Oligomeric and protofibrillar aggregates formed by the amyloid-β peptide (Aβ) are believed to be involved in the pathology of Alzheimer's disease. Central to Alzheimer pathology is also the fact that the longer Aβ42 peptide is more prone to aggregation than the more prevalent Aβ40 . Detailed structural studies of Aβ oligomers and protofibrils have been impeded by aggregate heterogeneity and instability. We previously engineered a variant of Aβ that forms stable protofibrils and here we use solid-state NMR spectroscopy and molecular modeling to derive a structural model of these. NMR data are consistent with packing of residues 16 to 42 of Aβ protomers into hexameric barrel-like oligomers within the protofibril. The core of the oligomers consists of all residues of the central and C-terminal hydrophobic regions of Aβ, and hairpin loops extend from the core. The model accounts for why Aβ42 forms oligomers and protofibrils more easily than Aβ40 .

The fibril structure formed by the amyloidogenic fragment SNNFGAILSS of the human islet amyloid polypeptide (hIAPP) is determined with 0.52 A resolution. Symmetry information contained in the easily obtainable resonance assignments from solid-state NMR spectra (see picture), along with long-range constraints, can be applied to uniquely identify the supramolecular organization of fibrils.

Die Fibrillenstruktur, die das amyloidogene Fragment SNNFGAILSS des menschlichen Insel‐Amyloid‐Polypeptids (hIAPP) bildet, wurde mit einer Auflösung von 0.52 Å bestimmt. Aus den Festkörper‐NMR‐Spektren einfach erhältliche Symmetrieinformationen (siehe Bild) können zusammen mit langreichweitigen Randbedingungen genutzt werden, um die supramolekulare Organisation von Fibrillen eindeutig zu identifizieren.magnified image

A minimally invasive liver biopsy technique was tested for its applicability to study the hepatic acute phase response (APR) in dairy cows with Escherichia coli lipopolysaccharide (LPS)-induced mastitis. The hepatic mRNA expression profiles of the inflammatory cytokines, tumor necrosis factor alpha (TNF-alpha), IL-1beta, IL-6, and IL-10, and the acute phase proteins serum amyloid A isoform 3 (SAA3), haptoglobin (Hp), and alpha(1)-acid glycoprotein (AGP) were determined by real-time reverse transcription-PCR. Fourteen primiparous cows in mid lactation were challenged with 200 microg of LPS (n = 8) or NaCl solution (n = 6) in 1 front quarter. Six repeated liver biopsies were collected at -22, 3, 6, 9, 12, and 48 h relative to LPS challenge in 4 LPS-infused cows and 3 NaCl-infused cows. The remaining cows had 3 liver biopsies taken at -22, 9, and 48 h. Production data and clinical signs were recorded and white blood cell counts and somatic cell counts (SCC) were analyzed to investigate the effect of repeated liver biopsies and verify the LPS model. Plasma concentrations of TNF-alpha, SAA3, Hp, and AGP were determined for comparison with the liver expression data. Repeated liver biopsies had no effects on the production data, clinical signs, or APR of dairy cows. Compared with the NaCl-infused cows the LPS-infused cows responded to the LPS treatment by increased body temperature (38.6 +/- 0.1 vs. 39.4 +/- 0.1 degrees C), short-term leukopenia followed by leukocytosis (6.44 +/- 0.4 vs. 5.69 +/- 0.3 x 10(6) cells/mL), an increased SCC (log(10) 2.1 +/- 0.1 vs. log(10) 2.8 +/- 0.1 x 10(3) cells/mL), heart rate (76 +/- 1 vs. 93 +/- 1 beats/min), and respiratory rate (32 +/- 2 vs. 36 +/- 1 breaths/min) in the acute phase of the disease. The LPS treatment upregulated the hepatic expression of TNF-alpha (103 +/- 24 vs. 255 +/- 18 units), IL-1beta (37 +/- 23 vs. 296 +/- 18 units), IL-6 (8 +/- 17 vs. 122 +/- 12 units), and IL-10 (130 +/- 66 vs. 541 +/- 50 units), and SAA3 (64 +/- 36 vs. 128 +/- 28 units) and Hp (9 +/- 82 vs. 762 +/- 65 units) reaching maximum levels at 3 to 6 h and 9 to 12 h postinfusion, respectively. Plasma concentrations of TNF-alpha (nondetectable vs. 1.9 +/- 0.3 ng/mL), SAA (19.8 +/- 19.4 vs. 149.7 +/- 15.5 microg/mL) and Hp (71.4 +/- 143.7 vs. 1,013.8 +/- 111.5 microg/mL) were elevated in the LPS-infused cows at 4 to 12 h, 8 to 120 h, and 24 to 120 h postinfusion, respectively. The hepatic expression of AGP and the AGP plasma concentration remained unaltered in LPS-induced cows. In conclusion, a minimally invasive liver biopsy technique can be used for studying the hepatic APR in diseased cattle. Lipopolysaccharide-induced mastitis resulted in a time-dependent production of inflammatory cytokines and SAA and Hp in the liver of dairy cows.

Systematic factors affecting the activities of L-lactate dehydrogenase (LDH) and N-acetyl-beta-D-glucosaminidase (NAGase) and somatic cell count (SCC), the association between the activities of LDH and NAGase and SCC with respect to udder health status, and the ability of LDH and NAGase to classify cows in udder health categories for early detection of mastitis were studied. A dataset of records from 74 Danish Holstein, 76 Danish Red and 47 Jersey cows on one research farm was used. Cows were grouped into healthy and clinically mastitic. A healthy cow was defined as having no veterinary treatment and SCC<100,000 cells/ml. A clinically infected cow was one receiving veterinary treatment after showing clinical signs of mastitis and SCC >800,000 cells/ml. Breed, month of production, and days in milk significantly influenced (P<0.001) LDH activity, NAGase activity and SCC in both healthy and clinically mastitic cows. In healthy cows, LDH activity, NAGase activity and SCC started at a high level immediately after calving and decreased to low levels approximately 30-40 d post partum. All the three parameters increased due to clinical mastitis. NAGase activity had numerically higher variation in healthy cows than in clinically mastitic cows (CV=56.2% v. CV=53.5%). The relationship between LDH activity and SCC was stronger in milk from clinically mastitic than from healthy cows (r=0.76 v. r=0.48 and r=0.67 v. r=0.44 for correlation of observed values and residuals, respectively). LDH activity had higher sensitivity than NAGase activity (73-95% v. 35-77%) while specificities were in a similar range (92-99%). Further, sensitivities for LDH activity were more robust to changes in the threshold value than those for NAGase activity. Opportunities for automated, in-line real-time mastitis detection are discussed.

Eleven Danish Holstein cows were used to examine the effects of quarter health (healthy vs. unhealthy), milking interval (12 vs. 6 h), and sampling time during milking on the concentration of 8 milk constituents [acetone, beta-hydroxybutyrate (BHBA), N-acetyl-beta-D-glucosaminidase (NAGase), somatic cell count (SCC), urea, fat, protein, and lactose]. The selection criterion was that each cow should have 2 or 3 healthy and 1 or 2 unhealthy quarters. Foremilk was collected before attaching the teat cups of the milking machinery, and thereafter, milk samples were collected automatically from each quarter every 45 s during milking. Compared with milk from healthy quarters, milk from unhealthy quarters had a higher concentration of BHBA, NAGase, SCC, and protein during the entire milking, whereas urea was higher in the last part of the milking process. Healthy quarters had a higher content of acetone and lactose during the whole milking, whereas fat was higher in the first part of the milking process. When the cows were milked at the 6-h interval, all milk constituents except lactose and protein were higher during the whole (NAGase, SCC, and urea) or part of the milking (acetone, BHBA, and fat) compared with when cows were milked at the 12-h interval. Lactose was higher in the first part of the milking at the 12-h compared with the 6-h interval, whereas protein was not affected by milking interval. beta-Hydroxybutyrate, NAGase, SCC, and fat increased during the milking process, whereas acetone, urea, protein, and lactose decreased. Foremilk was remarkably different for all constituents, except acetone, and should not be used as a representative milk sample to achieve the true level of a milk constituent. If these milk constituents are to be used in an inline management system, these effects should be taken into account.

This study tested a model for predicting reproductive status from in-line milk progesterone ;measurements. The model is that of Friggens and Chagunda [Theriogenology 64 (2005) 155]. Milk progesterone measurements (n = 55 036) representing 578 lactations from 380 cows were used to test the model. Two types of known oestrus were identified: (1) confirmed oestrus (at which insemination resulted in a confirmed pregnancy, n = 121) and (2) ratified oestrus (where the shape of the progesterone profile matched that of the average progesterone profile of a confirmed oestrus, n = 679). The model detected 99.2% of the confirmed oestruses. This included a number of cases (n = 16) where the smoothed progesterone did not decrease below 4 ng/ml. These cows had significantly greater concentrations of progesterone, both minimum and average, suggesting that between cow variation exists in the absolute level of the progesterone profile. Using ratified oestruses, model sensitivity was 93.3% and specificity was 93.7% for detection of oestrus. Examination of false positives showed that they were largely associated with low concentrations of progesterone, fluctuating around the 4 ng/ml threshold. The distribution of time from insemination until the model detected pregnancy failure had a median of 22 days post-insemination. In this test, the model was run using limited inputs, the potential benefits of including additional non-progesterone information were not evaluated. Despite this, the model performed at least as well as other oestrus detection systems.

We present the first solid-state NMR experiments developed using optimal control theory. Taking heteronuclear dipolar recoupling in magic-angle-spinning NMR as an example, it proves possible to significantly improve the efficiency of the experiments while introducing robustness toward instrumental imperfections such as radio frequency inhomogeneity. The improvements are demonstrated by numerical simulations as well as practical experiments on a 13Calpha,15N-labeled powder of glycine. The experiments demonstrate a gain of 53% in the efficiency for 15N to 13Calpha coherence transfer relative to the typically double-cross-polarization experiments.