Berberine is used in the treatment of metabolic syndrome and its low solubility and very poor oral bioavailability of berberine was one of the primary hurdles for its market approval. This study aimed to improve the solubility and bioavailability of berberine by preparing pellet formulations containing drug-excipient complex (obtained by solid dispersion). Berberine-excipient solid dispersion complexes were obtained with different ratios by the solvent evaporation method. The maximum saturation solubility test was performed as a key factor for choosing the optimal complex for the drug-excipient. The properties of these complexes were investigated by FTIR, DSC, XRD and dissolution tests. The obtained pellets were evaluated and compared in terms of pelletization efficiency, particle size, mechanical strength, sphericity and drug release profile in simulated media of gastric and intestine. Solid-state analysis showed complex formation between the drug and excipients used in solid dispersion. The optimal berberine-phospholipid complex showed a 2-fold increase and the optimal berberine-gelucire and berberine-citric acid complexes showed more than a 3-fold increase in the solubility of berberine compared to pure berberine powder. The evaluation of pellets from each of the optimal complexes showed that the rate and amount of drug released from all pellet formulations in the simulated gastric medium were significantly lower than in the intestine medium. The results of this study showed that the use of berberine-citric acid or berberine-gelucire complex could be considered a promising technique to increase the saturation solubility and improve the release characteristics of berberine from the pellet formulation.

Coordination compounds, like iron(II) triazole complexes, exhibit spin crossover (SCO) behavior at around room temperature. Therefore, they are interesting for a variety of possible applications, and it is convenient to integrate them into polymers. Due to a reduction of the iron content and thus also 57Fe content in the sample through integration in polymers, Mössbauer measurements are only possible with greater difficulty or very long measurement times without expensive enrichment of the samples with 57Fe. So, other ways of improving the Mössbauer signal for these composite materials are necessary. Therefore, we pressed these composite materials to improve the Mössbauer spectra. In this study, we synthesized an iron(II) triazole spin crossover complex and an electrospun polymer complex composite nanofiber material including the same complex. For both products, Mössbauer measurements were performed at room temperature before and after using a press to show that the complex composite is not harmed through pressing. We investigate the influence of the pressing impact on the Mössbauer measurements in the context of measurement statistics and the measured signals. We show that pressing is not connected to any changes in the sample regarding the spin and oxidation state. We present that pressing improves the statistics of the Mössbauer measurements significantly. Furthermore, we use SEM measurements and PXRD to investigate whether or not the obtained fiber mats are destroyed in the pressing process.

BACKGROUND: Members of the fungal kingdom are heterotrophic eukaryotes encased in a chitin containing cell wall. This polymer is vital for cell wall stiffness and, ultimately, cell shape. Most fungal genomes contain numerous putative chitin synthase encoding genes. However, systematic functional analysis of the full chitin synthase catalogue in a given species is rare. This greatly limits fundamental understanding and potential applications of manipulating chitin synthesis across the fungal kingdom.
RESULTS: In this study, we conducted in silico profiling and subsequently deleted all predicted chitin synthase encoding genes in the multipurpose cell factory Aspergillus niger. Phylogenetic analysis suggested nine chitin synthases evolved as three distinct groups. Transcript profiling and co-expression network construction revealed remarkably independent expression, strongly supporting specific role(s) for the respective chitin synthases. Deletion mutants confirmed all genes were dispensable for germination, yet impacted colony spore titres, chitin content at hyphal septa, and internal architecture of submerged fungal pellets. We were also able to assign specific roles to individual chitin synthases, including those impacting colony radial growth rates (ChsE, ChsF), lateral cell wall chitin content (CsmA), chemical genetic interactions with a secreted antifungal protein (CsmA, CsmB, ChsE, ChsF), resistance to therapeutics (ChsE), and those that modulated pellet diameter in liquid culture (ChsA, ChsB). From an applied perspective, we show chsF deletion increases total protein in culture supernatant over threefold compared to the control strain, indicating engineering filamentous fungal chitin content is a high priority yet underexplored strategy for strain optimization.
CONCLUSIONS: This study has conducted extensive analysis for the full chitin synthase encoding gene repertoire of A. niger. For the first time we reveal both redundant and non-redundant functional roles of chitin synthases in this fungus. Our data shed light on the complex, multifaceted, and dynamic role of chitin in fungal growth, morphology, survival, and secretion, thus improving fundamental understanding and opening new avenues for biotechnological applications in fungi.

This study evaluated the effect of feeding ergot contaminated grain continuously or intermittently through backgrounding (BG) and finishing (FN) in a mash or pelleted supplement on the growth performance, health and welfare parameters, and carcass characteristics of feedlot beef steers. Sixty black Angus steers (300 ± 29.4 kg BW) were used in a complete randomized 238-d study. Steers were stratified by weight and randomly assigned to four different diets (15 steers/treatment) and individually housed. Treatments included: (1) control [CON; no added ergot alkaloids (EA)], (2) continuous ergot mash (CEM; fed continuously at 2 mg total EA/kg of DM), (3) intermittent ergot mash (IEM; fed at 2 mg total EA/kg of DM, during the first week of each 21-d period and CON for the remaining 2 wk, this feeding pattern was repeated in each period), and (4) intermittent ergot pellet (IEP; fed at 2 mg of total EA/kg of DM as a pellet during the first week of each 21-d period and CON for the remaining 2 wk as described for IEM). Steers were fed barley based BG diets containing 40% concentrate:60% silage (DM basis) for 84 d (four 21-d periods), transitioned over 28 d (no ergot fed) to an FN diet (90% concentrate:10% silage DM basis) and fed for 126 d (six 21-d periods) before slaughter. In the BG phase, steer DMI (P < 0.01, 7.45 vs. 8.05 kg/d) and ADG (P < 0.01) were reduced for all EA diets compared to CON. The CEM fed steers had lower ADG (P < 0.01, 0.735 vs. 0.980 kg) and shrunk final BW (P < 0.01, 350 vs. 366 kg) than CON. CEM had lower gain:feed (P < 0.07, 0.130 vs. 0.142) than CON. In the FN phase, steer DMI (P < 0.01, 9.95 vs. 11.05 kg/d) and ADG (P = 0.04) were also decreased for all EA fed steers compared to CON. Total shrunk BW gain (P = 0.03, 202.5 vs. 225.2 kg), final BW (P = 0.03, 617.9 vs. 662.2 kg), and carcass weight (P = 0.06) decreased for all EA fed steers compared to CON. The percentage of AAA carcasses decreased for all EA fed steers (P < 0.01, 46.7 vs. 93.3%) compared to CON. EA fed steers had increased rectal temperatures (P < 0.01, 39.8 vs. 39.4 °C) compared to CON. Pelleting ergot contaminated grain did not reduce the impact of ergot alkaloids on any of the measured parameters during BG or FN. Continuously or intermittently feeding ergot contaminated diets (2 mg total EA/kg of DM) significantly reduced intake, growth performance, and carcass weight, with minimal impact on blood parameters in feedlot steers. Pelleting was not an effective method of reducing ergot toxicity.
Produced by the fungus Claviceps purpurea, ergot alkaloids (EA) are toxic to beef cattle when consumed and can lead to reduction in feed intake and growth performance, vasoconstriction of the blood vessels, hyperthermia, damage to extremities (ears, tails, and hooves) and in severe cases, death. Grain is often cleaned to meet quality standards, and the resulting screenings are often utilized for feeding livestock and can have high concentrations of EA. The application of heat during pelleting of EA contaminated grain has been suggested to reduce its toxicity. Backgrounding and finishing beef cattle feeding experiments were conducted to assess the effect of continuously or intermittently feeding EA contaminated grain (2 mg/kg of diet DM) either as a pellet or as mash on growth performance, health, and animal welfare. Feeding EA grain continuously or intermittently either as a mash or pellet drastically reduced growth performance of steers, with no difference between treatments.

The objective of this study was to describe the nutritional strategies used on Canadian dairy farms with automated milking systems (AMS), both at the feed bunk and the concentrate offered at the AMS, as well as to determine what dietary components and nutrients, as formulated, were associated with milk production and milking behaviors on those farms. Formulated diets (including ingredients and nutrient content) and AMS data were collected from April 1, 2019, until September 30, 2020, on 160 AMS farms (eastern Canada [East] = 8, Ontario [ON] = 76, Quebec [QC] = 22, and western Canada [West] = 54). Both partial mixed ration (PMR) and AMS concentrate samples were collected from May 1 to September 30, 2019, on 169 farms (East = 12, ON = 63, QC = 42, West = 52). We collected AMS milking data for 154 herds. For each farm (n = 161), milk recording data were collected and summarized by farm to calculate average milk yield and components. Multivariable regression models were used to associate herd-level formulated nutrient composition and feeding management practices with milk production and milking behavior. Milk yield (mean ± SD = 37.0 ± 0.3 kg/d) was positively associated with the PMR ether extract (EE) concentration (+0.97 kg/d per percentage point [p.p.] increase) and with farms that fed barley silage as their major forage source (n = 16; +2.18 kg/d) as compared with haylage (n = 42), whereas farms that fed corn silage (n = 96; +1.23 kg/d) tended to produce more milk than farms that fed haylage. Greater milk fat content (4.09 ± 0.28%) was associated with a greater PMR-to-AMS concentrate ratio (+0.02 p.p. per unit increase) and total diet net energy for lactation (+0.046 p.p. per 0.1 Mcal/kg increase), but a lesser percentage of NFC of the PMR (-0.016 p.p. per p.p. increase of NFC percentage). Milk protein content (3.38 ± 0.14%) was positively associated with the forage percentage of the PMR (+0.003 p.p. per p.p. increase of forage percentage) and the total diet starch percentage (+0.009 p.p. per p.p. increase of starch percentage), but was negatively associated with farms feeding corn silage (-0.1 p.p. compared with haylage) as their major forage. Greater milking frequency (2.77 ± 0.40 milkings/d) was observed on farms with free-flow cow traffic systems (+0.62 milkings/d) and was positively associated with feed push-up frequency (+0.013 milkings/d per additional feed push-up), but negatively associated with PMR NFC content and forage percentage of the total ration (-0.017 milkings/d per p.p. increase of forage percentage). Lastly, greater milking refusal frequency (1.49 ± 0.82 refusals/d) was observed on farms with free-flow cow traffic systems (+0.84 refusals/d) and farms feeding barley silage (+0.58 refusals/d) than with guided flow and farms feeding either corn silage or haylage, respectively. These data give insight into the ingredients, nutrient formulations and type of diets fed on AMS dairy farms across Canada and the association of those factors with milk production and milking behaviors.

UNASSIGNED: Testosterone treatment is employed in transgender men to help them affirm their gender. Our objective is to report the cases of 2 transgender men who received subdermal testosterone pellets as the mode of testosterone administration.
UNASSIGNED: Both patients presented for discussion of testosterone therapy. Patient 1 was a 47-year-old transgender male. He had bilateral mastectomy and total abdominal hysterectomy with bilateral salpingo-oophorectomy (TAH-BSO). Physical exam was significant for obesity. He was on injectable testosterone for 9 years and experienced voice deepening, facial hair growth, temporal hair thinning, and increased skin oiliness, but was interested in a long-acting testosterone formulation. Pellets were well tolerated, but the patient ultimately chose to return to injections. Patient 2 was a 20-year-old transgender male with no history of gender-affirming surgeries. Physical exam was pertinent for a thin, masculinized individual. He started on testosterone gel, but switched to weekly injections, on which he experienced voice deepening, increased skin oiliness and cessation of menses. Due to pain with injections and desire for a long-acting formulation, he elected to try pellets.
UNASSIGNED: The dose of testosterone pellets used in transgender men are similar to those employed for testosterone replacement in hypogonadal cisgender men.
UNASSIGNED: Subdermal testosterone pellets may be suitable as a means of delivering testosterone in transgender men, but the 2 cases reported here do not permit firm conclusions. Given the widespread use of testosterone for gender affirmation in transgender men, a prospective controlled study of subdermal testosterone pellets seems indicated.

Air pollution and the increasing production of greenhouse gases has prompted greater use of renewable energy sources; the EU has set a target that the use of green energy should be at 32 percent by 2030. With this in mind, in the last 10 years, the demand for pellets in Italy has more than doubled, making Italy the second largest consumer in Europe. The quality of the pellets burned in stoves is crucial to indoor and outdoor pollution. Among other parameters, moisture and ash are used to classify pellets according to EN ISO 17225:2014. This work involved the analysis of the organic and inorganic fraction of both some finished products on the Italian market and some raw materials (e.g., wood chips) sampled according to the technical standard EN 14778:2011. The analytical results showed the presence of some substances potentially harmful to human health such as formaldehyde, acetone, toluene and styrene for the organic fraction and nickel, lead and vanadium for the inorganic fraction. The chemometric approach showed that it is the inorganic fraction which is most responsible for the diversification of the samples under study. The detection of some substances may be a warning bell about the impact of such materials, both for the environment and for human health.

Few data are available evaluating the stability of direct-fed microbials (DFM) following their inclusion in different feed matrices. Therefore, six Exp. evaluated the recovery of bacilli spores (BOVACILLUSTM; Exp. 1 to 3) and an Enterococcus faecium DFM (LACTIFERM®; Exp. 4 to 6) when included in different feed preparations. The Bacillus-based DFM was included into pelleted feed prepared in different temperatures (75 to 95 °C), whereas both DFM were assessed in premix and milk replacer preparations. Bacillus spores and E. faecium recovery was evaluated through standard methodologies and data were reported as log10 colony forming units/gram of feed. The recovery of Bacillus spores was within the expected range and was not impacted by the temperature of pellet preparation (Exp. 1). Bacilli recovery was also stable up to 12 months in the premix and was not impacted by the temperature of milk replacer preparation. Regarding the Exp. with E. faecium (Exp. 4 to 6), its recoveries in the mineral premix and milk powder did not differ from T0 and were not impacted by the conditions of milk replacer preparation. These data are novel and demonstrate the stability of a Bacillus-based and an E. faecium-based DFM when included in different feed matrices often used in dairy production.

BACKGROUND: Filamentous fungi are used as industrial cell factories to produce a diverse portfolio of proteins, organic acids, and secondary metabolites in submerged fermentation. Generating optimized strains for maximum product titres relies on a complex interplay of molecular, cellular, morphological, and macromorphological factors that are not yet fully understood.
RESULTS: In this study, we generate six conditional expression mutants in the protein producing ascomycete Aspergillus niger and use them as tools to reverse engineer factors which impact total secreted protein during submerged growth. By harnessing gene coexpression network data, we bioinformatically predicted six morphology and productivity associated \'morphogenes\', and placed them under control of a conditional Tet-on gene switch using CRISPR-Cas genome editing. Strains were phenotypically screened on solid and liquid media following titration of morphogene expression, generating quantitative measurements of growth rate, filamentous morphology, response to various abiotic perturbations, Euclidean parameters of submerged macromorphologies, and total secreted protein. These data were built into a multiple linear regression model, which identified radial growth rate and fitness under heat stress as positively correlated with protein titres. In contrast, diameter of submerged pellets and cell wall integrity were negatively associated with productivity. Remarkably, our model predicts over 60% of variation in A. niger secreted protein titres is dependent on these four variables, suggesting that they play crucial roles in productivity and are high priority processes to be targeted in future engineering programs. Additionally, this study suggests A. niger dlpA and crzA genes are promising new leads for enhancing protein titres during fermentation.
CONCLUSIONS: Taken together this study has identified several potential genetic leads for maximizing protein titres, delivered a suite of chassis strains with user controllable macromorphologies during pilot fermentation studies, and has quantified four crucial factors which impact secreted protein titres in A. niger.

Delayed release of vaccine coupled with a soluble vaccine acts as a primer and a booster with only a single administration, which would be very beneficial to livestock producers. We developed a subdermal pellet consisting of solid-phase pure stearic acid (SA) or palmitic acid (PA) that was used to encapsulate a small volume liquid vaccine consisting of fluorescently labeled *Ovalbumin (Cy5-*OVA) formulated with Emulsigen-D +/- Poly I:C (EMP) adjuvants. Mice were also immunized via the subcutaneous route with Cy5-*OVA-EMP (soluble liquid). The vaccine leached out of the pellet with very little dissolution of the fat itself resulting in the sustained subdermal delivery of antigens and adjuvants. Cy5-*OVA was still visible 60 days post administration in mice immunized with stearic acid-coated or palmitic acid-coated pellets. In these mice, persistently high IgG1 and IgG2a antibody titres were detected as well as significant IFNγ production at least 60 days post-injection. These responses were significantly higher than those observed after a single subcutaneous injection of the vaccine. A repeat trial with the pellets alone +/- the soluble vaccine showed comparable immune responses after surgical implantation of the pellet, suggesting that pellet alone may be sufficient. The PA-coated vaccines led to dermal inflammation in the mice that would limit usefulness of this vehicle, but this was largely absent when SA was used to coat the pellets. These data suggest that the SA-coated adjuvanted vaccine prolonged the release of the vaccine and triggered a comparable immune response to the mice that received the two liquid injections, and a single pellet vaccine should be tested as a novel immunization method for livestock.