A scalable insect cell-based production process of the human recombinant BMX for in-vitro covalent ligand high-throughput screening.

Bone Marrow Tyrosine kinase in the chromosome X (BMX) is a TEC family kinase associated with numerous pathological pathways in cancer cells. Covalent inhibition of BMX activity holds promise as a therapeutic approach against cancer. To screen for potent and selective covalent BMX inhibitors, large quantities of highly pure BMX are normally required which is challenging with the currently available production and purification processes. Here, we developed a scalable production process for the human recombinant BMX (hrBMX) using the insect cell-baculovirus expression vector system. Comparable expression levels were obtained in small-scale shake flasks (13 mL) and in stirred-tank bioreactors (STB, 5 L). A two-step chromatographic-based process was implemented, reducing purification times by 75% when compared to traditional processes, while maintaining hrBMX stability. The final production yield was 24 mg of purified hrBMX per litter of cell culture, with a purity of > 99%. Product quality was assessed and confirmed through a series of biochemical and biophysical assays, including circular dichroism and dynamic light scattering. Overall, the platform herein developed was capable of generating 100 mg purified hrBMX from 5 L STB in just 34 days, thus having the potential to assist in-vitro covalent ligand high-throughput screening for BMX activity inhibition

A New Chanidae (Ostariophysii: Gonorynchiformes) from the Cretaceous of Brazil with Affinities to Laurasian Gonorynchiforms from Spain

Based on specimens originally referred to as “Dastilbe minor”, a nomem-nudum, we describe a new genus of Chanidae †Nanaichthys longipinnus nov. gen. and sp. which exhibits several diagnostic characters such as the absence of orbitosphenoid and basisphenoid, anteriorly displaced quadrate-mandibular articulation, laterally expanded supraneurals, an acute angle between the preopercular limbs, expansion at the angle between the preopercular limbs, and a curved maxillary articular process. Its occurrence and supposed relationship within the Chanidae reinforce the influence of the Mediterranean Tethys over the Gondwanan main rift system prior to the Aptian/Albian highstands

Consensus Recommendations of the Multiple Sclerosis Study Group and the Portuguese Neuroradiological Society for the Use of Magnetic Resonance Imaging in Multiple Sclerosis in Clinical Practice: Part 2

INTRODUCTION: Magnetic resonance imaging is recognized as the most important diagnostic test in the diagnosis of multiple sclerosis, differential diagnosis and evaluation of progression/therapeutic response. However, to make optimal use of magnetic resonance imaging in multiple sclerosis, the use of a standard, reproducible and comparable imaging protocol is of uttermost importance. In this context, the Portuguese Society of Neuroradiology and the Group of Studies of Multiple Sclerosis, after a joint discussion, appointed a committee of experts to create recommendations adapted to the national reality on the use of magnetic resonance imaging in multiple sclerosis. This document represents the second part of the first Portuguese consensus recommendations on the use of magnetic resonance imaging in multiple sclerosis in clinical practice. MATERIAL AND METHODS: The Portuguese Society of Neuroradiology and the Group of Studies of Multiple Sclerosis, after discussing the topic in national meetings and after a working group meeting held in Figueira da Foz, May 2017, appointed a committee of experts that have developed several standard protocols on the use of magnetic resonance imaging on multiple sclerosis by consensus. The document obtained was based on the best scientific evidence and expert opinion. Portuguese multiple sclerosis consultants and departments of neuroradiology scrutinized and reviewed the consensus paper; comments and suggestions were considered. Standardized strategies of magnetic resonance imaging referral in clinical practice for diagnosis and follow-up of multiple sclerosis were published in the first part of this paper. RESULTS: We provide magnetic resonance imaging acquisition protocols regarding multiple sclerosis diagnostic and monitoring and the information to be included in the report for application across Portuguese healthcare institutions. CONCLUSION: We hope that these first Portuguese magnetic resonance imaging guidelines will contribute to optimize multiple sclerosis management and improve patient care in Portugal.info:eu-repo/semantics/publishedVersio

Techniques for Arbuscular Mycorrhiza Inoculum Reduction

It is well established that arbuscular mycorrhizal (AM) fungi can play a significant role in sustainable crop production and environmental conservation. With the increasing awareness of the ecological significance of mycorrhizas and their diversity, research needs to be directed away from simple records of their occurrence or casual speculation of their function (Smith and Read 1997). Rather, the need is for empirical studies and investigations of the quantitative aspects of the distribution of different types and their contribution to the function of ecosystems. There is no such thing as a fungal effect or a plant effect, but there is an interaction between both symbionts. This results from the AM fungi and plant community size and structure, soil and climatic conditions, and the interplay between all these factors (Kahiluoto et al. 2000). Consequently, it is readily understood that it is the problems associated with methodology that limit our understanding of the functioning and effects of AM fungi within field communities. Given the ubiquous presence of AM fungi, a major constraint to the evaluation of the activity of AM colonisation has been the need to account for the indigenous soil native inoculum. This has to be controlled (i.e. reduced or eliminated) if we are to obtain a true control treatment for analysis of arbuscular mycorrhizas in natural substrates. There are various procedures possible for achieving such an objective, and the purpose of this chapter is to provide details of a number of techniques and present some evaluation of their advantages and disadvantages. Although there have been a large number of experiments to investigated the effectiveness of different sterilization procedures for reducing pathogenic soil fungi, little information is available on their impact on beneficial organisms such as AM fungi. Furthermore, some of the techniques have been shown to affect physical and chemical soil characteristics as well as eliminate soil microorganisms that can interfere with the development of mycorrhizas, and this creates difficulties in the interpretation of results simply in terms of possible mycorrhizal activity. An important subject is the differentiation of methods that involve sterilization from those focussed on indigenous inoculum reduction. Soil sterilization aims to destroy or eliminate microbial cells while maintaining the existing chemical and physical characteristics of the soil (Wolf and Skipper 1994). Consequently, it is often used for experiments focussed on specific AM fungi, or to establish a negative control in some other types of study. In contrast, the purpose of inoculum reduction techniques is to create a perturbation that will interfere with mycorrhizal formation, although not necessarily eliminating any component group within the inoculum. Such an approach allows the establishment of different degrees of mycorrhizal formation between treatments and the study of relative effects. Frequently the basic techniques used to achieve complete sterilization or just an inoculum reduction may be similar but the desired outcome is accomplished by adjustments of the dosage or intensity of the treatment. The ultimate choice of methodology for establishing an adequate non-mycorrhizal control depends on the design of the particular experiments, the facilities available and the amount of soil requiring treatment

Agronomic Management of Indigenous Mycorrhizas

Many of the advantages conferred to plants by arbuscular mycorrhiza (AM) are associated to the ability of AM plants to explore a greater volume of soil through the extraradical mycelium. Sieverding (1991) estimates that for each centimetre of colonized root there is an increase of 15 cm3 on the volume of soil explored, this value can increase to 200 cm3 depending on the circumstances. Due to the enhancement of the volume of soil explored and the ability of the extraradical mycelium to absorb and translocate nutrients to the plant, one of the most obvious and important advantages resulting from mycorrhization is the uptake of nutrients. Among of which the ones that have immobilized forms in soil, such as P, assume particular significance. Besides this, many other benefits are recognized for AM plants (Gupta et al, 2000): water stress alleviation (Augé, 2004; Cho et al, 2006), protection from root pathogens (Graham, 2001), tolerance to toxic heavy metals and phytoremediation (Audet and Charest, 2006; Göhre and Paszkowski, 2006), tolerance to adverse conditions such as very high or low temperature, high salinity (Sannazzaro et al, 2006), high or low pH (Yano and Takaki, 2005) or better performance during transplantation shock (Subhan et al, 1998). The extraradical hyphae also stabilize soil aggregates by both enmeshing soil particles (Miller e Jastrow, 1992) and producing a glycoprotein, golmalin, which may act as a glue-like substance to adhere soil particles together (Wright and Upadhyaya, 1998). Despite the ubiquous distribution of mycorrhizal fungi (Smith and Read, 2000) and only a relative specificity between host plants and fungal isolates (McGonigle and Fitter, 1990), the obligate nature of the symbiosis implies the establishment of a plant propagation system, either under greenhouse conditions or in vitro laboratory propagation. These techniques result in high inoculum production costs, which still remains a serious problem since they are not competitive with production costs of phosphorus fertilizer. Even if farmers understand the significance of sustainable agricultural systems, the reduction of phosphorus inputs by using AM fungal inocula alone cannot be justified except, perhaps, in the case of high value crops (Saioto and Marumoto, 2002). Nurseries, high income horticulture farmers and no-agricultural application such as rehabilitation of degraded or devegetated landscapes are examples of areas where the use of commercial inoculum is current. Another serious problem is quality of commercial available products concerning guarantee of phatogene free content, storage conditions, most effective application methods and what types to use. Besides the information provided by suppliers about its inoculum can be deceiving, as from the usually referred total counts, only a fraction may be effective for a particular plant or in specific soil conditions. Gianinazzi and Vosátka (2004) assume that progress should be made towards registration procedures that stimulate the development of the mycorrhizal industry. Some on-farm inoculum production and application methods have been studied, allowing farmers to produce locally adapted isolates and generate a taxonomically diverse inoculum (Mohandas et al, 2004; Douds et al, 2005). However the inocula produced this way are not readily processed for mechanical application to the fields, being an obstacle to the utilization in large scale agriculture, especially row crops, moreover it would represent an additional mechanical operation with the corresponding economic and soil compaction costs. It is well recognized that inoculation of AM fungi has a potential significance in not only sustainable crop production, but also environmental conservation. However, the status quo of inoculation is far from practical technology that can be widely used in the field. Together a further basic understanding of the biology and diversity of AM fungi is needed (Abbott at al, 1995; Saito and Marumoto, 2002). Advances in ecology during the past decade have led to a much more detailed understanding of the potential negative consequences of species introductions and the potential for negative ecological consequences of invasions by mycorrhizal fungi is poorly understood. Schwartz et al, (2006) recommend that a careful assessment documenting the need for inoculation, and the likelihood of success, should be conducted prior to inoculation because inoculations are not universally beneficial. Agricultural practices such as crop rotation, tillage, weed control and fertilizer apllication all produce changes in the chemical, physical and biological soil variables and affect the ecological niches available for occupancy by the soil biota, influencing in different ways the symbiosis performance and consequently the inoculum development, shaping changes and upset balance of native populations. The molecular biology tools developed in the latest years have been very important for our perception of these changes, ensuing awareness of management choice implications in AM development. In this context, for extensive farming systems and regarding environmental and economic costs, the identification of agronomic management practices that allow controlled manipulation of the fungal community and capitalization of AM mutualistic effect making use of local inoculum, seem to be a wise option for mycorrhiza promotion and development of sustainable crop production

Analysis of paternal lineages in Brazilian and African populations

The present-day Brazilian population is a consequence of the admixture of various peoples of very different origins, namely, Amerindians, Europeans and Africans. The proportion of each genetic contribution is known to be very heterogeneous throughout the country. The aim of the present study was to compare the male lineages present in two distinct Brazilian populations, as well as to evaluate the African contribution to their male genetic substrate. Thus, two Brazilian population samples from Manaus (State of Amazon) and Ribeirão Preto (State of São Paulo) and three African samples from Guinea Bissau, Angola and Mozambique were typed for a set of nine Y chromosome specific STRs. The data were compared with those from African, Amerindian and European populations. By using Y-STR haplotype information, low genetic distances were found between the Manaus and Ribeirão Preto populations, as well as between these and others from Iberia. Likewise, no significant distances were observed between any of the African samples from Angola, Mozambique and Guinea Bissau. Highly significant Rst values were found between both Brazilian samples and all the African and Amerindian populations. The absence of a significant Sub-Saharan African male component resulting from the slave trade, and the low frequency in Amerindian ancestry Y-lineages in the Manaus and Ribeirão Preto population samples are in accordance with the accentuated gender asymmetry in admixture processes that has been systematically reported in colonial South American populations

Results of a Zika Virus (ZIKV) Immunoglobulin M-Specific Diagnostic Assay Are Highly Correlated With Detection of Neutralizing Anti-ZIKV Antibodies in Neonates With Congenital Disease.

:  Usually, immunoglobulin M (IgM) serologic analysis is not sufficiently specific to confirm Zika virus (ZIKV) infection. However, since IgM does not cross the placenta, it may be a good marker of infection in neonates. :  We tested blood from 42 mothers and neonates with microcephaly and collected cerebrospinal fluid (CSF) specimens from 30 neonates. Molecular assays were performed for detection of ZIKV, dengue virus, and chikungunya virus; IgM enzyme-linked immunosorbent assays and plaque-reduction neutralization tests (PRNTs) were performed to detect ZIKV and dengue virus. No control neonates without microcephaly were evaluated. :  Among neonates, all 42 tested positive for ZIKV IgM: 38 of 42 serum specimens (90.5%) were positive, whereas 30 of 30 CSF specimens (100%) were positive. ZIKV IgM-specific ELISA ratios, calculated as the mean optical density (OD) of the test sample when reacted on viral antigen divided by the mean OD of the negative control when reacted with viral antigen, were higher in CSF specimens (median, 14.9 [range, 9.3-16.4]) than in serum (median, 8.9 [range, 2.1-20.6]; P = .0003). All ZIKV IgM-positive results among the neonates were confirmed by the detection of neutralizing antibodies. Mother/neonate pairs with primary ZIKV infection had neutralizing antibodies to ZIKV only, and mother/neonate pairs with ZIKV virus infection secondary to infection with another flavivirus had high titers of neutralizing antibodies to ZIKV. Among secondary infections, median titers in serum were 2072 (range, 232-12 980) for mothers and 2730 (range, 398-12 980) for neonates (P &lt; .0001), and the median titer in CSF was 93 (range, 40-578) among neonates (P &lt; .0001). :  Among neonates, detection of ZIKV IgM in serum is confirmatory of congenital ZIKV infection, and detection of ZIKV IgM in CSF is confirmatory of neurologic infection. Therefore, we recommend testing for ZIKV IgM in neonates suspected of having congenital ZIKV infection and performance of PRNTs in equivocal cases.<br/

Comparison of two modes of vitamin B12 supplementation on neuroconduction and cognitive function among older people living in Santiago, Chile: a cluster randomized controlled trial. a study protocol [ISRCTN 02694183]

BACKGROUND: Older people have a high risk of vitamin B12 deficiency; this can lead to varying degrees of cognitive and neurological impairment. CBL deficiency may present as macrocytic anemia, subacute combined degeneration of the spinal cord, or as neuropathy, but is often asymptomatic in older people. Less is known about subclinical vitamin B12 deficiency and concurrent neuroconduction and cognitive impairment. A Programme of Complementary Feeding for the Older Population (PACAM) in Chile delivers 2 complementary fortified foods that provide approximately 1.4 μg/day of vitamin B12 (2.4 μg/day elderly RDA). The aim of the present study is to assess whether supplementation with vitamin B12 will improve neuroconduction and cognitive function in older people who have biochemical evidence of vitamin B12 insufficiency in the absence of clinical deficiency. METHODS: We designed a cluster double-blind placebo-controlled trial involving community dwelling people aged 70-79 living in Santiago, Chile. We randomized 15 clusters (health centers) involving 300 people (20 per cluster). Each cluster will be randomly assigned to one of three arms: a) a 1 mg vitamin B12 pill taken daily and a routine PACAM food; b) a placebo pill and the milk-PACAM food fortified to provide 1 mg of vitamin B12; c) the routine PACAM food and a placebo pill.The study has been designed as an 18 month follow up period. The primary outcomes assessed at baseline, 4, 9 and 18 months will be: serum levels of vitamin B12, neuroconduction and cognitive function. CONCLUSIONS: In view of the high prevalence of vitamin B12 deficiency in later life, the present study has potential public health interest because since it will measure the impact of the existing program of complementary feeding as compared to two options that provide higher vitamin B12 intakes that might potentially may contribute in preserving neurophysiologic and cognitive function and thus improve quality of life for older people in Chile. TRIAL REGISTRATION: ISRCTN: ISRCTN02694183