Effect of α-Lipoic Acid on the Development of Human Skin Equivalents Using a Pumpless Skin-on-a-Chip Model

Owing to the prohibition of cosmetic animal testing, various attempts have recently been made using skin-on-a-chip (SOC) technology as a replacement for animal testing. Previously, we reported the development of a pumpless SOC capable of drug testing with a simple drive using the principle that the medium flows along the channel by gravity when the chip is tilted using a microfluidic channel. In this study, using pumpless SOC, instead of drug testing at the single-cell level, we evaluated the efficacy of α-lipoic acid (ALA), which is known as an anti-aging substance in skin equivalents, for skin tissue and epidermal structure formation. The expression of proteins and changes in genotyping were compared and evaluated. Hematoxylin and eosin staining for histological analysis showed a difference in the activity of fibroblasts in the dermis layer with respect to the presence or absence of ALA. We observed that the epidermis layer became increasingly prominent as the culture period was extended by treatment with 10 μM ALA. The expression of epidermal structural proteins of filaggrin, involucrin, keratin 10, and collagen IV increased because of the effect of ALA. Changes in the epidermis layer were noticeable after the ALA treatment. As a result of aging, damage to the skin-barrier function and structural integrity is reduced, indicating that ALA has an anti-aging effect. We performed a gene analysis of filaggrin, involucrin, keratin 10, integrin, and collagen I genes in ALA-treated human skin equivalents, which indicated an increase in filaggrin gene expression after ALA treatment. These results indicate that pumpless SOC can be used as an in vitro skin model similar to human skin, protein and gene expression can be analyzed, and it can be used for functional drug tests of cosmetic materials in the future. This technology is expected to contribute to the development of skin disease models.     

Farmers' preferences for adopting on-farm concentration of raw milk: Results from a discrete choice experiment in Germany

Milk concentrates are used in the manufacturing of dairy products such as yogurt and cheese or are processed into milk powder. Processes for the nonthermal separation of water and valuable milk ingredients are becoming increasingly widespread at farm level. The technical barriers to using farm-manufactured milk concentrate in dairies are minimal, hence the suspicion that the practice of on-farm raw milk concentration is still fairly uncommon for economic reasons. This study, therefore, set out to investigate farmers' potential willingness to adopt a raw milk concentration plant. The empirical analysis was based on discrete choice experiments with 75 German dairy farmers to identify preferences and the possible adoption of on-farm raw milk concentration. The results showed that, in particular, farmers who deemed the current milk price to be insufficient viewed on-farm concentration using membrane technology as an option for diversifying their milk sales. We found no indication that adoption would be impeded by a lack of trustworthy information on milk processing technologies or capital.     

Combined biotin,folic acid,and vitamin B12 supplementation given during the transition period to dairy cows: Part II. Effects on energy balance and fatty acid composition of colostrum and milk

Biotin (B₈), folate (B₉), and vitamin B₁₂ (B₁₂) are involved in several metabolic reactions related to energy metabolism. We hypothesized that a low supply of one of these vitamins during the transition period would impair metabolic status. This study was undertaken to assess the interaction between B₈ supplement and a supplementation of B₉ and B₁₂ regarding body weight (BW) change, dry matter intake, energy balance, and fatty acid (FA) compositions of colostrum and milk fat from d ?21 to 21 relative to calving. Thirty-two multiparous Holstein cows housed in tie stalls were randomly assigned, according to their previous 305-d milk yield, to 8 incomplete blocks in 4 treatments: (1) a 2-mL weekly i.m. injection of saline (0.9% NaCl; B₈?/B₉B₁₂?); (2) 20 mg/d of dietary B₈ (unprotected from ruminal degradation) and 2-mL weekly i.m. injection of 0.9% NaCl (B₈+/B₉B₁₂?); (3) 2.6 g/d of dietary B₉ (unprotected) and 2-mL weekly i.m. injection of 10 mg of B₁₂ (B₈?/B₉B₁₂+); (4) 20 mg/d of dietary B₈, 2.6 g/d of dietary B₉, and 2-mL weekly i.m. injection of 10 mg of B₁₂ (B₈+/B₉B₁₂+) in a 2 × 2 factorial arrangement. Colostrum was sampled at first milking. and milk samples were collected weekly on 2 consecutive milkings and analyzed for FA composition. Body condition score and BW were recorded every week throughout the trial. Within the first 21 d of lactation, B₈?/B₉B₁₂+ cows had an increased milk yield by 13.5% [45.5 (standard error, SE: 1.8) kg/d] compared with B₈?/B₉B₁₂? cows [40.1 (SE: 1.9)], whereas B₈ supplement had no effect. Even though body condition score was not affected by treatment, B₈?/B₉B₁₂+ cows had greater BW loss by 24 kg, suggesting higher mobilization of body reserves. Accordingly, milk de novo FA decreased and preformed FA concentration increased in B₈?/B₉B₁₂+ cows compared with B₈?/B₉B₁₂? cows. In addition, cows in the B₈+/B₉B₁₂? group had decreased milk de novo FA and increased preformed FA concentration compared with B₈?/B₉B₁₂? cows. Treatment had no effect on colostrum preformed FA concentration. Supplemental B₈ decreased concentrations of ruminal biohydrogenation intermediates and odd- and branched-chain FA in colostrum and milk fat. Moreover, postpartum dry matter intake for B₈+ cows tended to be lower by 1.6 kg/d. These results could indicate ruminal perturbation caused by the B₈ supplement, which was not protected from rumen degradation. Under the conditions of the current study, in contrast to B₈+/B₉B₁₂? cows, B₈?/B₉B₁₂+ cows produced more milk without increasing dry matter intake, although these cows had greater body fat mobilization in early lactation as suggested by the FA profile and BW loss.     

Lactobacillus reuteri-fortified camel milk infant formula: Effects of encapsulation,in vitro digestion,and storage conditions on probiotic cell viability and physicochemical characteristics of infant formula

Lactobacillus reuteri fortified camel milk infant formula (CMIF) was produced. The effect of encapsulation in different matrices (sodium alginate and galacto-oligosaccharides) via spray drying, simulated infant gastrointestinal digestion (SIGID), and storage conditions (temperature and humidity) on the viability of L. reuteri in CMIF and the physicochemical properties of CMIF were evaluated. Compared with free cells, probiotic cell viability was significantly enhanced against SIGID conditions upon encapsulation. However, L. reuteri viability in CMIF decreased after 60 d of storage, predominantly at higher storage humidity and temperature levels. At the end of the storage period, significant changes in the color values were observed in all CMIF, with a reduction in their greenness, an increase in yellowness, and a wide variation in their whiteness. Moreover, pH values and caking behavior of all CMIF stored at higher temperature (40°C) and humidity [water activity (a_w) = 0.52] levels were found to be significantly higher than the samples stored under other conditions. Over 30 d of storage at lower humidity conditions (a_w = 0.11 and 0.33) and room temperature (25°C), no significant increase in CMIF lipid oxidation rates was noted. Fourier-transform infrared spectroscopy analysis showed that, compared with the other storage conditions, CMIF experienced fewer changes in functional groups when stored at a_w = 0.11. Microscopic images showed typical morphological characteristics of milk powder, with round to spherical-shaped particles. Overall, camel milk fortified with encapsulated L. reuteri can be suggested as a promising alternative in infant formula industries, potentially able to maintain its physicochemical characteristics as well as viability of probiotic cells when stored at low humidity levels (a_w = 0.11) and temperature (25°C), over 60 d of storage.     

Macromineral and trace element concentrations in milk from Finnish Ayrshire cows fed microalgae (Spirulina platensis) and rapeseed (Brassica napus)

Given the lack of research regarding the effect of microalgal supplementation in dairy cows on milk mineral concentrations, this study investigated the effect of feeding different protein supplements in dairy cow diets on milk, feces, and blood plasma mineral concentrations, associated milk and blood plasma transfer efficiencies, and apparent digestibility. Lactating Finnish Ayrshire cows (n = 8) were allocated at the start of the trial to 4 diets used in a replicated 4 × 4 Latin square design experiment: (1) control diet (CON), (2) a pelleted rapeseed supplement (RSS; 2,550 g/d), (3) a mixture of rapeseed and Spirulina platensis (RSAL; 1,280 g of RSS + 570 g of S. platensis per day), and (4) S. platensis (ALG; 1,130 g of S. platensis per day). In each of the 4 experimental periods, a 2-wk adaptation to the experimental diets was followed by a 7-d sampling and measurement period. Feed samples were composited per measurement period, milk, and feed samples (4 consecutive days; d 17–20), and blood plasma samples (d 21) were composited for each cow period (n = 32). Data were statistically analyzed using a linear mixed effects model with diet, period within square, square and their interaction as fixed factors, and cow within square as a random factor. Cows fed ALG were not significantly different in their milk or blood plasma mineral concentrations compared with CON, although feeding ALG increased fecal concentrations of macrominerals (Ca and Mg) and trace elements (Co, Cu, Fe, I, Mn, and Zn), and reduced their apparent digestibility, compared with CON. When compared with CON and ALG, milk from cows fed RSAL and RSS had lower milk I concentrations (?69.6 and ?102.7 μg/kg of milk, respectively), but total plasma I concentrations were not affected significantly. Feeding S. platensis to dairy cows did not affect mineral concentrations in cows' blood or milk, but care should be taken when rapeseed is fed to avoid reducing milk I concentrations which may in turn reduce consumers' I intake from milk and dairy products.     

Shared human–robot path following control of an unmanned ground vehicle

This paper considers the shared path following control of an unmanned ground vehicle by a single person. A passive measure of human intent is used to blend the human and machine inputs in a mixed initiative approach. The blending law is combined with saturated super-twisting sliding mode speed and heading controllers, so that exogenous disturbances can be counteracted via equivalent control. It is proven that when the proposed blending law is used, the combined control signals from both the human and automatic controller respect the actuator magnitude constraints of the machine. To demonstrate the approach, shared control experiments are performed using an unmanned ground vehicle, which follows a lawn mower pattern shaped path.     

Transglycosylation Activity of Engineered Bifidobacterium Lacto-N-Biosidase Mutants at Donor Subsites for Lacto-N-Tetraose Synthesis

The health benefits of human milk oligosaccharides (HMOs) make them attractive targets as supplements for infant formula milks. However, HMO synthesis is still challenging and only two HMOs have been marketed. Engineering glycoside hydrolases into transglycosylases may provide biocatalytic routes to the synthesis of complex oligosaccharides. Lacto-N-biosidase from Bifidobacterium bifidum (LnbB) is a GH20 enzyme present in the gut microbiota of breast-fed infants that hydrolyzes lacto-N-tetraose (LNT), the core structure of the most abundant type I HMOs. Here we report a mutational study in the donor subsites of the substrate binding cleft with the aim of reducing hydrolytic activity and conferring transglycosylation activity for the synthesis of LNT from p-nitrophenyl β-lacto-N-bioside and lactose. As compared with the wt enzyme with negligible transglycosylation activity, mutants with residual hydrolase activity within 0.05% to 1.6% of the wild-type enzyme result in transglycosylating enzymes with LNT yields in the range of 10–30%. Mutations of Trp394, located in subsite -1 next to the catalytic residues, have a large impact on the transglycosylation/hydrolysis ratio, with W394F being the best mutant as a biocatalyst producing LNT at 32% yield. It is the first reported transglycosylating LnbB enzyme variant, amenable to further engineering for practical enzymatic synthesis of LNT.     

Effects of feed composition,protein denaturation and storage of milk serum protein/lactose powders on lactosylation

During whey powder production, the feed is subjected to several heat treatments which can cause lactosylation of proteins. In this study, lactosylation of whey proteins was evaluated in spray-dried powders before and after storage by varying the native protein fraction as well as the serum protein/lactose ratio in the powders. The lactosylation of native α-lactalbumin and β-lactoglobulin in the powders before storage was not affected to a large extent by the protein denaturation or if the feed had been heat treated in a high or low lactose environment. After storage (relative humidity of 23.5%, 30 °C, 25 days), the kinetic of lactosylation tended to increase with increasing native protein fraction and bulk protein content in the powders. An explanation could be that proteins dissolved in the lactose glassy structure might have a lower reactivity, while proteins present in the protein glassy structure with dissolved lactose may display higher lactosylation reactivity.     

The peptides in oat and malt extracts that are preferentially absorbed by Lactobacillus plantarum and stimulates its proliferation in milk

Lactobacillus plantarum proliferates inefficiently in milk, mainly because of its lack of cell envelope proteases and its inability to hydrolyse proteins in milk. Our previous study showed that this strain could grow well in milk with the addition of oat and malt extracts. To investigate the usage and preference for polypeptides and oligopeptides for this strain, sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), o-phthaldialdehyde (OPA), high-performance liquid chromatography (HPLC), plate counting and other methods were used in this study. The results showed that proteins in fermented milk cannot be absorbed and utilised by L. plantarum, whereas polypeptides and oligopeptides provide available nitrogen sources for their growth. Short-chain peptides were more conducive to absorption and utilisation than long-chain peptides. In particular, peptides with molecular weights in the range of 200–1400 Da in the oat extract and 100–700 Da in the malt extract were preferentially absorbed and utilised.