Background: Many enzymes of industrial interest are not in the market since they are bio-produced as bacterial inclusion bodies, believed to be biologically inert aggregates of insoluble protein. Results: By using two structurally and functionally different model enzymes and two fluorescent proteins we show that physiological aggregation in bacteria might only result in a moderate loss of biological activity and that inclusion bodies can be used in reaction mixtures for efficient catalysis. Conclusion: This observation offers promising possibilities for the exploration of inclusion bodies as catalysts for industrial purposes, without any previous protein-refolding step.

Fifteen Holstein male calves were randomly assigned to 1 of 3 dietary treatments according to age and body weight (BW) to determine the effects of feeding different forages sources on rumen fermentation and gastrointestinal tract (GIT) development. Treatments consisted of a starter (20% crude protein, 21% neutral detergent fiber) fed alone (CON) or supplemented with alfalfa (AH) or with oat hay (OH). All calves received 2L of milk replacer (MR) at 12.5% dry matter twice daily until 49 d of age. Calves received 2L of the same MR from 50 to 56 d of age and were weaned at 57 d of age. Individual starter, forage, and MR intakes were recorded daily and BW was recorded weekly. A rumen sample was taken weekly to determine rumen pH and volatile fatty acid concentrations. Three weeks after weaning, animals were harvested and each anatomical part of the GIT was separated and weighed with and without contents. Rumen pH was lower in CON than in OH and AH calves. Furthermore, acetate proportion in the rumen liquid tended to be greater in AH than in CON and OH treatments. Total GIT weight, expressed as a percentage of BW, tended to be greater in AH compared with the other 2 treatments. Rumen tissue tended to weigh more in CON than in OH animals. Animals with access to forage tended to have a greater expression of monocarboxylate transporter 1 than CON calves. In conclusion, calves supplemented with oat hay have a better rumen environment than calves offered no forage and do not have an increased gut fill.

Dairy cows suffer blood Ca losses as lactation begins and might be affected by hypocalcemia in its clinical (total serum Ca concentration <1.50 mM) or subclinical form (total serum Ca concentration ≤2.14 mM). Several studies have suggested that hypocalcemia is associated with different health problems of the cow but results from different studies are not consistent. The objective of this study was to assess potential associations between subclinical hypocalcemia (SCHC) and displaced abomasum, intramammary infections, metritis, retained placenta, and ketosis. Also, the associations between SCHC and milk yield and reproductive function were evaluated. After discarding cows (32) with clinical hypocalcemia, a total of 764 cows from 6 different commercial farms were enrolled in this study. Blood samples were collected at 24 to 48 h postcalving and analyzed for total Ca concentration. Odds ratios of the different afflictions potentially associated with SCHC were calculated. Seventy-eight percent of the analyzed cows incurred SCHC. The occurrence of displaced abomasum, ketosis, retained placenta, and metritis was 3.7, 5.5, 3.4, and 4.3 times more likely, respectively, in cows that had SCHC than in cows with normocalcemia. Furthermore, the risk of incurring retained placenta or metritis increased in multiparous cows as serum Ca concentrations decreased compared with that in primiparous cows. Normocalcemic cows, independent of parity, were more likely to show their first estrus sooner after calving than SCHC cows, but no correlation was found between SCHC and other reproductive parameters. Different serum Ca concentration cutoffs were identified for several postpartum afflictions (≤1.93, ≤2.05, ≤2.05, and ≤2.10 mM for ketosis, retained placenta, metritis, and displaced abomasum, respectively). In conclusion, SCHC, defined as serum Ca ≤2.14 mM, is a frequent illness affecting the majority of the dairy cows with important repercussions on health. However, if SCHC were to be used to predict postpartum disease, different serum Ca cutoff points are likely to be needed because best predictive cutoff values varied among postpartum ketosis, displaced abomasum, retained placenta, and metritis.

To study the effects of an extract of plant flavonoids [Bioflavex (FL)] in cattle fed high-concentrate diets, 2 experiments were designed. In the first experiment, the effects of Bioflavex on the development of rumen acidosis was evaluated in 8 Holstein-Friesian crossbreed heifers (451 kg; SEM 14.3 kg of BW) using a crossover design. Each experimental period lasted 22 d; from d 1 to 20, the animals were fed rye grass, on d 21 the animals were fasted, and on d 22, rumen acidosis was induced by applying 5 kg of wheat without [Control: (CTR) heifers who did not receive Bioflavex] or with flavonoids [heifers who received FL; 300 mg/kg DM] through a rumen cannula. Rumen pH was recorded continuously (from d 19 to d 22). On d 22, average rumen pH was significantly (P < 0.01) higher in the FL animals (6.29; SEM = 0.031) than it was in the CTR heifers (5.98; SEM = 0.029). After the wheat application, the rumen VFA concentration increased (P < 0.01), the proportion of acetic acid decreased (P < 0.01), and lactate concentration (mmol/L) increased, but the increase was not as great (P = 0.09) in the FL as it was in the CTR heifers (0.41 to 1.35 mmol/L; SEM = 0.24). On d 22, Streptococcus bovis and Selenomonas ruminantium titers increased after the wheat application, but Megasphaera elsdenii titers increased (P < 0.05) only in the FL heifers. In the second experiment, the effect of Bioflavex on the performance and rumen fermentation in finishing heifers was evaluated. Forty-eight Fleckvieh heifers (initial BW = 317 kg; SEM = 5.34) were used in a completely randomized design. Heifers were assigned to 1 of 4 blocks based on their BW and, within each block, assigned to 1 of 2 pens (6 heifers/pen). In addition, 16 heifers (2/pen) were rumen cannulated. Individual BW and group consumption of concentrate and straw were recorded weekly until the animals reached the target slaughter weight. Supplementation with FL did not affect ADG, feed consumption, or feed conversion ratio. Rumen pH and molar proportions of propionate were greater (P < 0.01) and acetate proportion was less in the FL (P < 0.01) than they were in the CTR heifers. Flavonoid supplementation might be effective in improving rumen fermentation and reducing the incidence of rumen acidosis. This effect of flavonoids may be partially explained by increasing the numbers of lactate-consuming microorganisms (e.g., M. elsdenii) in the rumen.

Bacterial inclusion bodies, while showing intriguing amyloid-like features, such as a β-sheet-based intermolecular organization, binding to amyloid-tropic dyes, and origin in a sequence-selective deposition process, hold an important amount of native-like secondary structure and significant amounts of functional polypeptides. The aggregation mechanics supporting the occurrence of both misfolded and properly folded protein is controversial. Single polypeptide chains might contain both misfolded stretches driving aggregation and properly folded protein domains that, if embracing the active site, would account for the biological activities displayed by inclusion bodies. Alternatively, soluble, functional polypeptides could be surface adsorbed by interactions weaker than those driving the formation of the intermolecular β-sheet architecture. To explore whether the fraction of properly folded active protein is a natural component or rather a mere contaminant of these aggregates, we have explored their localization by image analysis of inclusion bodies formed by green fluorescent protein. Since the fluorescence distribution is not homogeneous and the core of inclusion bodies is particularly rich in active protein forms, such protein species cannot be passively trapped components and their occurrence might be linked to the reconstruction dynamics steadily endured in vivo by such bacterial aggregates. Intriguingly, even functional protein species in inclusion bodies are not excluded from the interface with the solvent, probably because of the porous structure of these particular protein aggregates.