Isolation of Murine Peritoneal Macrophages to Carry Out Gene Expression Analysis Upon Toll-like Receptors Stimulation

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. Macrophages express surface Toll-like receptors (TLRs), which recognize molecular patterns conserved through evolution in a wide range of microorganisms. TLRs play a central role in macrophage activation which is usually associated with gene expression alteration. Macrophages are critical in many diseases and have emerged as attractive targets for therapy. In the following protocol, we describe a procedure to isolate murine peritoneal macrophages using Brewer’s thioglycollate medium. The latter will boost monocyte migration into the peritoneum, accordingly this will raise macrophage yield by 10-fold. Several studies have been carried out using bone marrow, spleen or peritoneal derived macrophages. However, peritoneal macrophages were shown to be more mature upon isolation and are more stable in their functionality and phenotype. Thus, macrophages isolated from murine peritoneal cavity present an important cell population that can serve in different immunological and metabolic studies. Once isolated, macrophages were stimulated with different TLR ligands and consequently gene expression was evaluated.     

Global markets,openness and co-operation to be emphasised at this year's GaAs IC symposium

No matter which area of GaAs (III–V) technology may be your specialty, the best of the best will be at this year's IEEE-sponsored GaAs IC Symposium. The 12th Annual event will be held October 7–10th in New Orleans, LA. The jam-packed program of 85 invited and contributed papers, and lively noontime panels, carries a distinctively global flavour, underscoring the relevance of this year's dramatic world changes to our critically important international industry.     

Acute and sub-acute effects of 2-butenoic acid-3-(diethoxy phosphinothioyl) methyl ester (RPR-II) on testis of albino rat

Acute and sub-acute toxic effects of a novel phosphorothionate coded as RPR-II on testis of albino rats were studied. In acute study rats received a single dose of 12.3 mg/kg of RPR-II and sacrificed after 24 hr. For sub-acute study 0.58 mg/kg/day was administered orally to rats for 10 and 21 days. Acute exposure of rats to RPR-II brought no change either in the gonadosomatic index (GSI) or in the structure of testis or in the serum levels of testosterone. Testis glutathione (GSH) level and glutathione S-transferase (GST) activity was significantly decreased whereas, acid phosphatase (AcP) levels increased significantly at 24 hr post-treatment. On 7th day (withdrawal period) after the cessation of the treatment the GSH, GST, AcP, and AkP levels reached to near control. The sub-acute study revealed a significant decrease in GSI on 10th and 21st day of the treatment. In contrast, a time-dependent and significant increased in GSH level and GST activity was observed on 100th and 21st day of post-treatment, except GSH level on 10th day, which was declined. Due to RPR-II treatment the testis AcP and alkaline phosphatase (AkP) levels were significant at both 10th and 21st day of medication but AcP levels were increased whereas AkP levels decreased. The histopathological studies on day 10th showed considerable loss of spermatozoids in testis and at 21st day complete derangement of cellular organization was observed. Testosterone levels decreased significantly on 10th day and remained significantly low at 21st day. However, withdrawal studies showed a recovery in testis of rat treated with RPR-II. GST, GSH, GSI, AcP and AkP values recovered, testosterone levels were also well recovered but recovery in testis structure remained at a low profile. The present study suggests that RPR-II may cause testicular toxicity in rats affecting the normal functioning of testis and it also gave some new information in withdrawal studies.     

Discovery of rare variants for complex phenotypes

With the rise of sequencing technologies, it is now feasible to assess the role rare variants play in the genetic contribution to complex trait variation. While some of the earlier targeted sequencing studies successfully identified rare variants of large effect, unbiased gene discovery using exome sequencing has experienced limited success for complex traits. Nevertheless, rare variant association studies have demonstrated that rare variants do contribute to phenotypic variability, but sample sizes will likely have to be even larger than those of common variant association studies to be powered for the detection of genes and loci. Large-scale sequencing efforts of tens of thousands of individuals, such as the UK10K Project and aggregation efforts such as the Exome Aggregation Consortium, have made great strides in advancing our knowledge of the landscape of rare variation, but there remain many considerations when studying rare variation in the context of complex traits. We discuss these considerations in this review, presenting a broad range of topics at a high level as an introduction to rare variant analysis in complex traits including the issues of power, study design, sample ascertainment, de novo variation, and statistical testing approaches. Ultimately, as sequencing costs continue to decline, larger sequencing studies will yield clearer insights into the biological consequence of rare mutations and may reveal which genes play a role in the etiology of complex traits.     

Neuronal labeling patterns in the spinal cord of adult transgenic Zebrafish

We describe neuronal patterns in the spinal cord of adult zebrafish. We studied the distribution of cells and processes in the three spinal regions reported in the literature: the 8th vertebra used as a transection injury site, the 15th vertebra mainly used for motor cell recordings and also for crush injury, and the 24th vertebra used to record motor nerve activity. We used well‐known transgenic lines in which expression of green fluorescent protein (GFP) is driven by promoters to hb9 and isl1 in motoneurons, alx/chx10 and evx1 interneurons, ngn1 in sensory neurons and olig2 in oligodendrocytes, as well as antibodies for neurons (HuC/D, NF and SV2) and glia (GFAP). In isl1:GFP fish, GFP‐positive processes are retained in the upper part of ventral horns and two subsets of cell bodies are observed. The pattern of the transgene in hb9:GFP adults is more diffuse and fibers are present broadly through the adult spinal cord. In alx/chx10 and evx1 lines we respectively observed two and three different GFP‐positive populations. Finally, the ngn1:GFP transgene identifies dorsal root ganglion and some cells in dorsal horns. Interestingly some GFP positive fibers in ngn1:GFP fish are located around Mauthner axons and their density seems to be related to a rostrocaudal gradient. Many other cell types have been described in embryos and need to be studied in adults. Our findings provide a reference for further studies on spinal cytoarchitecture. Combined with physiological, histological and pathological/traumatic approaches, these studies will help clarify the operation of spinal locomotor circuits of adult zebrafish. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 642–660, 2016     

An interview with José Xavier Neto. Interviewed by Eva Amsen

The Latin American Society for Developmental Biology (LASDB) is getting ready for their Sixth International Meeting, which will be held in Montevideo, Uruguay, from April 26th to 29th, 2012. To find out more about the society, and about developmental biology in Latin America, we talked to LASDB president José Xavier Neto, who studies heart morphogenesis at the Laboratório Nacional de Biociências in Sao Paulo, Brazil.     

A method for rearing the yellow-spotted longicorn beetle, <Emphasis Type="Italic">Psacothea hilaris</Emphasis> (Coleoptera: Cerambycidae), to stabilize the last larval instar

The larvae of Psacothea hilaris (Pascoe) grown continuously under a long day in the laboratory pupate after the 4th, 5th, or 6th instar. This developmental polymorphism has complicated studies on the control of metamorphosis in P. hilaris. Since pupation in P. hilaris is known to be suppressed under a short day, a change in the photoperiod from a short to a long day with appropriate timing may assist in obtaining physiologically homogeneous larvae that pupate at the next molt. When the photoperiod was changed from 12-h light:12-h dark to 15-h light:9-h dark at the beginning of the 5th instar, the pupation rate at the next (5th) molt reached 90%, which was significantly higher than the proportion of larvae that pupated after the 5th instar when the larvae were grown continuously under a long day (52%). This rearing technique will expedite studies on the control of metamorphosis in P. hilaris.     

欧洲伪叶甲Lagria hirta L.种群季节变化和幼虫生长发育(鞘翅目:伪叶甲科)(英)

欧洲伪叶甲Lagria hirta L.野外种群季节变化特点鲜明:成虫仅发生于6月底至8月初,幼虫除6月份外全年可见。基于野外观察和测量幼虫头宽,发现这种甲虫的生殖季节很短,在6至8月份;卵孵化后幼虫生长发育,在越冬前可达第3、4或5龄(个别达6龄);幼虫越冬后,经过总共8—10龄,于6月份化蛹。冬季幼虫发育减慢但不完全停滞(非参数型中数检验,P<0.01)。这项研究改变了以前的观点,即认为该甲虫的幼虫发育只有4或5龄,同时也对这种甲虫的生命周期研究有促进作用。     

Global gene expression during the human organogenesis: from transcription profiles to function predictions

Human embryogenesis includes an integrated set of complex yet coordinated development of different organs and tissues, which is regulated by the spatiotemporal expression of many genes. Deciphering the gene regulation profile is essential for understanding the molecular basis of human embryo development. While molecular and genetic studies in mouse have served as a valuable tool to understand mammalian development, significant differences exists in human and mouse development at morphological and genomic levels. Thus it is important to carry out research directly on human embryonic development. Here we will review some recent studies on gene regulation during human embryogenesis with particular focus on the period of organogenesis, which had not been well studied previously. We will highlight a gene expression database of human embryos from the 4(th) to the 9(th) week. The analysis of gene regulation during this period reveals that genes functioning in a given developmental process tend to be coordinately regulated during human embryogenesis. This feature allows us to use this database to identify new genes important for a particular developmental process/pathway and deduce the potential function of a novel gene during organogenesis. Such a gene expression atlas should serve as an important resource for molecular study of human development and pathogenesis.     

pUC12-Wl: a plastnid vector for the study of cloned DNA conformation

pUC12-Wl is a new cloning vector for the study of torsion-induced structural transitions of insert DNA. It was derived from pUC12 by deleting three A + T-rich sequences which can undergo structural transitions when torsionally stressed. Transitions at these sites have low energy of activation and undefined structures. They complicate studies on transitions of DNA inserts by diverting torsional force and causing the vector to be undefined in helical and energetic terms. The new vector pUC12-Wl, from which these segments have been deleted, will facilitate studies of torsion-induced structural transitions of insert DNA.     

Neuroscience and psychological studies sustain the cognitive benefits of print reading

On behalf of the Journal of Cell Communication and Signaling Editorial board it is my great pleasure to present through this message of peace and love our warmest wishes of health, happiness and professional success. We sincerely hope that 2017 will be a peaceful year worldwide and that solutions will be brought to resolve the great tensions that crystalized last year into terrible acts of violence which reflected the inability of the political powers to bring satisfactory solutions to human dispair and fear. The year 2017 will be the time for celebration of the 10th JCCS anniversary and 9th International Workshop on the CCN family of Genes. Both events should allow us to meet in a productive interactive way. I have had the opportunity to express several times in these columns my deep belief in the power of communication at all levels of human biological and social interactions. Indeed, « Communication is the key » at large.     

Water footprint benchmarks for crop production: A first global assessment

In the coming few decades, global freshwater demand will increase to meet the growing demand for food, fibre and biofuel crops. Raising water productivity in agriculture, that is reducing the water footprint (WF) per unit of production, will contribute to reducing the pressure on the limited global freshwater resources. This study establishes a set of global WF benchmark values for a large number of crops grown in the world. The study distinguishes between benchmarks for the green–blue WF (the sum of rain- and irrigation water consumption) and the grey WF (volume of polluted water). The reference period is 1996–2005. We analysed the spatial distribution of the green–blue and grey WFs of different crops as calculated at a spatial resolution of 5 by 5′ with a dynamic water balance and crop yield model. Per crop, we ranked the WF values for all relevant grid cells from smallest to largest and plotted these values against the cumulative percentage of the corresponding production. The study shows that if we would reduce the green–blue WF of crop production everywhere in the world to the level of the best 25th percentile of current global production, global water saving in crop production would be 39% compared to the reference water consumption. With a reduction to the WF levels of the best 10th percentile of current global production, the water saving would be 52%. In the case that nitrogen-related grey WFs in crop production are reduced, worldwide, to the level of the best 25th percentile of current global production, water pollution is reduced by 54%. If grey WFs per ton of crop are further reduced to the level of the best 10th percentile of current production, water pollution is reduced by 79%. The benchmark values provide valuable information for formulating WF reduction targets in crop production. Further studies will be required to test the sensitivity of the benchmark values to the underlying model assumptions, to see whether regionalization of benchmarks is necessary and how certain WF benchmark levels relate to specific technology and agricultural practices.     

Fifteen years of Servitude et Grandeur to the application of a biophysical technique in medicine: The tale of AFMBioMed

AFMBioMed is the founding name under which international conferences and summer schools are organized around the application of atomic force microscopy in life sciences and nanomedicine. From its inception at the Atomic Energy Commission in Marcoule near 2004 to its creation in 2007 and to its 10th anniversary conference in Krakow, a brief narrative history of its birth and rise will demonstrate how and what such an organization brings to laboratories and the AFM community. With the current planning of the next AFMBioMed conference in Münster in 2019, it will be 15 years of commitment to these events.     

Effects of plant sterols and stanols on intestinal cholesterol metabolism: suggested mechanisms from past to present

Plant sterols and stanols are natural food ingredients found in plants. It was already shown in 1950 that they lower serum low-density lipoprotein cholesterol (LDL-C) concentrations. Meta-analysis has reported that a daily intake of 2.5 g plant sterols/stanols reduced serum LDL-C concentrations up to 10%. Despite many studies, the underlying mechanism remains to be elucidated. Therefore, the proposed mechanisms that have been presented over the past decades will be described and discussed in the context of the current knowledge. In the early days, it was suggested that plant sterols/stanols compete with intestinal cholesterol for incorporation into mixed micelles as well as into chylomicrons. Next, the focus shifted toward cellular processes. In particular, a role for sterol transporters localized in the membranes of enterocytes was suggested. All these processes ultimately lowered intestinal cholesterol absorption. More recently, the existence of a direct secretion of cholesterol from the circulation into the intestinal lumen was described. First results in animal studies suggested that plant sterols/stanols activate this pathway, which also explains the increased fecal neutral sterol content and as such could explain the cholesterol-lowering activity of plant sterols/stanols.     

Oka株水痘病毒的生物学特性研究

将日本大阪大学微生物病研究会（BIKEN）提供的Oka水痘病毒第28代减毒株,通过MRC－5人二倍体细胞连续传递10代,建立主代及工作代毒种批,并对其后的32代,34代,36代及38代病毒按WHO规程进行全面检定,证实其生物学特性相似,免疫原性良好,确保制备水痘减毒活疫苗毒种的稳定性和一致性。     

O-GlcNAc glycosylation: a signal for the nuclear transport of cytosolic proteins?

Year 2004 marks the 20th anniversary of the discovery of O-linked N-acetylglucosamine (O-GlcNAc) by Gerald W. Hart. Despite interest for O-GlcNAc, the functions played by this single monosaccharide remain poorly understood, though numerous roles have been suggested, among which is the involvement of O-GlcNAc in the nuclear transport of cytosolic proteins. This idea was first sustained by studies on bovine serum albumin that showed that the protein could be actively carried to the nucleus when it was modified with sugars. In this paper, we will review data on this puzzling problem. We will first describe the well-established nuclear localisation signal (NLS)-dependent nuclear transport by presenting the different factors involved, and then, we will examine where and how O-GlcNAc could be involved in nuclear transport. Whereas it has been suggested that O-GlcNAc could interfere at two levels in the nuclear transport both by modifying proteins to be translocated to the nucleus and by modifying the nucleoporins of the nuclear pore complex, according to us, this second idea seems unlikely. Part of this study will also be dedicated to a relatively new concept in the nuclear transport: the role of the 70-kDa heat shock proteins (HSP70). The action of the chaperone in nuclear translocation was put forward 10 years ago, but new findings suggest that this mechanism could be linked to O-GlcNAc glycosylation.     

Limits of Adaptation: The Evolution of Selective Neutrality

Many enzymes in intermediary metabolism manifest saturation kinetics in which flux is a concave function of enzyme activity and often of the Michaelis-Menten form. The result is that, when natural selection favors increased enzyme activity so as to maximize flux, a point of diminishing returns will be attained in which any increase in flux results in a disproportionately small increase in fitness. Enzyme activity ultimately will reach a level at which the favorable effect of an increase in activity is of the order 1/(4Ne) or smaller, where Ne is the effective population number. At this point, many mutations that result in small changes in activity will result in negligible changes in fitness and will be selectively nearly neutral. We propose that this process is a mechanism whereby conditions for the occurrence of nearly neutral mutations and gene substitutions can be brought about by the long-continued action of natural selection. Evidence for the hypothesis derives from metabolic theory, direct studies of flux, studies of null and other types of alleles in Drosophila melanogaster and chemostat studies in Escherichia coli. Limitations and complications of the theory include changes in environment or genetic background, enzymes with sharply defined optima of activity, overdominance, pleiotropy, multifunctional enzymes and branched metabolic pathways. We conclude that the theory is a useful synthesis that unites many seemingly unrelated observations. The principal theoretical conclusion is that the conditions for the occurrence of neutral evolution can be brought about as an indirect result of the action of natural selection.     

野生稻的遗传学研究——Ⅱ、普通野生稻过氧化物酶同工酶的多态性研究

本文利用电泳方法,研究了普通野生稻,尤其是中国和印度普通野生稻的过氧化物酶同工酶的多态性,结果表明: 1).普通野生稻中,过氧化物酶同工酶能分离出24条酶带,各酶带的出现频率不同,其中第3、5、6、9、10、11、19、20、21和22号酶带出现频率超过50%,又以第3、20和22三条酶带出现频率最高,这10条带可以做为普通野生稻过氧化物酶同工酶的基本酶谱,而第3、20和22三条带可能是其原始酶带。2).中国和印度的普通野生稻过氧化物酶同工酶有一定差异,但在墓本酶谱和原始酶带的表现上是一致的。因此,尽管普通野生稻过氧化物酶同工酶具有高度的分子多态性,但仍能反映种的分子特征。本文还讨论了过氧化物酶同工酶与普通野生稻的演化,和我国栽培稻的起源及其在进化学研究中的价值。     

Effects of inserting a pressensor film into articular joints on the actual contact mechanics.

Fuji film has been widely used in studies aimed at obtaining the contact mechanics of articular joints. Once sealed for practical use in biological joints, Fuji Pressensor film has a total effective thickness of 0.30 mm, which is comparable to the cartilage thickness in the joints of many small animals. The average effective elastic modulus of Fuji film is approximately 100 MPa in compression, which is larger by a factor of 100-300 compared to that of normal articular cartilage. Therefore, inserting a Pressensor film into an articular joint will change the contact mechanics of the joint. The measurement precision of the Pressensor film has been determined systematically; however, the changes in contact mechanics associated with inserting the film into joints have not been investigated. This study was aimed at quantifying the changes in the contact mechanics associated with inserting sealed Fuji Pressensor film into joints. Spherical and cylindrical articular joint contact mechanics with and without Pressensor film and for varying degrees of surface congruency were analyzed and compared by using finite element models. The Pressensor film was taken as linearly elastic and the cartilage was assumed to be biphasic, composed of a linear elastic solid phase and an inviscid fluid phase. The present analyses showed that measurements of the joint contact pressures with Fuji Pressensor film will change the maximum true contact pressures by 10-26 percent depending on the loading, geometry of the joints, and the mechanical properties of cartilage. Considering this effect plus the measurement precision of the film (approximately 10 percent), the measured joint contact pressures in a joint may contain errors as large as 14-28 percent.