Determination of the optimal priming interval of rumen fluids used as inocula for the in vitro digestibility trials through radial enzyme diffusion method

Context: Determination of the neutral detergent fibre digestibility is one of the important parameters to consider when formulating diets. However, the in vitro determination shows low repeatability because of the source of rumen-fluid inoculum. Priming of the rumen fluid inocula, obtained through an oesophageal probe, has been proposed to overcome this issue. Aim: The objective of the study was to investigate the evolution of the microbial enzymatic activities of different rumen fluids during a priming procedure, to establish the fermentation interval that minimises the differences among rumen-fluid degradative potentials. Methods: Three farms for each type of diet were involved in the study. Rumen fluids were obtained from dry and lactating cows fed the following four diet types: 100% hay or a diet with 80: 20 forage: Concentrate ratio (F: C) as dry-cow diets, and ad libitum hay and concentrate, or a total mixed ration (both at 60: 40 F: C) as lactating-cow diets. On each farm, rumen fluid was collected from three Holstein cows by using an oesophageal probe, and mixed. Two aliquots of each rumen fluid mix were added to the medium containing the same priming substrate in an in vitro batch-fermentation system. During the incubation, the fermentation fluids were sampled in duplicate at 0-, 1-, 2-, 4-, 8-, 24- A nd 48-h intervals. Enzymatic activities of amylase, cellulase and xylanase were determined by radial enzyme diffusion method. Key results: Initial enzymatic activities were quite variable and increased with an increasing incubation time. By 24 h, amylase showed similar values among high-concentrate diet fermentation fluids, and a lower data dispersion in comparison to the other intervals cellulase was characterised by similar values in all the fermentation fluids derived from diets including concentrates, and xylanase showed similar activity in the fermentation fluids derived from high-concentrate diets. Development of the enzymatic activity of the fermentation fluids derived from the 100% hay diet differed from the others. Conclusions: A 24-h priming procedure was needed to stabilise and equalise the enzymatic activity of the rumen fluid from cows fed high-concentrate diets. This was not observed in rumen fluid from cows fed hay-based diets. Implications: The 24-h-primed rumen fluid can be used to increase the repeatability of neutral detergent fibre digestibility determination

A short-term comparison of wheat straw and poplar wood chips used as litter in tiestalls on hygiene, milk, and behavior of lactating dairy cows

A short-term study was conducted to compare the effect of using poplar wood chips (PWC) instead of wheat straw (WS) litter in dairy cows. A total of 38 lactating Holstein cows (204 ± 119 days in milk, 26.9 ± 6.5 kg of milk yield [MY]) were housed in a tiestall farm for a 10-d trial including 5 d of adaptation followed by 5 sampling days (from d 5 to 10). Cows were divided into 2 homogeneous groups: one group was bedded with WS, and the second with PWC. Both litter materials were provided in the amount of 7 kg/stall per d. Each group was composed of 3 subgroups of 6 or 7 cows; the subgroups were physically separated along the feeding line by wooden boards. During the sampling days, fecal composition, used litter composition, and bacterial count (Clostridium spp., Salmonella spp., Escherichia coli, Lactobacillus, and total bacterial count) were analyzed by subgroup twice a day. On d 1 and from d 5 to 10, udder hygiene score and cow cleanliness score were also evaluated individually twice a day. Meanwhile MY, milk hygiene (total bacterial count [TBC], coliform bacterial count [CBC], and spore-forming unit [SFU]) and quality were measured and analyzed from 9 animals per group. Moreover, individual animal behavior (body position and behavioral traits) and subgroup dry matter intake were measured on d 9 and 10. Fecal dry matter did not differ between groups, PWC had the lowest used litter moisture and N content favoring the highest clean cow frequency, but also gave rise to the greatest used litter microbial contamination. The MY, milk quality, TBC, SFU, and CBC were similar. The lying behavior frequency was similar between groups. However, the PWC group showed the lowest sleeping frequency, the highest frequency of other behaviors (including discomfort signs), and the lowest dry matter intake. However, despite this apparent reduction in cow comfort, no biologically important differences were observed in this short-term study between cows on PWC and WS in milk production or hygiene

Increased risk of bone fractures in hemodialysis patients treated with proton pump inhibitors in real world: results from the Dialysis Outcomes and Practice Patterns Study (DOPPS)

Long-term treatment with Proton Pump Inhibitors (PPIs) is associated with an increased risk of fractures in the general population. PPIs are widely prescribed to dialysis patients but to date no study specifically tested, by state-of-art statistical methods, the relationship between PPIs use and fractures in this patient-population. This study aimed to assess whether PPIs use is associated with bone fractures (i.e. hip fractures and fractures other than hip fractures) in a large international cohort of hemodialysis patients. We considered an observational prospective cohort of 27097 hemodialysis patients from the DOPPS study. Data analysis was performed by the Fine & Gray method, considering the competitive risk of mortality, as well as by a cause-specific hazards Cox model dealing death as a censoring event and matching patients according to the prescription time. Out of 27,097 hemodialysis patients, 13,283 patients (49%) were on PPI treatment. Across the follow-up (median:19\u2009months), 3.8 bone fractures x 100 person-years and 1.2 hip fractures x 100 person-years occurred. In multiple Cox models, considering the competitive risk of mortality, the incidence rate of bone (SHR: 1.22, 95% CI: 1.10-1.36, P\u2009<\u20090.001) and hip fractures (SHR: 1.35, 95% CI: 1.13-1.62, P = 0.001) was significantly higher in PPI treated than in PPI untreated patients. These findings held true also in multiple, cause-specific, hazards Cox models matching patients according to the prescription time (bone fractures, HR: 1.47, 95% CI: 1.23-1.76, P\u2009<\u20090.001, hip fractures (HR: 1.85, 95% CI: 1.37-2.50, P\u2009<\u20090.001). The use of PPIs requires caution and a careful evaluation of risks/benefits ratio in hemodialysis patients

Use of hardwood mulch applications to improve soil characteristics of Alfisols used in Florida citrus production

IntroductionImproving soil fertility is a top priority in Florida’s citrus growing regions, especially in the age of Huanglongbing (HLB; also known as citrus greening). This disease severely reduces fine root mass, causes higher incidences of nutrient deficiencies, and eventually results in the death of affected trees. Additionally, the soils commonly found in Florida’s citrus growing regions are sandy (greater than 98%) and naturally low in fertility, making the nutrient management of HLB-affected trees even more challenging. As a result, interest in organic amendments to increase soil fertility are being tested. Although hardwood chip mulches are successfully used in other regions of the country, no studies exist observing their use on the soils in Florida’s citrus growing regions; therefore, the objectives of this study were to measure the impacts of hardwood oak mulch on (i) Florida Alfisols characteristics and (ii) HLB-affected citrus trees.MethodsA two-treatment field study using 6-year-old ‘Valencia’ sweet orange trees (Citrus × sinensis) grafted on US-812 (C. reticulata × C. trifoliata) rootstock was conducted in Florida’s Indian River District (IRD). The experimental treatment consisted of 0.08 m of hardwood chip mulch sourced from oak trees applied every September for 3 years (2020, 2021, and 2022) while the control treatment had no mulch applied. Soil chemical and physical properties, leaf nutrient concentration, and leaf Candidatus Liberibacter asiaticus (CLas) titer was collected in the fall (October), winter (January), spring (April), and summer (July).Results and discussionOverall, after 3 years, oak mulch applications increased soil available phosphorus (32%), potassium (66%), magnesium (71%), organic matter (49%), and moisture (25-88%, depending on the season); however, oak mulch inconsistently impacted leaf nutrient concentrations and was not effective at suppressing HLB. The results show that annual applications of hardwood oak mulch can improve the chemical and physical properties of sandy soils within three years, however, these improvements did not reduce the severity of HLB

Osteocalcin (bone GLA protein) levels, vascular calcifications, vertebral fractures and mortality in hemodialysis patients with diabetes mellitus

Background and aims: Diabetes mellitus is recognized as one of the major causes of end stage kidney disease. Bone Gla protein (BGP) is a vitamin K-dependent protein involved in bone mineralization and vascular calcifications (VC). Our goal was to characterize BGP and undercarboxylated BGP (ucBGP) in DM patients on HD, compared to HD patients without DM, and their association with vascular and bone disease. Methods: 387 HD patients from 18 dialysis centers in Italy. Associations of DM, levels of BGP, vitamin D and VC were evaluated. Time-to-event analysis for all-cause mortality was performed by the Kaplan\u2013Meier. Results: Patients with DM had lower levels of total BGP (139.00 vs. 202.50 mcg/L, p < 0.001), 25(OH)D (23.4 vs. 30.2 ng/ml, p < 0.001), and ucBGP (9.24 vs. 11.32 mcg/L, p = 0.022). In regression models, the geometric means of total BGP and ucBGP were 19% (p = 0.009) and 26% (p = 0.034) lower in diabetic patients. In univariate Cox regression analysis, DM patients had a higher risk of all-cause mortality (HR:1.83, 95% CI 1.13\u20132.96, p = 0.014). Adjustment for confounders confirmed the significant DM-mortality link. We included VC and warfarin into the Cox model, the DM-mortality link was no longer significant, suggesting a role of these risk factors as causal mediators leading to increased mortality in dialysis patients. Conclusions: HD patients have an increased mortality risk associated with DM. Furthermore, we found an association between DM and decreased BGP levels. Although our study does not support the notion that BGP levels act as mediator in the DM-mortality link, to our knowledge this is the first study in HD patients suggesting a potential protective role of BGP in the bone, endocrine and vascular pathway

E46K-like α-synuclein mutants increase lipid interactions and disrupt membrane selectivity

Parkinson's disease (PD) is one of the most common neurodegenerative disorders and both genetic and histopathological evidence have implicated the ubiquitous presynaptic protein α-synuclein (αSyn) in its pathogenesis. Recent work has investigated how disrupting αSyn's interaction with membranes triggers trafficking defects, cellular stress, and apoptosis. Special interest has been devoted to a series of mutants exacerbating the effects of the E46K mutation (associated with autosomal dominant PD) through homologous E-to-K substitutions in αSyn's N-terminal region (i.e. E35K, E61K). Such E46K-like mutants have been shown to cause dopaminergic neuron loss and severe, yet L-DOPA-responsive, motor defects in mouse overexpression models, presenting enormous translational potential for PD and other "synucleinopathies". In this work, using a variety of biophysical techniques, we characterize the molecular pathology of E46K-like αSyn mutants by studying their structure and membrane-binding and remodeling abilities. We find that, although a slight increase in the mutants' avidity for synaptic vesicle-like membranes can be detected, most of their deleterious effects are connected to their complete disruption of αSyn's curvature selectivity. Indiscriminate binding can shift αSyn's subcellular localization away from its physiological interactants at the synaptic bouton toward trafficking vesicles and organelles, as observed in E46K-like cellular and murine models, as well as in human pathology. In conclusion, our findings suggest that a loss of curvature selectivity, rather than increased membrane affinity, could be the critical dyshomeostasis in synucleinopathies

In vitro and in vivo modeling of lipid bioaccessibility and digestion from almond muffins: the importance of the cell-wall barrier mechanism

This study compares in vitro and in vivo models of lipid digestion from almond particles within a complex food matrix (muffins) investigating whether the cell-wall barrier regulates the bioaccessibility of nutrients within this matrix. Muffins containing small (AF) or large (AP) particles of almond were digested in triplicate using an in vitro dynamic gastric model (DGM, 1 h) followed by a static duodenal digestion (8 h). AF muffins had 97.1 ± 1.7% of their lipid digested, whereas AP muffins had 57.6 ± 1.1% digested. In vivo digestion of these muffins by an ileostomy volunteer (0–10 h) gave similar results with 96.5% and 56.5% lipid digested, respectively. The AF muffins produced a higher postprandial triacylglycerol iAUC response (by 61%) than the AP muffins. Microstructural analysis showed that some lipid remained encapsulated within the plant tissue throughout digestion. The cell-wall barrier mechanism is the main factor in regulating lipid bioaccessibility from almond particles

Tobacco Rattle Virus Vector: A Rapid and Transient Means of Silencing Manduca sexta Genes by Plant Mediated RNA Interference

Background: RNAi can be achieved in insect herbivores by feeding them host plants stably transformed to express double stranded RNA (dsRNA) of selected midgut-expressed genes. However, the development of stably transformed plants is a slow and laborious process and here we developed a rapid, reliable and transient method. We used viral vectors to produce dsRNA in the host plant Nicotiana attenuata to transiently silence midgut genes of the plant’s lepidopteran specialist herbivore, Manduca sexta. To compare the efficacy of longer, undiced dsRNA for insect gene silencing, we silenced N. attenuata’s dicer genes (NaDCL1- 4) in all combinations in a plant stably transformed to express dsRNA targeting an insect gene. Methodology/Principal Findings: Stable transgenic N. attenuata plants harboring a 312 bp fragment of MsCYP6B46 in an inverted repeat orientation (ir-CYP6B46) were generated to produce CYP6B46 dsRNA. After consuming these plants, transcripts of CYP6B46 were significantly reduced in M. sexta larval midguts. The same 312 bp cDNA was cloned in an antisense orientation into a TRV vector and Agro-infiltrated into N. attenuata plants. When larvae ingested these plants, similar reductions in CYP6B46 transcripts were observed without reducing transcripts of the most closely related MsCYP6B45. We used this transient method to rapidly silence the expression of two additional midgut-expressed MsCYPs. CYP6B46 transcripts were further reduced in midguts, when the larvae fed on ir-CYP6B46 plants transiently silenced for tw