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The adipose tissue has been recognized as an active endocrine organ which can modulate numerous physiological processes such as metabolism, appetite, immunity, and reproduction. The aim of this study was to look for differentially abundant proteins and their biological functions in the abdominal adipose tissue between pre- and postpubertal Brahman heifers. Twelve Brahman heifers were divided into 2 groups and paired on slaughter day. Prepubertal heifers had never ovulated and postpubertal heifers were slaughtered on the luteal phase of their second estrous cycle. After ensuring the occurrence of puberty in postpubertal heifers, abdominal adipose tissue samples were collected. Mass spectrometry proteomic analysis identified 646 proteins and revealed that 171 proteins showed differential abundance in adipose tissue between the pre- and postpuberty groups (adjusted P-value < 0.05). Data are available via ProteomeXchange with identifier PXD009452. Using a list of 51 highly differentially abundant proteins as the target (adjusted P-value < 10−5), we found 14 enriched pathways. The results indicated that gluconeogenesis was enhanced when puberty approached. The metabolism of glucose, lipids, and AA in the adipose tissue mainly participated in oxidation and energy supply for heifers when puberty occurred. Our study also revealed the differentially abundant proteins were enriched for estrogen signaling and PI3K-Akt signaling pathways, which are known integrators of metabolism and reproduction. These results suggest new candidate proteins that may contribute to a better understanding of the signaling mechanisms that relate adipose tissue function to puberty. Protein–protein interaction network analysis identified 4 hub proteins that had the highest degrees of connection: PGK1, ALDH5A1, EEF2, and LDHB. Highly connected proteins are likely to influence the functions of all differentially abundant proteins identified, directly or indirectly.

To understand genes, pathways, and networks related to puberty, we characterized the transcriptome of two tissues: the pituitary gland and ovaries. Samples were harvested from pre- and postpubertal Brahman heifers (same age group). Brahman heifers (Bos indicus) are older at puberty compared with Bos taurus, a productivity issue. With RNA sequencing, we identified differentially expressed (DEx) genes and important transcription factors (TF) and predicted coexpression networks. The number of DEx genes detected in the pituitary gland was 284 (P< 0.05), and VWC2Lwas the most DEx gene (fold change = 4.12, P= 0.01). The gene VWC2Lpromotes bone mineralization through transforming growth factor-β (TGFβ) signaling. Further studies of the link between bone mineralization and puberty could target VWC2L. In ovaries, 3,871 genes were DEx (P< 0.05). Four highly DEx genes were noteworthy for their function: SLC6A13(a γ-aminobutyric acid [GABA] transporter), OXT(oxytocin), and NPY(neuropeptide Y) and its receptor NPY2R. These genes had higher ovarian expression in postpubertal heifers. The GABA and its receptors and transporters were expressed in the ovaries of many mammals, suggesting a role for this pathway beyond the brain. The OXTpathway has been known to influence the timing of puberty in rats, via modulation of GnRH. The effects of NPYat the hypothalamus, pituitary gland, and ovaries have been documented. Neuropeptide Yand its receptors are known factors in the release of GnRH, similar to OXTand GABA, although their roles in ovarian tissue are less clear. Pathways previously related to puberty such as TGFβ signaling (P= 6.71 × 10−5), Wnt signaling (P= 4.1 × 10−2), and peroxisome proliferator-activated receptor (PPAR) signaling (P= 4.84 × 10−2) were enriched in our data set. Seven genes were identified as key TF in both tissues: HIC2, ZIC4, ZNF219, ZSCAN26, LHX1, OLIG1, and a novel gene. An ovarian subnetwork created with TF and significant ovarian DEx genes revealed five zinc fingers as regulators: ZNF507, ZNF12, ZNF512, ZNF184, and ZNF432. Recent work of hypothalamic gene expression also pointed to zinc fingers as TF for bovine puberty. Although some zinc fingers may be ubiquitously expressed, the identification of DEx genes in common across tissues points to key regulators of puberty. The hypothalamus and pituitary gland had eight DEx genes in common. The hypothalamus and ovaries had 89 DEx genes in common. The pituitary gland and ovaries had 48 DEx genes in common. Our study confirmed the complexity of puberty and suggested further investigation on genes that code zinc fingers.

Puberty onset is a developmental process influenced by genetic determinants, environment, and nutrition. Mutations and regulatory gene networks constitute the molecular basis for the genetic determinants of puberty onset. The emerging knowledge of these genetic determinants presents opportunities for innovation in the breeding of early pubertal cattle. This paper presents new data on hypothalamic gene expression related to puberty in Bos indicus(Brahman) in age- and weight-matched heifers. Six postpubertal heifers were compared with 6 prepubertal heifers using whole-genome RNA sequencing methodology for quantification of global gene expression in the hypothalamus. Five transcription factors (TF) with potential regulatory roles in the hypothalamus were identified in this experiment: E2F8, NFAT5, SIX5, ZBTB38, and ZNF605. These TF genes were significantly differentially expressed in the hypothalamus of postpubertal versus prepubertal heifers and were also identified as significant according to the applied regulatory impact factor metric (P< 0.05). Two of these 5 TF, ZBTB38and ZNF605, were zinc fingers, belonging to a gene family previously reported to have a central regulatory role in mammalian puberty. The SIX5gene belongs to the family of homologues of Drosophila sine oculis (SIX) genes implicated in transcriptional regulation of gonadotrope gene expression. Tumor-related genes such as E2F8and NFAT5are known to affect basic cellular processes that are relevant in both cancer and developmental processes. Mutations in NFAT5were associated with puberty in humans. Mutations in these TF, together with other genetic determinants previously discovered, could be used in genomic selection to predict the genetic merit of cattle (i.e., the likelihood of the offspring presenting earlier than average puberty for Brahman). Knowledge of key mutations involved in genetic traits is an advantage for genomic prediction because it can increase its accuracy.

Six visits were conducted to four dairy farms to collect swab, liquid, and solid dairy farm environmental samples (165 to 180/farm; 15 sample types). The objective of the study was to determine on-farm sources of Campylobacter jejuni, Salmonella spp., Listeria monocytogenes, and Shiga toxin-producing Escherichia coli (STEC), which might serve as reservoirs for transmission of pathogens. Samples were analyzed using mostly U.S. Food and Drug Administration's Bacteriological Analytical Manual protocols; however, Salmonella spp., L. monocytogenes and STEC were co-enriched in universal pre-enrichment broth. Campylobacter jejuni were enriched in Bolton broth containing Bolton broth supplement. Pathogens were isolated on agar media, typed biochemically, and confirmed using multiplex polymerase chain reaction protocols. Campylobacter jejuni, Salmonella spp., L. monocytogenes, Sorbitol-negative (SN)-STEC O157:H7, and sorbitol-positive (SP)-STEC, respectively, were isolated from 5.06%, 3.76%, 6.51%, 0.72%, and 17.3% of samples evaluated. Whereas other pathogens were isolated from all four farms, SN-STEC O157:H7 were isolated from only two farms. Diverse serotypes of SP-STEC including O157:H7, O26:H11, O111, and O103 were isolated. None of the five pathogen groups studied were isolated from bulk tank milk (BTM). Most pathogens (44.2%) were isolated directly from fecal samples. Bovine fecal samples, lagoon water, bedding, bird droppings, and rat intestinal contents constituted areas of major concern on dairy farms. Although in-line milk filters from two farms tested positive for Salmonella or L. monocytogenes, none of the pathogens were detected in the corresponding BTM samples. Good manure management practices, including control of feral animals, are critical in assuring dairy farm hygiene. Identification of on-farm pathogen reservoirs could aid with implementation of farm-specific pathogen reduction programs.

1Mechanically skinned skeletal muscle fibres from the twitch region of the iliofibularis muscle of cane toads were used to investigate the relationship between fibre glycogen content and fibre capacity to respond to transverse tubular (T‐) system depolarization.2A large proportion of total fibre glycogen remained in mechanically skinned muscle fibres exposed to aqueous solutions. This glycogen pool (about 80 % of total fibre glycogen) was very stable when the preparation was incubated in a rigor solution (pH 7.0) but decreased gradually at a rate of 0.59 ± 0.20 % min−1in a relaxing solution (200 nM [Ca2+]). The rate was considerably higher (2.66 ± 0.38 % min−1) when the preparations were exposed to 30 μM [Ca2+]. An even greater rate of glycogen loss was found after T‐system depolarization‐induced contractions. The Ca2+‐dependent loss of fibre glycogen was caused by endogenous glycogenolytic processes.3Silver stained SDS gels of components eluted into relaxing solution from single skinned fibres revealed a rapid (2 min) loss of parvalbumin and at least 10 other proteins varying in molecular mass between 10 and 80 kDa but there was essentially no loss of myosin heavy and light chains and actin. Subsequent elution for a further 30 min in either relaxing or maximally Ca2+‐activating solution did not result in additional, appreciable detectable loss of fibre protein.4Depletion of fibre glycogen was associated with loss of fibre ability to respond to T‐system depolarization even though the bathing solutions contained high levels of ATP (8 mM) and creatine phosphate (10 mM).5The capacity of mechanically skinned fibres to respond to T‐system depolarization was highly positively correlated (P< 0.0001) with initial fibre glycogen concentration.6In conclusion, the results show that (i) the capacity of skeletal muscle to respond to T‐system depolarization is related directly or indirectly to the non‐washable glycogen pool in fibres, (ii) this relationship holds for conditions where glycogen is not required as a source of energy and (iii) the mechanically skinned fibre preparation is well suited to study the regulation of endogenous glycogenolytic enzymes.

Tablets (100 mg), containing microdose quantities of micronized model drug (1 μg-10 mg), were produced from interactive powder mixtures which had been fluid bed granulated. Granulation prevents adhesion unit and constituent segregation occurring during compression. Single tablet assays were performed on 100 tablets from each batch. The content of tablets fell within ±15% of the mean, and the coefficient of variation was <5% (99% confidence) for all batches. Skewness in the content distribution showed little evidence of superpotent tablets and indicated the absence of significant agglomerates of particles of the micronized component. However, there was some indication that the mixtures produced were not totally agglomerate free. These results demonstrate that the true potential of interactive mixing may be realized, provided segregation in the mixture can be eliminated. The distribution of micronized model drug, as a function of carrier particle size, was determined for the different concentration mixtures before and after granulation. When 0.1–2% interactive mixtures were sieved gently, the proportions of micronized material adhering to the different size fractions of carrier were similar. However, the very low concentration mixtures (0.01–0.03%) proved to be relatively unstable, a significant proportion of the micronized component being transferred from the mixtures to the metal surfaces of the sieves. Granulation of the mixtures in a fluidized bed produced a uniform distribution of micronized material throughout the different sized granules. These granules were stable during vibration on the sieves and when compressed. All samples of tablets met the United States Pharmacopeia XXI content uniformity requirements.

Very finely divided particulate salicylic acid (2–5 μm) was microencapsulated using conventional fluidized bed coating techniques. The process involved spray coating a preformed ordered mixture, in which the micronized particles are adsorbed on the surface of a coarser carrier material. The mixtures contained 0·1, 1·0 and 5·0% weight of microfine model drug (salicylic acid). Between 75 and 95% of the micronized particles were retained on the carrier surface beneath the film. This process offers a novel method of microencapsulating very fine particulate materials, which may find an application in the production of enteric coated and sustained release microdose products. A recent publication on electrostatic charge interactions in ordered mixtures reported that the addition of a third component can affect the stability of a binary ordered mixture. Addition of 2% talc increased the stability of some mixtures during coating and resulted in the microencapsulation of a significantly greater amount of micronized model drug. A 0·1% salicylic acid binary ordered mixture retained 74% of model drug beneath the film, the 0.1% ternary mixture (2% talc) gave a mean retention of 99%.

The phenomenon of ordered unit segregation is of paramount importance in determining the homogeneity of an ordered mixture. This problem can be avoided by using a monodisperse carrier, but it is uneconomic. The fluidized bed granulation of a 0.1% ordered mixture has been studied as a method of reducing the effects of segregation. The ordered units were stable when fluidized and no significant loss of the microfine adsorbed material occurred during processing. The distribution of the minor component in the granules was significantly more uniform than in the original ordered mixtures. The demixing potential(where p = proportion of cohesive minor component and w is the weight % of carrier material in a particular size range) was used to quantify the distribution of the minor component as a function of particle size. DP is a coefficient of variation and can be directly compared with the specification standard deviation σA. Batches of granules were compressed into 100 or 350 mg tablets, containing approximately 100 and 350 μg of model drug substance. The weight and content coefficients of variation (c.v.) were determined for 20 tablets. All batches had a weight c.v. ≤1.5% and a content c.v. ≤4.6%. Granulation of an ordered mixture greatly reduces the potential for segregation to decrease the mixture homogeneity. It also provides a solution to the problems which may be encountered when the mixing of small quantities of cohesive materials during conventional fluidized bed granulation is attempted.

This paper discusses the current reliability issues involved with typical postmolded IC packages. Four major topics are presented, namely, stress, moisture permeation and corrosion-related problems, adhesion, and outgassing impurities. As the trend moves toward higher lead count, with increasingly larger dies which require more power dissipation within smaller package outlines, stress reduction will become a main concern. Methods to quantify package stresses in situ are outlined, together with the advantages and drawbacks of various schemes for decoupling the silicon die from the molding compound. Since the typical molding compound is permeable to moisture, the package collects water during stringent qualification tests (autoclave, T/H, and HAST), and even under normal ambient conditions. Any ionic impurities in the molding compound will combine with the absorbed water to form an electrolytic pool. This increases the likelihood of corrosion in the presence of passivation defects or exposed metal lines. Device structures designed to follow moisture permeation and detect water-induced damage are discussed. The integrity of the interface between the molding compound and the device components is crucial to the long term reliability of the package. Delamination at the die interface produces complex stress profiles that can result in various defects ranging from metal line shift, passivation cracking, corner die chipping, to ball bond fatigue. Poor adhesion along the leads also opens potential paths for moisture ingress. The effects of assembly conditions and various adhesion enhancing schemes on the package integrity are evaluated. Finally, outgassing of halogenated (bromine and antimony oxide) byproducts added for flame retardancy during high temperature storage life testing results in ball bond degradation. The interaction yields typically porous intermetallic structures which grow in size with exposure time, leading ultimately to bond lifting. Possible reaction mechanisms are presented.