Aminothiazoles inhibit RANKL‐ and LPS‐mediated osteoclastogenesis and PGE2 production in RAW 264.7 cells

Periodontitis is characterized by chronic inflammation and osteoclast‐mediated bone loss regulated by the receptor activator of nuclear factor‐κB (RANK), RANK ligand (RANKL) and osteoprotegerin (OPG). The aim of this study was to investigate the effect of aminothiazoles targeting prostaglandin E synthase‐1 (mPGES‐1) on RANKL‐ and lipopolysaccharide (LPS)‐mediated osteoclastogenesis and prostaglandin E₂ (PGE₂) production in vitro using the osteoclast precursor RAW 264.7 cells. RAW 264.7 cells were treated with RANKL or LPS alone or in combination with the aminothiazoles 4‐([4‐(2‐naphthyl)‐1,3‐thiazol‐2‐yl]amino)phenol (TH‐848) or 4‐(3‐fluoro‐4‐methoxyphenyl)‐N‐(4‐phenoxyphenyl)‐1,3‐thiazol‐2‐amine (TH‐644). Aminothiazoles significantly decreased the number of multinucleated tartrate‐resistant acid phosphatase (TRAP)‐positive osteoclast‐like cells in cultures of RANKL‐ and LPS‐stimulated RAW 264.7 cells, as well as reduced the production of PGE₂ in culture supernatants. LPS‐treatment induced mPGES‐1 mRNA expression at 16 hrs and the subsequent PGE₂ production at 72 hrs. Conversely, RANKL did not affect PGE₂ secretion but markedly reduced mPGES‐1 at mRNA level. Furthermore, mRNA expression of TRAP and cathepsin K (CTSK) was reduced by aminothiazoles in RAW 264.7 cells activated by LPS, whereas RANK, OPG or tumour necrosis factor α mRNA expression was not significantly affected. In RANKL‐activated RAW 264.7 cells, TH‐848 and TH‐644 down‐regulated CTSK but not TRAP mRNA expression. Moreover, the inhibitory effect of aminothiazoles on PGE₂ production was also confirmed in LPS‐stimulated human peripheral blood mononuclear cell cultures. In conclusion, the aminothiazoles reduced both LPS‐ and RANKL‐mediated osteoclastogenesis and PGE₂ production in RAW 264.7 cells, suggesting these compounds as potential inhibitors for treatment of chronic inflammatory bone resorption, such as periodontitis.     

Characterization of commercial laminin preparations from human placenta in comparison to recombinant laminins 2 (alpha2beta1gamma1), 8 (alpha4beta1gamma1), 10 (alpha5beta1gamma1).

Laminins, a family of large heterotrimeric (alphabetagamma) proteins, are major components of basement membranes implicated in a variety of cellular functions. Different commercial laminin preparations isolated from human placenta have been widely used in functional studies but their molecular properties are poorly known. In the present study, we characterized several of these preparations by ELISA, silver staining and Western blotting, in comparison to mouse laminin 1 (alpha1beta1gamma1), and recombinant human laminins 2 (alpha2beta1gamma1), 8 (alpha4beta1gamma1) and 10 (alpha5beta1gamma1). The cell migration-promoting activity of different batches was also tested. The placenta laminin preparations differed from one another and consisted of highly fragmented proteins, a mixture of laminin isoforms, and/or contaminating fibronectin. Major functional differences between batches were also observed, reflecting molecular heterogeneity. Previous data obtained in functional studies using these preparations need to be interpreted with caution and may require revision, and future functional studies demand prior molecular characterization of the laminins, particularly their alpha-chain.     

Robust reference intervals for Liver function test (LFT) analytes in newborns and infants

ABSTRACT: Back groundReference intervals (RIs) are ranges of upper and lower limits of a given analyte which are used for a laboratory test to determine whether a disease is present or absent or to know if the patient is at risk for future disease states. In Ethiopia, a country with highly diversified population groups and geographical sites, there are no established RIs to metabolic analytes including the liver function test (LFT) analytes for the pediatric population though it has been known that liver function assessment in this population is vital as a result of varied vulnerability to both endogenous and xenobiotic substances. METHODS: A cross sectional study was conducted in Tikur Anbessa Specialized Hospital (TASH) and Teklehaymanot Health Center (THC) from November 2010 to April 2011. 117 cord blood (from newborns) and venous blood samples (from infants) were collected and analyzed using HumaStar 300. All pre-analytical, analytical and post-analytical aspects were thoroughly controlled. A robust, CLSI/ IFCC recommended, method was used for the determination of upper and lower end points covering 95% of the reference values of each analyte with respective 90% CIs using MedCalc(R) software. RESULTS: Combined RIs for newborns and infants were established for albumin, AST, ALP, direct bilirubin and total bilirubin to be 3.88-5.82 g/dl, 16.1-55.4U/l, 130-831U/l, <0.41 mg/dl and <1.37 mg/dl respectively. But, separated RIs were indicated for ALT and GGT as 1.2-23.1U/l and 6.94-24.8U/l ALT; and 30.6-160.7U/L and 10--28.2U/l GGT for newborns and infants respectively. Some maternal and infantile factors were identified to affect the values of analytes. CONCLUSION: Almost all analytes were different from previously reported values for other target population of similar age group, kit insert values and adult values. So, interpretation of values of these analytes in newborns and infants of Ethiopian population sounds better to be performed by using such RIs taking the effect of some maternal and infantile factors in to account.     

Localization of laminin alpha4-chain in developing and adult human tissues.

Recent studies suggest important functions for laminin-8 (Ln-8; alpha4beta1gamma1) in vascular and blood cell biology, but its distribution in human tissues has remained elusive. We have raised a monoclonal antibody (MAb) FC10, and by enzyme-linked immunoassay (EIA) and Western blotting techniques we show that it recognizes the human Ln alpha4-chain. Immunoreactivity for the Ln alpha4-chain was localized in tissues of mesodermal origin, such as basement membranes (BMs) of endothelia, adipocytes, and skeletal, smooth, and cardiac muscle cells. In addition, the Ln alpha4-chain was found in regions of some epithelial BMs, including epidermis, salivary glands, pancreas, esophageal and gastric glands, intestinal crypts, and some renal medullary tubules. Developmental differences in the distribution of Ln alpha4-chain were detected in skeletal muscle, walls of vessels, and intestinal crypts. Ln alpha4- and Ln alpha2-chains co-localized in BMs of fetal skeletal muscle cells and in some epithelial BMs, e.g., in gastric glands and acini of pancreas. Cultured human pulmonary artery endothelial (HPAE) cells produced Ln alpha4-chain as M(r) 180,000 and 200,000 doublet and rapidly deposited it to the growth substratum. In cell-free extracellular matrices of human kidney and lung, Ln alpha4-chain was found as M(r) 180,000 protein.     

Analysis of household's energy consumption,forest degradation and plantation requirements in Eastern Tigray,Northern Ethiopia

This study examines the link between household's energy consumption, forest degradation and plantation requirements in Eastern Tigray. Data on household's fuelwood, cow‐dung, kerosene and electricity consumption in the study sites were collected from 557 urban and 114 rural households. Deforestation rate was estimated following FAO and Woody Biomass Inventory Strategy Planning Project (WBISPP's) conversion factors. The study showed that household's fuelwood consumption in urban areas (mean = 2.5 kg/day) was about 52% lower as compared to the rural villages (mean = 3.8 kg/day). The estimated area abandoned annually in the vicinity of urban areas as the result of urban fuelwood consumption varies from 217 ha around Maichew town to 12047 ha around Mekelle City. The estimated area abandoned in rural areas annually as the result of tree cutting for fuelwood also varies from 6.5 ha around Sinkata to 127 ha around Dergajen village. It is therefore recommended that an estimated annual tree plantation of area ranging from 286 ha around Maichew town to 21,684 ha around Mekelle city is required. Moreover, an estimated annual tree plantation of area ranging from 16 ha around Sinkata village to 229 ha around the Dergajen village is required taking the existing population into account.     

Pro-fibrotic pathway activation in trabecular meshwork and lamina cribrosa is the main driving force of glaucoma

While primary open-angle glaucoma (POAG) is a leading cause of blindness worldwide, it still does not have a clear mechanism that can explain all clinical cases of the disease. Elevated IOP is associated with increased accumulation of extracellular matrix (ECM) proteins in the trabecular meshwork (TM) that prevents normal outflow of aqueous humor (AH) and has damaging effects on the fine mesh-like lamina cribrosa (LC) through which the optic nerve fibers pass. Applying a pathway analysis algorithm, we discovered that an elevated level of TGFβ observed in glaucoma-affected tissues could lead to pro-fibrotic pathway activation in TM and in LC. In turn, activated pro-fibrotic pathways lead to ECM remodeling in TM and LC, making TM less efficient in AH drainage and making LC more susceptible to damage from elevated IOP via ECM transformation in LC. We propose pathway targets for potential therapeutic interventions to delay or avoid fibrosis initiation in TM and LC tissues.     

Neuronal Spike Timing Adaptation Described with a Fractional Leaky Integrate-and-Fire Model

The voltage trace of neuronal activities can follow multiple timescale dynamics that arise from correlated membrane conductances. Such processes can result in power-law behavior in which the membrane voltage cannot be characterized with a single time constant. The emergent effect of these membrane correlations is a non-Markovian process that can be modeled with a fractional derivative. A fractional derivative is a non-local process in which the value of the variable is determined by integrating a temporal weighted voltage trace, also called the memory trace. Here we developed and analyzed a fractional leaky integrate-and-fire model in which the exponent of the fractional derivative can vary from 0 to 1, with 1 representing the normal derivative. As the exponent of the fractional derivative decreases, the weights of the voltage trace increase. Thus, the value of the voltage is increasingly correlated with the trajectory of the voltage in the past. By varying only the fractional exponent, our model can reproduce upward and downward spike adaptations found experimentally in neocortical pyramidal cells and tectal neurons in vitro. The model also produces spikes with longer first-spike latency and high inter-spike variability with power-law distribution. We further analyze spike adaptation and the responses to noisy and oscillatory input. The fractional model generates reliable spike patterns in response to noisy input. Overall, the spiking activity of the fractional leaky integrate-and-fire model deviates from the spiking activity of the Markovian model and reflects the temporal accumulated intrinsic membrane dynamics that affect the response of the neuron to external stimulation.     

Spotted hyena (Crocuta crocuta) coexisting at high density with people in Wukro district,northern Ethiopia

We surveyed density and abundance of spotted hyena (Crocuta crocuta) in the highly degraded and prey depleted Wukro district, northern Ethiopia, with a human population density of 98 persons per square kilometer. A total of 117 spotted hyenas responded to callups, giving a hyena density of 52 hyenas per 100 km² or a total population of 535 hyenas in the district. We quantified the economic impact of spotted hyena predation on livestock using semi structured interviews with randomly selected households. Respondents indicated a total loss of 203 domestic animals to hyena depredation over the past five years. Average annual depredation per household was 0.13 livestock worth US$ 6.1. The diet of spotted hyenas was assessed in three sub-districts by scat analysis and showed 99% prey items of domestic origin, only three of 211 scat contained hair of Ethiopian hare (Lepus fagani) and porcupine (Hystrix cristata). We conclude that hyenas in northern Ethiopia live at high density and eat almost exclusively anthropogenic food and are not dependent on conservation areas.     

The deubiquitinating gene Usp29 is dispensable for fertility in male mice

The balanced actions between ubiquitination and deubiquitination precisely control the levels of various proteins vital for spermatogenesis. Ubiquitin-specific processing proteases(USPs) are the largest family of deubiquitinatingenzymes(DUBs),containing more than 50 members. So far, the functions of only a few USPs in male fertility have been studied, the roles of the majority are yet unknown. The present study aimed to explore the function of Usp29(ubiquitin-specific protease 29) in male fertility. We found that Usp29 showed predominant expression in mouse testis, and its m RNA expression started to increase at 14 days postpartum(dpp), with a peak at 28 and 35 dpp. Using CRISPR/Cas9 technology, we generated Usp29 knockout mice(Usp29~(–/–)). Usp29~(–/–)mice exhibited no overt developmental anomalies. Further examination revealed that Usp29~(–/–)mice had normal fertility and showed no detectable difference in the testis/body weight ratio, testicular and epididymal histology as well as epididymal sperm count from the wild-type littermates. Moreover, Usp29 is not a pseudogene in mice. Taken together, our study first reported that though Usp29 is predominantly expressed in the testis, it is not essential for male fertility in mice.