Tumor necrosis factor alpha and palmitate simulate bovine fatty liver disease in vitro when using abattoir-derived primary bovine hepatocytes isolated by a novel nonperfusion method.

Hepatic lipidosis (i.e., fatty liver) is a common periparturient disease in high-producing dairy cattle affecting nearly 50% of cows to some degree and costing an estimated 60 million dollars annually. Large animal studies are costly, labor intensive, and are not well suited to mechanistic studies. Traditionally, mechanistic studies employ in vitro methodologies, utilizing established cell lines or primary cell culture methods. However, with dairy cattle, established hepatic cell lines do not exist, and methods for primary cell culture studies typically involve complicated procedures that often utilize very young animals (typically bull calves). Several previously published papers have used abattoir-derived tissues as a source of primary cells; however, a simple method utilizing simple culture media has yet to be presented. In addition, we sought to develop a way to replicate the syndrome of fatty liver disease "in a dish" using adult cattle that should more closely represent the physiology of the periparturient dairy cow. Herein we present a non-perfusion-based method that results in robust growth and proliferation of abattoir-derived bovine hepatocytes that demonstrate lipid loading, elevated lactate dehydrogenase leakage, and cytotoxicity as demonstrated by elevated caspase 3/7 expression consistent with in vivo physiology of the periparturient dairy cow with fatty liver disease.

Hypogalactia in mammary quarters adjacent to lipopolysaccharide-infused quarters is associated with transcriptional changes in immune genes.

Infusion of lipopolysaccharides (LPS) into a mammary gland can provoke inflammatory responses and impair lactation in both the infused gland and neighboring glands. To gain insight into the mechanisms controlling the spatiotemporal response to localized mastitis in lactating dairy cows, we performed RNA sequencing on mammary tissue from quarters infused with LPS, neighboring quarters in the same animals, and control quarters from untreated animals at 3 and 12 h postinfusion. Differences in gene expression were annotated to Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Comparing mammary transcriptomes from all 3 treatments revealed 3,088 and 1,644 differentially expressed (DE) genes at 3 and 12 h, respectively. Of these genes, >95% were DE only in LPS-infused quarters and represented classical responses to LPS: inflammation, apoptosis, tissue remodeling, and altered cell signaling and metabolism. Although relatively few genes were DE in neighboring quarters (56 at 3 h; 74 at 12 h), these represented several common pathways. At 3 h, tumor necrosis factor (TNF), nuclear factor-κB, and nucleotide-binding and oligomerization domain (NOD)-like receptor signaling pathways were identified by the upregulation of anti-inflammatory (NFKBIA, TNFAIP3) and cell adhesion molecule (VCAM1, ICAM1) genes in neighboring glands. Additionally, at 12 h, several genes linked to 1-carbon and serine metabolism were upregulated. Some responses were also regulated over time. The proinflammatory response in LPS-infused glands diminished between 3 and 12 h, indicating tight control over transcription to re-establish homeostasis. In contrast, 2 glucocorticoid-responsive genes, FKBP5 and ZBTB16, were among the top DE genes upregulated in neighboring quarters at both time points, indicating potential regulation by glucocorticoids. We conclude that a transient, systemic immune response was sufficient to disrupt lactation in neighboring glands. This response may be mediated directly by proinflammatory factors from the LPS-infused gland or indirectly by secondary factors released in response to systemic inflammatory signals.

Intramammary lipopolysaccharide infusion induces local and systemic effects on milk components in lactating bovine mammary glands.

Each quarter of the bovine mammary gland is an anatomically and functionally distinct gland. However, mastitis in one quarter may affect function of adjacent, uninfected glands. To investigate the mechanisms and potential mediators of these effects, we quantified early responses of the mammary gland to intramammary lipopolysaccharide (LPS) challenge, distinguishing between local and systemic effects. Ten multiparous cows over 70 d in milk were blocked into pairs by breed, cow-level somatic cell count (SCC), and milk yield. Within block, one cow was assigned to LPS treatment (T) such that both the front and the rear quarter of a randomly selected udder half received an infusion of 50 µg of LPS in 10 mL of saline (T-L); the contralateral quarters received only 10 mL of saline (T-S). Similarly, each paired control cow (C) received either 10 mL of saline (C-S) or no infusion (C-N) into udder halves. Cows were quarter milked twice daily, with foremilk samples (∼30 mL, front quarters) taken at -24, 0, 3, 6, 12, and 24 h relative to infusions. At 24 h, average milk yield in T-L and T-S quarters fell to 23 and 32% of pre-infusion levels, respectively. For T cows, systemic effects were observed by 3 h post-infusion as rectal temperature was elevated and foremilk fat concentration was reduced in both T-L and T-S. However, SCC and concentrations of l-lactate and total protein in foremilk indicated a local response to LPS: protein was transiently higher at 3 h, whereas SCC and lactate were higher at 6 h in T-L compared with T-S. Lactose concentration showed a local effect at 6 h, being lower in T-L than in T-S, and then a systemic effect at 12 h, being lower in both T-L and T-S than C quarters. Concomitant with changes in milk, systemic effects were also observed in blood. Plasma antioxidant potential and glucose concentration were lower in T cows than in C cows at 6 or 12 h, respectively, although neither variable remained different at 24 h. In summary, unilateral LPS infusion induced distinct, time-dependent effects on each milk component. Depending on the component, effects were local, systemic, or both, suggesting involvement of multiple different mediators that collectively result in systemic inhibition of milk production.

Practical consensus recommendations on management of triple-negative metastatic breast cancer.

Patients with breast cancer along with metastatic estrogen and progesterone receptor (ER/PR)- and human epidermal growth factor receptor 2 (HER2)-negative tumors are referred to as having metastatic triple-negative breast cancer (mTNBC) disease. Resistance to current standard therapies such as anthracyclines or taxanes limits the available options for previously treated patients with metastatic TNBC to a small number of non-cross-resistant regimens, and there is currently no preferred standard chemotherapy. Clinical experience suggests that many women with triple-negative metastatic breast cancer (MBC) relapse quickly. Expert oncologist discussed about new chemotherapeutic strategies and agents used in treatment of mTNBC and the expert group used data from published literature, practical experience and opinion of a large group of academic oncologists to arrive at this practical consensus recommendations for the benefit of community oncologists.

Identification of the MicroRNA Repertoire in TLR-Ligand Challenged Bubaline PBMCs as a Model of Bacterial and Viral Infection.

In the present study, we used high-throughput sequencing, miRNA-seq, to discover and explore the expression profiles of known and novel miRNAs in TLR ligand-stimulated vis-à-vis non-stimulated (i.e. Control) peripheral blood mononuclear cells (PBMCs) isolated from blood of healthy Murrah buffaloes. Six small RNA (sRNA) libraries were multiplexed in Ion Torrent PI chip and sequenced on Ion Proton System. The reads obtained were aligned to the Bos taurus genome (UMD3.1 assembly), which is phylogenetically closest species to buffalo (Bubalus bubalis). A total of 160 bovine miRNAs were biocomputationally identified in buffalo PBMCs and 130 putatively novel miRNAs (not enlisted in the bovine mirBase) were identified. All of these 290 miRNAs identified across the six treatment and control samples represent the repertoire of novel miRNAs for the buffalo species. The expression profiles of these miRNAs across the samples have been represented by sample dendrogram and heatmap plots. The uniquely expressed miRNAs in each treatment and control groups were identified. A few miRNAs were expressed at very high levels while the majority of them were moderately expressed. The miRNAs bta-miR-103 and -191 were found to be highly abundant and expressed in all the samples. Other abundantly expressed miRNAs include bta-miR-19b, -29b, -15a, -19a, -30d, -30b-5p and members of let family (let 7a-5p, let 7g & let 7f) in LPS and CpG treated PBMCS and bta-miR-191, -103 & -19b in Poly I:C stimulated PBMCs. Only one novel miRNA (bta-miR-11039) out of 130 identified putatively novel miRNAs, was expressed in all the six samples and differentially expressed (>2- fold) miRNAs were identified. Six of the differentially expressed miRNAs across the groups (bta-miR-421, bta-let-7i, bta-miR-138, bta-miR-21-5p, bta-miR-222 and bta-miR-27b) were subsequently confirmed by TaqMan quantitative reverse transcription polymerase chain reaction (qRT-PCR). Furthermore, the target genes of differentially expressed miRNAs were enriched for the roles in innate immunity and TLR signaling pathways. This maiden study on profiling and cataloguing of bubaline miRNAs expressed in TLR-ligand stimulated PBMCs will provide an important reference point for future studies on regulatory roles of miRNAs in immune system of buffaloes.

Phylogeny and systematics of Indian Polygonum sensu lato in the subfamily Polygonoideae based on ITS sequences of nuclear ribosomal DNA.

The nuclear ribosomal DNA internal transcribed spacer (ITS) sequences from 44 Indian Polygonum taxa were examined to investigate relationships among various sections proposed by earlier researchers. The maximum parsimony trees obtained from analysis of the ITS sequences suggested eight major groups of the Indian Polygonum spp. The relationships among different sections were largely congruent with those inferred from morphological characters as described by Hooker. Also, the treatment of the Persicaria suggested by Haraldson on the basis of anatomical characters proved to be nearly in line with that based on our molecular data. We provide a high resolution of phylogeny of the Himalayan Polygonum sensu lato and support merger of the section Amblygonon in the section Persicaria. Moreover, we made the first phylogenetic analysis of many of the less known Himalayan Polygonums, including Polygonum microcephalum, P. assamicum, P. recumbens, and P. effusum. Molecular differences were detected among Persicaria barbata collected from different geographical locations of India, although these were not differentiated at the morphological level.

Reticulate acropigmentation of dohi: a case report with insight into genodermatoses with mottled pigmentation.

Reticulate acropigmentation of Dohi also called dyschromatosis symmetrica hereditaria or symmetrical dyschromatosis of the extremities is an autosomal dominant inherited disorder. It is characterized by mottled pigmentation with patchy depigmentation commonly over the back of the hands and feet and sometimes on the arms and legs.

Bovine mammary stem cells: cell biology meets production agriculture.

Mammary stem cells (MaSC) provide for net growth, renewal and turnover of mammary epithelial cells, and are therefore potential targets for strategies to increase production efficiency. Appropriate regulation of MaSC can potentially benefit milk yield, persistency, dry period management and tissue repair. Accordingly, we and others have attempted to characterize and alter the function of bovine MaSC. In this review, we provide an overview of current knowledge of MaSC gained from studies using mouse and human model systems and present research on bovine MaSC within that context. Recent data indicate that MaSC retain labeled DNA for extended periods because of their selective segregation of template DNA strands during mitosis. Relying on this long-term retention of bromodeoxyuridine-labeled DNA, we identified putative bovine MaSC. These label-retaining epithelial cells (LREC) are in low abundance within mammary epithelium (<1%). They are predominantly estrogen receptor (ER)-negative and localized in a basal or suprabasal layer of the epithelium throughout the gland. Thus, the response of MaSC to estrogen, the major mitogen in mammary gland, is likely mediated by paracrine factors released by cells that are ER-positive. This is consistent with considerable evidence for cross-talk within and between epithelial cells and surrounding stromal cells. Excision of classes of cells by laser microdissection and subsequent microarray analysis will hopefully provide markers for MaSC and insights into their regulation. Preliminary analyses of gene expression in laser-microdissected LREC and non-LREC are consistent with the concept that LREC represent populations of stem cells and progenitor cells that differ with regard to their properties and location within the epithelial layer. We have attempted to modulate the MaSC number by infusing a solution of xanthosine through the teat canal and into the ductal network of the mammary glands of prepubertal heifers. This treatment increased the number of putative stem cells, as evidenced by an increase in the percentage of LREC and increased telomerase activity within the tissue. The exciting possibility that stem cell expansion can influence milk production is currently under investigation.

Antioxidant activity of phenolic and flavonoid compounds in some medicinal plants of India.

In this study, total phenolics, flavonoids and vitamin C content vis-a-vis antioxidant activities were assayed in leaves and stem bark of Azadirachta indica, Butea monosperma, Cassia fistula, Mangifera indica, Syzygium cumini and Tamarindus indica using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and superoxide radical scavenging method. The DPPH radical scavenging activity positively correlated with the total phenolic content in both stem bark and leaf. Superoxide radical scavenging activity increased with increasing flavonoid contents. However, the vitamin C content could not be correlated with DPPH and superoxide radical scavenging capacity.

Studies on genetic diversity among populations of Persicaria barbata (L.) H. Hara from India based on internal transcribed spacer sequences of nuclear ribosomal DNA.

Internal transcribed spacer (ITS) region of nuclear ribosomal DNA from 16 populations of Persicaria barbata (L.) H. Hara (Polygonaceae) belonging to five geographical locations of India (Arunachal Pradesh, Himachal Pradesh, Bihar, Karnataka and Andaman Island) was sequenced. Analysis of nucleotide sequences reveals polymorphism among the populations. UPGMA analysis conducted on the ITS datasets shows that the sampled populations of P. barbata are grouped according to their geographic locations and are supposed to be evolved under reproductive isolation which most probably are due to the long distance distribution and population fragmentation.

Technical note: A rapid method for 5-bromo-2'-deoxyuridine (BrdU) immunostaining in bovine mammary cryosections that retains RNA quality.

A rapid method of 5-bromo-2'-deoxyuridine (BrdU) immunostaining was developed in cryosections of bovine mammary tissue while preserving RNA quality of the stained section. A thymidine analog that is incorporated into DNA of proliferating cells, BrdU serves as a proliferation marker. Immunostaining of BrdU-labeled cells within a histological section requires heat, enzymatic or chemical-mediated antigen retrieval to open double-stranded DNA, and exposure to the BrdU antigen. Although these established treatments permit staining, they preclude use of cells within the tissue section for further gene expression experiments. Additionally, long antibody incubations and washing steps lead to extensive RNA degradation and elution. A protocol was developed for immunolocalization of BrdU-labeled cells in cryosections of bovine mammary tissue, which does not require harsh DNA denaturation and preserved RNA integrity and quantity. This protocol used an initial acetone:polyethylene glycol 300 [9:1 (vol/vol)] fixation (2 min) followed by staining with methyl green (0.5% aqueous; 2 min) to stabilize macromolecules, antigen retrieval with deionized formamide (70% in nuclease-free phosphate buffered saline; 4 min incubation), antibody incubation in the presence of RNase inhibitors (5 min), and minimal washing to facilitate recovery of RNA from cells from the stained sections. Applicability of this protocol to other nuclear antigens was evaluated by testing its suitability for staining estrogen receptor alpha and Ki-67 antigen. In both cases, use of the protocol provided good immunostaining and tissue morphology. The RNA quality of estrogen receptor alpha- and Ki-67-stained sections was not evaluated. Quality of the isolated RNA from BrdU-stained sections was evaluated by micro-fluidic electrophoresis and its utility was confirmed using quantitative reverse transcription-PCR. Staining intensity obtained with this labeling protocol was similar to that obtained using conventional immunohistochemistry protocols. When coupled with laser microdissection and RNA or cDNA amplification, this immunostaining protocol provided a means for future transcriptome analysis of BrdU-labeled cells within a complex tissue.

Dietary genistein stimulates mammary hyperplasia in gilts.

The possible role of the phytoestrogen genistein on prepubertal development of mammary glands, hormonal status and bone resorption was investigated in gilts. Forty-five gilts were fed a control diet containing soya (CTLS, n = 15), a control diet without soya (CTL0, n = 15) or the CTLS diet supplemented with 2.3 g of genistein daily (GEN, n = 15) from 90 days of age until slaughter (day 183 ± 1). Both basal diets were isonitrogenous and isocaloric. Jugular blood samples were obtained on days 89 and 176 to determine concentrations of isoflavone metabolites (on day 176 only), prolactin, estradiol, progesterone, insulin-like growth factor 1 (IGF1), and N-telopeptide of type I collagen (NTx; on day 176 only). At slaughter, mammary glands were excised, parenchymal and extraparenchymal tissues were dissected, and composition of parenchymal tissue (protein, fat, dry matter (DM), DNA) was determined. Histochemical analyses of mammary parenchyma were performed. Dietary genistein increased parenchymal protein (P < 0.05) while decreasing DM (P < 0.05) and tending to lower fat content compared with the CTLS, but not the CTL0, diet. There was more parenchymal DNA (1.26 v. 0.92 mg/g, P < 0.05) in GEN than CTLS gilts, likely reflecting an increase in the quantity of mammary epithelial cells. Circulating concentrations of genistein were increased in GEN gilts (P < 0.001) but concentrations of hormones or NTx (indicator of bone collagen resorption) were not affected by GEN (P > 0.1). Percentage of estradiol receptor alpha (ERα)-positive epithelial cells was lower (P < 0.05) in GEN than CTLS gilts, whereas 5-bromo-2'-deoxyuridine labeling index was unaltered (P > 0.1). Transcript levels for ERα, ERß, IGF1, epidermal growth factor (EGF), epidermal growth factor receptor and transforming growth factor alpha were not altered by treatments. Supplementation of the diet with genistein during the growing phase in gilts, therefore, led to hyperplasia of mammary parenchymal tissue after puberty; yet, even though circulating genistein was increased, this was not accompanied by changes in mammary expression of selected genes or circulating hormone levels.

Differential activation of mitogen-activated protein kinases following high and low LET radiation in murine macrophage cell line.

Mitogen-activated protein kinases have been shown to respond to various stimuli including cytokines, mitogens and gamma irradiation, leading to cell proliferation, differentiation, or death. The duration of their activation determines the specificity of response to each stimulus in various cells. In this study, the crucial intracellular kinases, ERK, JNK, and p38 kinase involved in cell survival, death, or damage and repair were examined for their activity in RAW 264.7 cells at various time points after irradiation with 2 Gy doses of proton ions or X-rays. This is the first report that shows that the MAPK signaling induced after heavy ion or X-ray exposure is not the same. Unlike gamma irradiation, there was prolonged but marginal activation of prosurvival ERK pathway and significant activation of proapoptotic p38 pathway in response to high LET radiation.

Flexible Bronchoscopy in the Evaluation of Mediastinal and Hilar Lymphadenopathy.

BACKGROUND: The diagnosis in cases of mediastinal and/or hilar lymphadenopathy with no lung parenchymal involvement is often difficult. We undertook this study to assess the diagnostic value of flexible bronchoscopy (FOB) especially transbronchial needle aspiration (TBNA) and transbronchial lung biopsy (TBLB) in these patients. METHODS: Forty eight patients with hilar and/or mediastinal lymphadenopathy without any parenchymal lung lesions, managed between 2000 to 2004 at a tertiary care centre who underwent FOB were evaluated retrospectively. RESULTS: Out of 48 patients, FOB showed widening of carina in six, widening of secondary carina in four, bulge in airways because of extrinsic compression in seven and endobronchial nodule in two patients. It was normal in rest 29 patients. TBNA was done in all patients and TBLB in 13 patients where clinico-radiologic findings were consistent with stage 1 sarcoidosis. FOB established diagnosis in 18 patients (caseating granuloma in eight, noncaseating granuloma in nine, and AFB culture positive in one). It was inconclusive in other patients. One patient developed pneumothorax requiring intercostal tube drainage. CONCLUSION: FOB especially TBNA has an important role in the diagnosis of hilar and mediastinal lymphadenopathy and should be considered before other invasive procedures.

Utilisation of redundant time on trauma list for carpal tunnel decompression.

This audit reviewed the trauma theatre time utilisation during April 2000 to March 2001. Instead of a scheduled 8 30 am start, first patient was on the table by only 9 40 am because of various reasons. To use this redundant time carpal tunnel release was started under local anaesthesia, as first case. On re-auditing, it was found that the patient for carpal tunnel release was on the table at 8 44 am. The first trauma case was on the table at 9 46 am. This simple idea has helped in the performing of an additional case every day with a delay to the trauma list of only six minutes (p<0.05).