD has great potential in cell transplantation therapy, especially for large-area or irregular skin wounds.

Host-guest interactions between oleic acid and ß-cyclodextrin: A combined experimental and theoretical study.

The physicochemical properties and interactions of the complex of oleic acid (OA) and ß-cyclodextrin (ß-CD) were studied by experimental and computational methods. Differential scanning calorimetry and X-ray diffraction confirmed the successful preparation of OA/ß-CD inclusion complex. The stability of the complex was improved, exhibiting a 61.2 °C higher degradation temperature and a lower peroxide value than OA. Raman and Fourier transform infrared spectroscopy studies revealed that the carboxyl group of OA entered into the ß-CD cavity to form hydrogen bonds, which was confirmed by conformational search and weak interactions analysis. Dispersion energy from van der Waals (-290.79 kJ/mol) contributed 87.3% to total interaction energy (-253.88 kJ/mol). Topological analysis showed that four moderate hydrogen bonds were formed between OA and ß-CD with the bond energy ranging from -76.05 to -30.25 kJ/mol. This work provided theoretical basis for the development of nutritional supplements containing unsaturated fatty acids encapsulated by ß-CD.

Performance of simultaneous nitrification-denitrification and denitrifying phosphorus and manganese removal by driving a single-stage moving bed biofilm reactor based on manganese redox cycling.

Simultaneous removal of NH4+-N, NO3--N, COD, and P by manganese redox cycling in nutrient wastewater was established with a single-stage moving bed biofilm reactor (MBBR) under low C/N ratio. When sodium succinate replaced the conventional denitrifying carbon source, removal efficiencies of TN, NO3--N, NH4+-N, TP, and Mn2+ were 65.13 %, 79.63 %, 92.79 %, 51.57 %, and 68.10 %, respectively. Based on modified Stover-Kincannon model, 11.03 and 10.05 mg TN·L-1·h-1 of Umax values were obtained with sodium acetate and sodium succinate as substrates. Extracellular polymeric substances were used to evaluate the characteristics of biofilm, and microbial community of biofilm was identified. Transformation processes of NO3--N, NH4+-N, Mn2+, and P were investigated, suggesting that the main functional groups (e.g., CO, Mn-O, and CN bonds) participated in N, P, and Mn2+ removal, and MnO2 was the main component of biogenic manganese oxides. This study provides a new strategy for nutrients removal by Mn2+ driven MBBR.

Performance and mechanism of simultaneous nitrification-denitrification and denitrifying phosphorus removal in long-term moving bed biofilm reactor (MBBR).

The long-term moving bed biofilm reactor (MBBR) with carrier-attached biofilm was successfully operated for simultaneous removal of nitrogen, phosphorus, and COD at various C/N ratios. Results indicated that 99.60%, 63.58%, 78.94%, and 59.64% of NH4+-N, NO3--N, TN, and TP were removed at C/N ratio, hydraulic retention time (HRT), and carrier film amount of 5, 40 h, and 1.2 mg·g-1. Nitrogen balance analysis showed that more than 89% of nitrogen (C/N = 20, 15, 10, 5) was converted to gas products. Extracellular polymeric substances (EPS), electron transport system activity (ETSA), and enzyme activity of biofilm were evaluated. Protein (PN)/polysaccharose (PS) values and ETSA decreased with the decrease of C/N ratios. Metagenomics sequencing further revealed that the prominent phyla for nitrogen and phosphorus removal were identified including Proteobacteria, Acidobacteria, Nitrospirae, and Chloroflexi. Proteobacteriaand Gammaproteobacteria were identified as the dominant denitrifying phosphate accumulating organisms (PAO) at the phylum and class level, respectively.