Use of milk proteins as biomarkers of changes in the rumen metaproteome of Holstein cows fed low-fiber, high-starch diets.

Dietary levels of undegraded neutral detergent fiber (uNDF240) and rumen-fermentable starch (RFS) can affect the rumen microbiome and milk composition. The objective of the study is to investigate the use of milk proteins as biomarkers of rumen microbial activity through a comparative evaluation of the rumen microbial and milk protein profiles produced by Holstein cows fed diets with varying contents of physically effective uNDF240 (peuNDF240) and RFS. Eight ruminally cannulated lactating Holstein cows were included in a larger study as part of a 4 × 4 Latin square design with 4 28-d periods to assess 4 diets varying in peuNDF240 and RFS content. For this experiment, cows received one of 2 dietary treatments: (1) low-peuNDF240, high-RFS (LNHR) diet or (2) high-peuNDF240, low-RFS (HNLR) diet. Within each period, rumen fluid samples were collected from each cow on d 26 (1400 h) and d 27 (0600 h and 1000 h), and milk samples were collected from each cow on d 25 (2030 h), d 26 (0430 h, 1230 h, and 2030 h), and d 27 (0430 h and 1230 h). Microbial proteins were isolated from each rumen fluid sample. For milk samples, milk proteins were fractionated, and the whey fraction was subsequently isolated. Isolated proteins within each rumen fluid or milk sample were isobarically labeled and analyzed by liquid chromatography-tandem mass spectrometry. Product ion spectra acquired from rumen fluid samples were searched using SEQUEST against 71 composite databases. In contrast, product ion spectra acquired from milk samples were searched against the Bos taurus database. Data were analyzed using the PROC MIXED procedure in SAS 9.4 to assess the effect of diet and time of sampling. To increase stringency, the false discovery rate-adjusted P-value (PFDR) was also calculated to account for multiple comparisons. Using the mixed procedure, a total of 129 rumen microbial proteins were quantified across 24 searched microbial species. Of these, the abundance of 14 proteins across 9 microbial species was affected due to diet and diet × time interaction, including 7 proteins associated with energetics pathways. Among the 159 quantified milk proteins, the abundance of 21 proteins was affected due to the diet and diet × time interaction. The abundance of 19 of these milk proteins was affected due to diet × time interactions. Of these, 16 proteins had the disparity across diets at the 0430 h sampling time, including proteins involved in host defense, nutrient synthesis, and transportation, suggesting that biological shifts resulting from diet-induced rumen changes are not diurnally uniform across milkings. The concentration of lipoprotein lipase (LPL) was statistically higher in the milk from the cows fed with the LNHR diet, which was numerically confirmed with an ELISA. Further, as determined by ELISA, the LPL concentration was significantly higher in the milk from the cows fed with the LNHR diet at 0430 h sampling point, suggesting that LPL concentration may indicate dietary carbohydrate-induced ruminal changes. The results of this study suggest that diet-induced rumen changes can be reflected in milk in a diurnal pattern, further highlighting the need to consider sampling time points for using milk proteins as a representative biomarker of rumen microbial activity.

Influence of dietary carbohydrate profile on the dairy cow rumen meta-proteome.

Diet starch and fiber contents influence the rumen microbial profile and its fermentation products, yet no information exists about the effects of these dietary carbohydrate fractions on the metabolic activity of these microbes. The objective of this experiment was to evaluate the effects of dietary carbohydrate profile changes on the rumen meta-proteome profile. Eight cannulated Holstein cows were assigned to the study as part of a 4 × 4 Latin square design with a 2 × 2 factorial treatment arrangement including four 28-d periods. Cows received 1 of 4 dietary treatments on a dry matter (DM) basis. Diets included different concentrations of rumen fermentable starch (RFS) and physically effective undigested NDF (peuNDF240) content in the diet: (1) low peuNDF240, low RFS (LNLS); (2) high peuNDF240, low RFS (HNLS); (3) low peuNDF240, high RFS (LNHS); and (4) high peuNDF240, high RFS (HNHS). Rumen fluid samples were collected from each cow on the last 2 d of each period at 3 time points (0600, 1000, and 1400 h). The microbial protein fraction was isolated, isobarically labeled, and analyzed using liquid chromatography combined with tandem mass spectrometry techniques. Product ion spectra were searched using the SEQUEST search on Proteome Discoverer 2.4 (Thermo Scientific) against 71 curated microbe-specific databases. Data were analyzed using PROC MIXED procedure in SAS 9.4 (SAS Institute Inc.). A total of 138 proteins were characterized across 26 of the searched microbial species. In total, 46 proteins were affected by treatments across 17 of the searched microbial species. Of these 46 proteins, 28 were affected by RFS content across 13 microbial species, with 20 proteins having higher abundance with higher dietary RFS and 8 proteins having higher abundance with lower dietary RFS. The majority of these proteins have roles in energetics, carbon metabolism, and protein synthesis. Examples include pyruvate, phosphate dikinase (Ruminococcus albus SY3), 30S ribosomal protein S11 (Clostridium aminophilum), and methyl-coenzyme M reductase subunit α (Methanobrevibacter ruminantium strain 35063), which had higher abundances with higher dietary RFS. Conversely, glutamate dehydrogenase (Butyrivibrio fibrisolvens) and 50S ribosomal protein L5 (Pseudobutyrivibrio ruminis) and L15 (Ruminococcus bromii) had lower abundances with higher dietary RFS content. Among the remaining 18 proteins unaffected by RFS content alone, 5 proteins were affected by peuNDF240 content, and 13 were affected by peuNDF240 × RFS interactions. Our results suggest that the RFS content of the diet may have a greater influence on rumen microbial protein abundances than dietary peuNDF240 content or peuNDF240 × RFS interactions. This research highlights that dietary carbohydrate profile changes can influence rumen microbial protein abundances. Further research is needed to fully characterize the effects of diet on the rumen meta-proteome and manipulate the various roles of rumen microbes. This will aid in designing the strategies to maximize the efficiency of nutrient use in the rumen.

Assessment of dietary protein supplementation on milk productivity of commercial organic dairy farms during the grazing season.

Variability of protein and energy supply from pasture during the grazing season is a primary factor that can influence milk production of grazing organic dairy herds in the Northeast United States. This study evaluated the effects of altering the crude protein (CP) content of dietary supplements included in dairy rations fed to grazing organic dairy herds, on milk production and composition. Six commercial organic farms participated in a 6-wk trial, consisting of a 2-wk baseline period and 4-wk experimental period. Farms were paired by their summer 2017 milk urea nitrogen profile, and farms within each pair were assigned by restricted randomization to (1) continuation of their regular supplements (n = 3, control group, CON), or (2) supplement with altered CP as percentage of dry matter, formulated using an organic barley and roasted soybean mix (n = 3, treatment group, TRT). Throughout the 6-wk trial, individual milk samples were collected at 2 consecutive milkings weekly, while pasture and supplement samples, pasture measurements, and management information were collected twice weekly per farm. Data were statistically analyzed using the MIXED procedure of SAS (version 9.4, SAS Institute Inc.) for all parameters, and effects of treatment, week, and their interaction (treatment × week) were determined. The supplement CP (percentage of dry matter) during the baseline period was 13.5% for CON and 15.3% for TRT and 14.8% for CON and 19.3% for TRT during the experimental period. Milk production was 21% higher during the experimental period for TRT compared with CON (24.1 vs. 19.9 kg of milk per day, respectively). Milk production decreased for CON from wk 1 to wk 6 (23.6 vs. 20.4 kg of milk per day), whereas TRT maintained milked production from wk 1 to wk 6 (22.8 vs. 22.7 kg of milk per day). Milk composition was different between groups, with CON having higher fat percent (4.21 vs. 3.73%, respectively) and protein percent (3.15 vs. 3.05%, respectively) compared with TRT for the 6 wk. The milk urea nitrogen concentrations were similar between TRT and CON for the baseline period (11.9 vs. 12.1 mg/dL) and the final week of the experimental period (14.5 vs. 14.2 mg/dL). Although the effects of different diet CP fractions, particularly rumen undegradable protein and soluble protein, must be further delineated, these results indicate that altering the CP content of dietary supplements fed to grazing organic dairy cattle during the summer period in the Northeast US could be a useful mechanism to maintain milk production.

Training the assessors for the General Medical Council's Performance Procedures.

From July 1997, the General Medical Council (GMC) has had the power to investigate doctors whose performance is considered to be seriously deficient. Assessment procedures have been developed for all medical specialties to include peer review of performance in practice and tests of competence. Peer review is conducted by teams of at least two medical assessors and one lay assessor. A comprehensive training programme for assessors has been developed that simulates the context of a typical practice-based assessment and has been tailored for 12 medical specialties. The training includes the principles of assessment, familiarization with the assessment instruments and supervised practice in assessment methods used during the peer review visit. High fidelity is achieved through the use of actors who simulate third party interviewees and trained doctors who role play the assessee. A subgroup of assessors, selected to lead the assessment teams, undergo training in handling group dynamics, report writing and in defending the assessment report against legal challenge. Debriefing of assessors following real assessments has been strongly positive with regard to their preparedness and confidence in undertaking the assessment.

Phospholipase D1 is required for efficient mating projection formation in Saccharomyces cerevisiae.

Phospholipase D1 (PLD1) is an important enzyme involved in lipid signal transduction in eukaryotes. A role for PLD1 in signaling in Saccharomyces cerevisiae was examined. Pheromone response in yeast is controlled by a well-characterized protein kinase cascade. Loss of PLD1 activity was found to impair pheromone-induced changes in cellular morphology that result in formation of mating projections. The rate at which projections appeared following pheromone treatment was delayed, suggesting that PLD1 facilitates the execution of a rate-limiting step in morphogenesis. Mutants were found to be less sensitive to pheromone, again arguing that PLD1 is acting at a rate-limiting step. The fact that morphogenesis is most dramatically affected indicates that PLD1 functions primarily in the morphogenic branch of the pheromone response pathway.

Effects of reversible inactivation of the primate mesencephalic reticular formation. I. Hypermetric goal-directed saccades.

Single-neuron recording and electrical microstimulation suggest three roles for the mesencephalic reticular formation (MRF) in oculomotor control: 1) saccade triggering, 2) computation of the horizontal component of saccade amplitude (a feed-forward function), and 3) feedback of an eye velocity signal from the paramedian zone of the pontine reticular formation (PPRF) to higher structures. These ideas were tested using reversible inactivation of the MRF with pressure microinjection of muscimol, a GABA(A) agonist, in four rhesus monkeys prepared for chronic single-neuron and eye movement recording. Reversible inactivation revealed two subregions of the MRF: ventral-caudal and rostral. The ventral-caudal region, which corresponds to the central MRF, the cMRF, or nucleus subcuneiformis, is the focus of this paper and is located lateral to the oculomotor nucleus and caudal to the posterior commissure (PC). Inactivation of the cMRF produced contraversive, upward saccade hypermetria. In three of eight injections, the velocity of hypermetric saccades was too fast for a given saccade amplitude, and saccade duration was shorter. The latency for initiation of most contraversive saccades was markedly reduced. Fixation was also destabilized with the development of macrosaccadic square-wave jerks that were directed toward a contraversive goal in the hypermetric direction. Spontaneous saccades collected in total darkness were also directed toward the same orbital goal, up and to the contraversive side. Three of eight muscimol injections were associated with a shift in the initial position of the eyes. A contralateral head tilt was also observed in 5 out of 8 caudal injections. All ventral-caudal injections with head tilt showed no evidence of vertical postsaccadic drift. This suggested that the observed changes in head movement and posture resulted from inactivation of the caudal MRF and not spread of the muscimol to the interstitial nucleus of Cajal (INC). Evidence of hypermetria strongly supports the idea that the ventral-caudal MRF participates in the feedback control of saccade accuracy. However, development of goal-directed eye movements, as well as a shift in the initial position following some of the cMRF injections, suggest that this region also contributes to the generation of an estimate of target or eye position coded in craniotopic coordinates. Last, the observed reduction in contraversive saccade latency and development of macrosaccadic square-wave jerks supports a role of the MRF in saccade triggering.

Effects of reversible inactivation of the primate mesencephalic reticular formation. II. Hypometric vertical saccades.

Electrical microstimulation and single-unit recording have suggested that a group of long-lead burst neurons (LLBNs) in the mesencephalic reticular formation (MRF) just lateral to the interstitial nucleus of Cajal (INC) (the peri-INC MRF, piMRF) may play a role in the generation of vertical rapid eye movements. Inactivation of this region with muscimol (a GABA(A) agonist) rapidly produced vertical saccade hypometria (6 injections). In three of six injections, there was a marked reduction in the velocity of vertical saccades out of proportion to saccade amplitude (i.e., saccades fell below the main sequence). This was associated with a moderate increase in saccade duration. Inadvertent inactivation of the INC could not account for these observations because vertical, postsaccadic drift was not observed. Similarly, pure downward saccade hypometria, the hallmark of rostral interstitial nucleus of the medial longitudinal fasciculus (riMLF) inactivation, was always preceded by loss of upward saccades in our experiments. We also found a downward and ipsiversive displacement of initial eye position and evidence of a contraversive head tilt following piMRF injections. Saccade latency was shorter after two of six injections. Simulation of a local feedback model provided three possible explanations for vertical saccade hypometria: 1) a shift in the input to the model to request smaller saccades, 2) a reduction of LLBN input to the vertical saccade medium lead burst neurons (MLBNs), or 3) an increase in the gain of the feedback pathway. However, when the second hypothesis was coupled to a shortened duration of the saccade trigger (i.e., the discharge of the omnipause neurons), the physiological observations of piMRF inactivation could be replicated. This suggested that muscimol had targeted structures that provided both long-lead burst activity to the MLBNs in the riMLF and were critical for reactivation of the omnipause neurons. Evidence of markedly reduced vertical saccade amplitude, curved saccade trajectories, increased saccade duration, and saccades that fall below the amplitude/velocity main sequence in these monkeys closely parallels the oculomotor findings of patients with progressive supranuclear palsy (PSP).

Azido derivatives of low-valent group 14 elements: synthesis, characterization, and electronic structure of [(n-Pr)2ATI]GeN3 and [(n-Pr)2ATI]SnN3 featuring heterobicyclic 10-pi-electron ring systems.

Treatment of THF solutions of [(n-Pr)2ATI]MCl (where [(n-Pr)2ATI]- = N-(n-propyl)-2-(n-propylamino)troponiminate; M = Ge and Sn) with sodium azide affords the compounds [(n-Pr)2ATI]MN3 in excellent yield. X-ray analyses revealed that these Ge(II) and Sn(II) compounds feature linear azide moieties and planar heterobicyclic C7N2M ring systems. Germanium and tin atoms adopt a pyramidal geometry. IR spectra of [(n-Pr)2ATI]GeN3 and [(n-Pr)2ATI]SnN3 display a nu asym(N3) band at 2048 and 2039 cm-1, respectively. DFT calculations on the corresponding methyl-substituted species demonstrate that the geometrical and electronic structure of these two species are very similar, and the dominant canonical form of the metal-azide moiety is M-N-N identical to N. The tin system is, as expected, slightly more ionic. A comparative CASSCF/DFT study on the model system H-Sn-N3 illustrates that the DFT approach is viable for the calculation of the structures of these species.

Evaluation of head examinations produced with a mobile CT unit.

Recent years have seen the development of mobile CT units, designed for use in operating theatres, intensive care units and accident and emergency departments. One such unit is the Tomoscan M (Philips, Utrecht, The Netherlands). It operates with a maximum tube voltage of 130 kV, and a maximum tube current of only 50 mA. This study tested whether acceptable quality CT images of the brain could be produced on the mobile unit with these parameters. 44 consecutive normal head examinations performed on the mobile scanner were compared with 35 examinations from two conventional CT units. Two independent readers scored the examinations for noise and artefact. CT dose index (CTDI) values for the three CT units were obtained in free air as an estimate of patient dose. Differences in artefact score between CT units were generally small, but noise scores were worse when using the Tomoscan M with a 2 s slice time. The lowest CTDI values were obtained with the Somatom DRH (Siemens, Erlangen, Germany) unit and the highest with the SR 7000 (Philips, Utrecht, The Netherlands), with values from Tomoscan M, in all except one case, falling between these values for the protocols used in the study. The measured scattered radiation doses from the Tomoscan M are presented.

Dose-dependent effects of multiple acute cocaine injections on maternal behavior and aggression in Sprague-Dawley rats.

Rat dams, which had no prior drug treatment, were either nontreated controls or were injected subcutaneously 4 times during a 10-day period with a single dose of 30, 15 or 7.5 mg/kg of cocaine hydrochloride HCl, or normal saline. Injections were given immediately postpartum following delivery of their final pup (PPD 1), and again on postpartum day 3 (PPD 3), postpartum day 6 (PPD 6) and postpartum day 10 (PPD 10). Dams were observed 30 min following injections for maternal behavior (MB) towards 8 surrogate male pups on PPD 1 and PPD 3 and for aggression towards a male or female intruder in the presence of their litter on PPD 6 and PPD 10. Compared to saline and untreated controls, cocaine-treated dams exhibited more disruptions in MB on both PPD 1 and PPD 3 and were less aggressive towards an intruder, regardless of intruder sex, on PPD 6 and PPD 10. In most cases MB was altered in a dose-dependent manner with the higher doses of cocaine resulting in a greater disruption of behavior.

A dose-response study of chronic cocaine on maternal behavior in rats.

To determine if there was a dose-response relationship with regard to cocaine treatment and maternal behavior exhibited by lactating rats at doses that had not been previously investigated, we examined the effects of three doses of chronic cocaine administration throughout gestation on both onset and established maternal behavior. Dams were injected (SC) with 6.3, 13, or 25 mg/kg cocaine HCl or an equivalent volume of saline throughout gestation; maternal behavior was tested on postpartum days 1 and 3. At the doses employed, cocaine disrupted the onset of only one pup-directed component of maternal behavior significantly in a dose-response manner, although there were several statistically nonsignificant dose-dependent trends of behavioral disruptions. No pup-directed behaviors were disrupted during testing for established maternal behavior. These results indicate that gestational cocaine treatment at doses of 25 mg/kg and less have only minimal effects on the onset and no effect on the maintenance of maternal behavior using our paradigm. The relationship of the present findings to previous work is discussed.

Imaging the thoracic manifestations of AIDS.

Pulmonary complications occur in over half of patients with AIDS at some point in their illness. A chest X-ray is a valuable first-line investigation. The plain film and computed tomographic features of these complications are described and several examples illustrated. The role of computed tomography, including high resolution scanning techniques in further investigation and management, is discussed.

Magnetic resonance renography: optimisation of pulse sequence parameters and Gd-DTPA dose, and comparison with radionuclide renography.

The aim of this study was to assess the feasibility of magnetic resonance renography (MRR) using gadolinium diethylenetriaminepentaacetic acid (Gd-DTPA) in comparison with conventional radionuclide renography (RR) using technetium-99m-DTPA (99mTc-DTPA). MRR has many advantages over RR, including lack of ionising radiation, increased spatial resolution, and visible background anatomy. By optimising the pulse sequence, we developed an MRR protocol in which signal intensity is linear with Gd-DTPA concentration over a clinically relevant range. Twenty-nine patients and a volunteer were studied using this protocol. Magnetic resonance renography was performed using three different doses of Gd-DTPA: 0.1 mmol kg-1 (n = 13), 0.05 mmol kg-1 (n = 7), and 0.025 mmol kg-1 (n = 9). Each patient was also assessed using radionuclide renography. The resulting renograms were assessed in terms of time to peak signal intensity, signal decrease after peak, and kidney function ratios calculated from both the areas underneath and the slopes of the uptake curves. We have shown that the MR renograms obtained using low dose Gd-DTPA correlate best with the radionuclide renograms. Remaining discrepancies may be explained by variations in the injection procedures (hence in arterial input functions) and the limited coverage of the three MRR slices compared to the whole body projection of RR. Furthermore, at high local concentrations, signal becomes independent of T1 and is dominated by T2.

The use of antibiotic prophylaxis in radiology departments for patients at high-risk of infective endocarditis.

Patients with a prosthetic heart valve, or a history of endocarditis are at particular risk of developing infective endocarditis during a bacteraemia which may follow many gastrointestinal and genito-urinary radiological procedures. The current British Society for Antimicrobial Chemotherapy guidelines (1992) do recommend antibiotic prophylaxis for these high-risk patients prior to instrumentation of the gastrointestinal or genito-urinary tracts. Routine antibiotic prophylaxis for patients with damaged native valves or heart murmurs is recommended prior to dental procedures only. A questionnaire enquiring about prescribing practice prior to several radiological investigations known to be associated with bacteraemia was sent to 192 radiology departments and 126 replied. Only 38 of the 126 respondents have a prescribing policy for antibiotic prophylaxis in high-risk groups. Fifty-eight departments employ antibiotic prophylaxis prior to one or more of the procedures named in the questionnaire, but only 30 of these have mechanisms for identifying at-risk patients. Forty-eight departments keep recommended antibiotics in the department.

Posterior pituitary ectopia and hypopituitarism--magnetic resonance appearances of four cases and a review of the literature.

In four patients presenting in childhood with varying degrees of hypopituitarism, magnetic resonance imaging (MRI) showed a reduction in size of the normal pituitary fossa contents and an absent or very narrow stalk. A high signal intensity, enhancing area at the base of the stalk, having the appearances and signal characteristics of the posterior pituitary, was seen in each case. We discuss the case histories and MR findings in our patients and review the relevant literature.

Computed tomography in the evaluation of cutaneous T-cell lymphoma.

The computed tomography (CT) scans performed in 28 patients with cutaneous T-cell lymphoma (CTCL) were reviewed. Fifteen patients had clinically advanced cutaneous mycosis fungoides, six patients Sézary syndrome and seven variant CTCL. Of the 40 scans available 12 were normal, 15 indeterminate and 13 abnormal. Indeterminate and abnormal nodes showed a predilection for inguinal and axillary sites with a relative sparing of deep nodal regions. Visceral involvement was infrequent. In six patients CT detected abnormalities not obvious clinically and upstaged the disease. CT should be performed as part of the initial staging and as a baseline for follow-up in patients with advanced mycosis fungoides, Sézary syndrome and variant CTCL.

Lymphocytic adenohypophysitis: magnetic resonance imaging features of two new cases and a review of the literature.

Lymphocytic adenohypophysitis can cause pituitary expansion and hypopituitarism closely mimicking the features of a pituitary adenoma. Discrimination between these two conditions is of importance since, despite the similarity of their presentation, there are significant differences in pathophysiology. In contrast to pituitary adenoma, lymphocytic adenohypophysitis occurs almost exclusively in young women in relation to pregnancy, there is a preference for destruction of ACTH and TSH secreting cells and computed tomographic scanning shows uniform contrast enhancement in a proportion of cases. There is, as yet, no proven specific non-surgical treatment. There are anecdotal reports of a beneficial effect of steroids but there is also evidence that spontaneous resolution may occur. We have reviewed the literature and report two new cases of lymphocytic adenohypophysitis both of whom exhibited early striking diffuse homogeneous contrast enhancement on magnetic resonance imaging scanning which we suggest may be a diagnostic feature of this condition.