An assessment of feral horse impacts on treeless drainage lines in the Australian Alps

The feral Horse (Equus caballus) is widespread across the Australian Alps. Feral horses degrade alpine and sub‐alpine ecosystems and damage habitat of a range of threatened species. Despite this, there is little published work to document the extent and severity of these impacts. This study investigated impacts of feral horses on treeless drainage lines at 186 sites across the Australian Alps. The study included sites in the Australian Capital Territory, New South Wales and Victoria. We assessed nine variables related to soil and stream stability and vegetation cover, which in turn influence ecosystem function and habitat quality. We found significant differences among horse‐occupied and horse‐free sites for all soil and stream stability variables assessed. For all variables assessed, the average score (and hence, condition) was worse in horse‐occupied areas. The sites in poorest condition were occupied by horses. Impacts from other mammalian herbivores species appeared to be minor. Management intervention is necessary if these impacts of feral horses are to be addressed.     

Basal cell adenocarcinoma of the salivary gland: report of a case with morphology on fine needle aspiration cytology

BACKGROUND: Basal cell adenocarcinoma of the parotid is rare and prone to recur. CASE: A 54-year-old woman had a history of afacial mass 12 years earlier that had been excised and was diagnosed as low grade adenocarcinoma of the parotid. Over the years, the patient had multiple local and lymph node recurrences. Histology of the excised local recurrent tumor showed basal cell adenocarcinoma, and FNAC of a separate recurrent nodule was performed. The aspirate showed moderate cellularity of basaloid cells with mildly pleomorphic nuclei, small nucleoli and occasional mitotic figures. The cells were mostly single, but some formed clusters with a rosettelike pattern of tumor cells surrounding central eosinophilic globules. A second, less prominent population of smaller cells with dark-staining nuclei was also noted. The differential diagnosis included adenoid cystic carcinoma, polymorphous low grade adenocarcinoma, and basal cell and pleomorphic adenoma. CONCLUSION: The cytologic features of basal cell adenocarcinoma are not distinctive, but the presence of two cell populations with moderate pleomorphism and a rosettelike pattern with central, eosinophilic globules may assist with its differentiation from other salivary gland neoplasms.     

A high‐throughput capillary isoelectric focusing immunoassay for fingerprinting protein sialylation

The serum half‐life, biological activity, and solubility of many recombinant glycoproteins depend on their sialylation. Monitoring glycoprotein sialylation during cell culture manufacturing is, therefore, critical to ensure product efficacy and safety. Here a high‐throughput method for semi‐quantitative fingerprinting of glycoprotein sialylation using capillary isoelectric focusing immunoassay on NanoPro (Protein Simple) platform was developed. The method was specific, sensitive, precise, and robust. It could analyze 2 μL of crude cell culture samples without protein purification, and could automatically analyze from 8 samples in 4 h to 96 samples in 14 h without analyst supervision. Furthermore, its capability to detect various changes in sialylation fingerprints during cell culture manufacturing process was indispensable to ensure process robustness and consistency. Moreover, the changes in the sialylation fingerprints analyzed by this method showed strong correlations with intact mass analysis using liquid chromatography and mass spectrometry. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:235–241, 2016     

Electrical stimulation influences satellite cell proliferation and apoptosis in unloading-induced muscle atrophy in mice

Muscle atrophy caused by disuse is accompanied by adverse physiological and functional consequences. Satellite cells are the primary source of skeletal muscle regeneration. Satellite cell dysfunction, as a result of impaired proliferative potential and/or increased apoptosis, is thought to be one of the causes contributing to the decreased muscle regeneration capacity in atrophy. We have previously shown that electrical stimulation improved satellite cell dysfunction. Here we test whether electrical stimulation can also enhance satellite cell proliferative potential as well as suppress apoptotic cell death in disuse-induced muscle atrophy. Eight-week-old male BALB/c mice were subjected to a 14-day hindlimb unloading procedure. During that period, one limb (HU-ES) received electrical stimulation (frequency: 20 Hz; duration: 3 h, twice daily) while the contralateral limb served as control (HU). Immunohistochemistry and western blotting techniques were used to characterize specific proteins in cell proliferation and apoptosis. The HU-ES soleus muscles showed significant improvement in muscle mass, cross-sectional area, and peak tetanic force relative to the HU limb (p<0.05). The satellite cell proliferative activity as detected within the BrdU+/Pax7+ population was significantly higher (p<0.05). The apoptotic myonuclei (detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) and the apoptotic satellite cells (detected by cleaved Poly [ADP-ribose] polymerase co-labeled with Pax7) were reduced (p<0.05) in the HU-ES limb. Furthermore the apoptosis-inducing factor and cleaved caspase-3 were down-regulated while the anti-apoptotic Bcl-2 protein was up-regulated (p<0.05), in the HU-ES limb. These findings suggest that the electrical stimulation paradigm provides an effective stimulus to rescue the loss of myonuclei and satellite cells in disuse muscle atrophy, thus maintaining a viable satellite cell pool for subsequent muscle regeneration. Optimization of stimulation parameters may enhance the outcome of the intervention.     

The cysteine protease α-clostripain is not essential for the pathogenesis of Clostridium perfringens-mediated myonecrosis

Clostridium perfringens is the causative agent of clostridial myonecrosis or gas gangrene and produces many different extracellular toxins and enzymes, including the cysteine protease α-clostripain. Mutation of the α-clostripain structural gene, ccp, alters the turnover of secreted extracellular proteins in C. perfringens, but the role of α-clostripain in disease pathogenesis is not known. We insertionally inactivated the ccp gene C. perfringens strain 13 using TargeTron technology, constructing a strain that was no longer proteolytic on skim milk agar. Quantitative protease assays confirmed the absence of extracellular protease activity, which was restored by complementation with the wild-type ccp gene. The role of α-clostripain in virulence was assessed by analysing the isogenic wild-type, mutant and complemented strains in a mouse myonecrosis model. The results showed that although α-clostripain was the major extracellular protease, mutation of the ccp gene did not alter either the progression or the development of disease. These results do not rule out the possibility that this extracellular enzyme may still have a role in the early stages of the disease process.     

Influenza virus evades innate and adaptive immunity via the NS1 protein

Both antibodies and T cells contribute to immunity against influenza virus infection. However, the generation of strong Th1 immunity is crucial for viral clearance. Interestingly, we found that human dendritic cells (DCs) infected with influenza A virus have lower allospecific Th1-cell stimulatory abilities than DCs activated by other stimuli, such as lipopolysaccharide and Newcastle disease virus infection. This weak stimulatory activity correlates with a suboptimal maturation of the DCs following infection with influenza A virus. We next investigated whether the influenza A virus NS1 protein could be responsible for the low levels of DC maturation after influenza virus infection. The NS1 protein is an important virulence factor associated with the suppression of innate immunity via the inhibition of type I interferon (IFN) production in infected cells. Using recombinant influenza and Newcastle disease viruses, with or without the NS1 gene from influenza virus, we found that the induction of a genetic program underlying DC maturation, migration, and T-cell stimulatory activity is specifically suppressed by the expression of the NS1 protein. Among the genes affected by NS1 are those coding for macrophage inflammatory protein 1beta, interleukin-12 p35 (IL-12 p35), IL-23 p19, RANTES, IL-8, IFN-alpha/beta, and CCR7. These results indicate that the influenza A virus NS1 protein is a bifunctional viral immunosuppressor which inhibits innate immunity by preventing type I IFN release and inhibits adaptive immunity by attenuating human DC maturation and the capacity of DCs to induce T-cell responses. Our observations also support the potential use of NS1 mutant influenza viruses as live attenuated influenza virus vaccines.     

The Glycerol‐3‐Phosphate Acyltransferase TbGAT is Dispensable for Viability and the Synthesis of Glycerolipids in Trypanosoma brucei

Glycerolipids are the main constituents of biological membranes in Trypanosoma brucei, which causes sleeping sickness in humans. Importantly, they occur as a structural component of the glycosylphosphatidylinositol lipid anchor of the abundant cell surface glycoproteins procyclin in procyclic forms and variant surface glycoprotein in bloodstream form, that play crucial roles for the development of the parasite in the insect vector and the mammalian host, respectively. The present work reports the characterization of the glycerol‐3‐phosphate acyltransferase TbGAT that initiates the biosynthesis of ester glycerolipids. TbGAT restored glycerol‐3‐phosphate acyltransferase activity when expressed in a Leishmania major deletion strain lacking this activity and exhibited preference for medium length, unsaturated fatty acyl‐CoAs. TbGAT localized to the endoplasmic reticulum membrane with its N‐terminal domain facing the cytosol. Despite that a TbGAT null mutant in T. brucei procyclic forms lacked glycerol‐3‐phosphate acyltransferase activity, it remained viable and exhibited similar growth rate as the wild type. TbGAT was dispensable for the biosynthesis of phosphatidylcholine, phosphatidylinositol, phosphatidylserine, and GPI‐anchored protein procyclin. However, the null mutant exhibited a slight decrease in phosphatidylethanolamine biosynthesis that was compensated with a modest increase in production of ether phosphatidylcholine. Our data suggest that an alternative initial acyltransferase takes over TbGAT's function in its absence.     

Post-Operative Plasma Osteopontin Predicts Distant Metastasis in Human Colorectal Cancer

BackgroundThe overall prognosis of colorectal cancer (CRC) patients is unsatisfactory due to cancer metastasis after operation. This study aims to investigate the clinical significance of plasma osteopontin (OPN) levels as minimally invasive, predictive, and surrogate biomarkers for prognosis of CRC patients.MethodsThis randomized study design consists of pre-operative and post-operative plasma samples from a total of 79 patients. We determined plasma levels of OPN by ELISA and examined their correlation with the clinicopathological parameters of CRC patients. The effects of endogenous and exogenous OPN on CRC metastasis were investigated by examination of the effect on regulators of epithelial to messenchymal transition and migration assay.ResultsOur findings demonstrated for the first time the clinical correlation of plasma OPN with metastasis of CRC patients. High post-operative plasma OPN level (>153.02 ng/ml) associated with development of metastasis after curative resection (p<0.001). Moreover, post-operative plasma OPN level correlated with disease-free survival of CRC patients (p=0.009) and was an independent factor for predicting development of metastasis in CRC patients after curative resection (p=0.036). Our in vitro model showed that OPN ectopic expression induced DLD1 cell migration through Snail and Twist1 overexpression and E-cadherin repression, and secretory OPN level enhanced cell migration.ConclusionsThe results of the current study suggest that post-operative plasma OPN correlated with post-operative metastasis, suggesting that it is a potential non-invasive biomarker for the development of future metastasis in CRC patients. In addition, OPN was shown to be involved in the metastatic process and thus inhibition of OPN is a potential therapeutic approach to treat CRC patients.     

A rapid quantitative method for measuring UTP, UDP, and UMP in the picomole range.

A procedure for the determination of picomole amounts of uracil nucleotides is described. The key reaction is the condensation of UTP and [¹⁴C]glucose 1-phosphate catalyzed by uridine 5′-diphosphoglucose pyrophosphorylase yielding UDP-[¹⁴C]glucose. The product is determined by selective adsorption onto charcoal in the presence of 0.8 m Trizma Base. UDP is measured as UTP after its conversion in an incubation with excess ATP and nucleoside diphosphate kinase. Similarly, UMP is analyzed after it is converted to UDP by nucleoside monophosphate kinase. The uracil nucleotide content of germinated wheat embryos had been determined with this method.