In the frame of modern agriculture facing the predicted increase of population and general environmental changes, the securement of high quality food remains a major challenge to deal with. Vegetable crops include a large number of species, characterized by multiple geographical origins, large genetic variability and diverse reproductive features. Due to their nutritional value, they have an important place in human diet. In recent years, many crop genomes have been sequenced permitting the identification of genes and superior alleles associated with desirable traits. Furthermore, innovative biotechnological approaches allow to take a step forward towards the development of new improved cultivars harboring precise genome modifications. Sequence-based knowledge coupled with advanced biotechnologies is supporting the widespread application of new plant breeding techniques to enhance the success in modification and transfer of useful alleles into target varieties. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 system, zinc-finger nucleases, and transcription activator-like effector nucleases represent the main methods available for plant genome engineering through targeted modifications. Such technologies, however, require efficient transformation protocols as well as extensive genomic resources and accurate knowledge before they can be efficiently exploited in practical breeding programs. In this review, we revise the state of the art in relation to availability of such scientific and technological resources in various groups of vegetables, describe genome editing results obtained so far and discuss the implications for future applications. Vegetable crops include a large number of species belonging to various families, characterized by multiple geographical origins, large genetic variability and diverse reproductive features. As result of natural and artificial selection, various vegetables are differentially used worldwide for many purposes, either as fresh or processed products. Due to their nutritional value, vegetables have an important place in human diet, providing, in combination with freshness and taste, protection against various non-transmissible diseases and reduction of the so-called “hidden hunger” especially in developing countries .in situ and ex situ by private or public institutions. In addition, local vegetable varieties (landraces) are still grown and reproduced by farmers, based on their adaptation to specific environments and on the presence of valuable traits. New genomics and phenomics tools allow the throughout characterization of such resources and should facilitate their better use in breeding. Nonetheless, the overall level of biodiversity in horticultural systems is decreasing, with threats to their economic, social, and environmental sustainability . Such approaches, however, require efficient transformation protocols as well as extensive genomic resources and accurate knowledge before they can be efficiently exploited in practical breeding programs. In this review, we revise the state of the art in relation to availability of such scientific and technological resources in various groups of vegetables, describe genome editing results obtained so far and discuss the implications for future applications.New plant breeding techniques, in particular gene transfer based on cisgenic approaches and next-generation precision genome engineering relying on genome editing technologies, can play a key role in accessing genetic resources and using them in functional studies and streamlined breeding strategies   and direct methods  or somatic embryogenesis  and, to a lesser extent, tomato (S. lycopersicum) have been successfully subjected to genetic transformation with various purposes using different approaches (in vitro systems is well known (Rg1 gene involved in regeneration capacity has been transferred from S. peruvianum into S. lycopersicum and subsequently mapped on chromosome 3 (Capsicum spp.) is considered a recalcitrant species due to several issues that jeopardize the in vitro response  followed by regeneration through organogenesis, is strongly dependent on the genotype and various other factors, being B. rapa genotypes more difficult to transform than B. oleracea ones, and cabbage (B. oleracea var. capitata) the most difficult type within the latter. A strong genetic component with significant additive effects both for A. tumefaciens susceptibility and regeneration ability from cotiledonary explants as well as from leaf protoplasts has been found in B. oleracea . Due toin vitro , includiin vitro , the Agrin vitro , the inoin vitro .in vitro regeneration and not amenable to Agrobacterium-mediated genetic transformation. Subsequently, however, several approaches have been developed . In onithe year . Using tthe year . Agrobaclantlets . Finallyolistics , but regin vitro culture and genetic transformation protocols for various vegetables is summarized in Table 1.The availability of Next-Generation Sequencing technologies have determined a significant advancement in data generation. Indeed, large datasets are now being generated across various model and non-model plant species by sequencing whole genomes and/or by applying genome reduction strategies . The list of vegetable crops with publicly available complete or draft genome sequences is getting rich very quickly .1 (S. pimpinellifolium) were published in 2012  as well as of the cultivated pepper Zunla-1 (C. annuum L.) and its wild progenitor Chiltepin (C. annuum var. glabriusculum) have been released into the public domain together with re-sequencing data of cultivated, semi-wild/wild accessions  and melon (C. melo L.) as well as the draft genome of watermelon (C. lanatus) and zucchini (C. pepo) are already available  genome sequence3.Within the Cucurbitaceae family, the complete genome of cucumber (vailable 2. In caserformed  providinerformed  and meloerformed . The latC. cardunculus var. scolymus), a species belonging to the Asteraceae, was released in 2016 (Lactuca sativa) and chicory (Cichorium intybus) genomes has been announced4  genome is now available  genomes of two genotypes from Andean and Mesoamerican gene pools (5). Several GWAS have been performed in common bean to explore broader genetic diversity in order to establish marker-trait associations for future application in breeding programs (Common bean (ne pools  have beeprograms .Allium sativum L.), onion (A. cepa L.), and asparagus  are limited because of their large, extremely complex, repetitive, and often polyploid genomes and long generation times. Nevertheless, the Sequon – Onion Genome Sequencing project is underway6 with the main goal to generate a high quality sequence for the gene-rich regions of a doubled haploid (DH) onion line as a reference for the Amaryllidaceae family.Knowledge on Amaryllidaceae and Asparagaceae genomes is scarce compared to Solanaceae and Cucurbitaceae. Genomic resources for garlic  or clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 endonuclease system (CRISPR/Cas9).Current genome editing approaches rely on the induction of cuts in double-strand DNA , which are then “repaired” through two different processes: non-homologous end joining (NHEJ) or homology-directed repair HDR; . In the PRO) involved in negative regulation of gibberellic acid (GA) signaling. Consistently with the induction of frameshift mutations, loss of DELLA function and increased GA response, plants carrying two different mutant alleles  or the same mutation in homozygous condition showed longer internodes than the wild type and lighter green leaves with smoother margins. Induced pro mutations were transmitted to the progenies according to Mendelian inheritance, while the transgene encoding the TALE nuclease segregated away from the TALEN-induced mutations and was not present in some of the plants showing the mutant phenotype (ARGONAUTE7 (SlAGO7) gene, which through the synthesis of short interfering RNAs causes post-transcriptional silencing of AUXIN RESPONSE FACTOR genes and regulates organ polarity , the remaining being homozygous, biallelic or chimeric for small insertions and deletions. Induced mutations could be transmitted through the germline and the loss of the Cas9 transgene was shown in some progenies. A similar approach has been used in the same work to carry out functional studies in three homologs of the gene Solyc11g064850 involved in multiple aspects of tomato reproductive development. CRISPR/Cas has also been used in functional studies aiming to understand, in a hairy root tissue culture system, the role of the tomato homolog of the transcription factor SHORT-ROOT (SHR), which in Arabidopsis is known to regulate the expression of another transcription factor  and induce a short root phenotype  and sp (self-pruning), involved in photoperiod response, flowering and control of determinate growth. Regardless of day length, “double-determinate CR-sp/sp5g plants” showed an earlier burst of flowering and earliness for fruit set when compared to indeterminate and determinate sp-classic controls  frequency, template and nuclease sequences (either TALEN or CRISPR/Cas9) have been cloned in place of endogenous viral genes encoding coat and movement proteins in a modified Bean Yellow Dwarf Virus (BeYDV) genome. As in previous reports in tomato, DNA delivery in plant cells has been accomplished by Agrobacterium-mediated transformation of cotyledon explants. By employing non-integrating geminivirus replicons GT frequency achieved by TALEN and CRISPR/Cas9 systems was comparable and about one order of magnitude higher than using non-replicating T-DNA vectors. Furthermore, no off-target mutations could be detected.In tomato, TALE nucleases under the control of an estrogen-inducible promoter have been used to knockout one DELLA gene  which, compared with the GA4.a gene, showed two mismatches in target region 2 .In ynthesis . Regenervia organogenesis from mutant calli transmitted the mutations to the progeny.An alternative innovative approach for delivering editing reagents in plant cells has been recently reported in a number of species, including lettuce . In the eIF4E) gene in two sites: in the first case, the gene was completely knocked-down, whereas, in the second, translation of two-thirds of the protein product was still possible (0 plants containing indel and SNP mutations were regenerated and T3 progenies were submitted to virus tests. Non-transgenic homozygous mutant plants showed either immunity or resistance to Cucumber vein yellowing virus (CVYV), Zucchini yellow mosaic virus (ZYMV), and Papaya ring spot mosaic virus-W (PRSV-W), although resistance breaking was observed in some cases. The same plants had no resistance to Cucumber mosaic virus (CMV) and Cucumber green mottle mosaic virus (CGMMV). After specific analyses, no off-target activity could be detected.Finally, the virus resistance of cucumber plants has been investigated after mutating the Eukaryotic translation initiation factor 4E (possible . Chimerivia MAB (marker assisted breeding) programs. These approaches, however, are not free from limitations due to the large number of crosses and derived plants to manage. Artificial mutagenesis, developed in the ‘60, allowed to increase the range of variability available for direct or indirect use, with beneficial effects particularly in vegetatively propagated crops, but the necessity to recover the desirable trait in very large populations composed of plants carrying also many undesirable mutations limits its use.Vegetables include a broad number of species with different reproductive features and genetic structures, which largely affect the access to and the exploitation of genetic variability as well as the efficiency of breeding efforts. In the past decades, genetic improvement programs relied on the selection of superior families or plants from intra-specific or inter-specific cross-derived populations, involving the combination of favorable traits from different genetic backgrounds. The low efficiency of phenotypic selection, especially for quantitatively inherited agronomic traits, has been overcome by the introduction of molecular marker technologies that have improved the efficiency of selection, allowing the detection of specific regions and/or genes to introgress Priorities in the development of novel vegetable cultivars include the improvement of quality traits, the introduction of resistance to a broad range of pests and pathogens, and tolerance to abiotic stresses such as drought, salinity, and low/high temperature. Most of these resistance/tolerance traits need often to be transferred from wild germplasm, taking many generations to remove the deleterious genes that go along with the desirable traits due to linkage drag . In someFigure 2). The possibility to easily recover in regenerated plants or first generation progenies homozygous/biallelic mutations induced by targeted mutagenesis or other editing approaches will ease the production of novel sets of parental lines to be used in F1 hybrid production. An additional advantage consists in the possibility to produce modified plants eventually non-transgenic, by expressing editing reagents transiently or inducing sorting-out of transgenic sequences by segregation or by the use of recombinases  and BeYDV are particularly interesting considering their possible transfer in vegetable host species  with large and small effects on the phenotype. Editing quantitative traits is feasible once the availability of datasets of sequences and phenotypes will enable to discover large numbers of QTNs . A possiTC, NDA, and PT conceived and wrote the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Recently, macropinocytosis has been determined to function as a nutrient-scavenging pathway in Ras-driven cancer cells. Macropinocytic uptake of extracellular proteins, and their further degradation within endolysosomes, provides the much-needed amino acids that fuel cancer cell metabolism and tumor growth. Here, we review the molecular mechanisms that govern the process of macropinocytosis, as well as discuss recent work that provides evidence of the important role of macropinocytosis as a nutrient supply pathway in cancer cells.Oncogenic mutations, such as Ras mutations, drive not only enhanced proliferation but also the metabolic adaptations that confer to cancer cells the ability to sustain cell growth in a harsh tumor microenvironment. These adaptations might represent metabolic vulnerabilities that can be exploited to develop novel and more efficient cancer therapies. Macropinocytosis is an evolutionarily conserved endocytic pathway that permits the internalization of extracellular fluid  Sustained rapid proliferation represents a major metabolic hurdle for cancer cells. The challenge lies in balancing the towering energy and nutrient demands required for biomass production with the harsh nutrient-depleted conditions of the tumor microenvironment. Hence, it is not surprising that tumors have evolved the capacity to employ the same oncogenic signaling pathways  that trigger aberrant growth to also control the metabolic rewiring that is necessary to adapt to a nutrient-deprived ecosystem .Dictyostelium discoideum utilizes macropinocytic uptake to engulf fluid and nutrients during axenic growth P3, and the phospholipid kinases and phosphatases that interconvert them, are important players in the spatiotemporal regulation of macropinocytosis   , 28, as a (PDAC) .Ras activation leads to the stimulation of a plethora of different signal transduction pathways, including Rac, Cdc42, PI3K, and Raf/Erk activation. Activation of Rac by Ras has been shown to have an important role in inducing both membrane ruffling and macropinocytosis in different cell types . Both Ravia p21-activated kinase 1 (Pak1), which has been shown to co-localize with macropinosomes in 3T3 fibroblasts, and can drive macropinocytosis through circular dorsal ruffles when expressed in a constitutively active form , PI3K activity seems to be specifically required for macropinosome closure in tumor cells and macrophages , 20, 21.The fate of macropinosomes after internalization varies depending on the cell type, as they can be recycled to the cell membrane as is the case in A431 cells  or they +/H+ exchangers (NHEs) by amiloride, dramatically inhibit macropinocytosis (N-ethyl-N-isopropyl) amiloride (EIPA) and HOE-694, has been used as a distinctive feature of macropinocytosis  function to maintain the acidic pH of different intracellular organelles such as late endosomes and lysosomes and these proton pumps play a critical role in vesicular trafficking and protein degradation , a key regulator of cell growth that responds to nutrient availability (in vitro, and in hypovascularized, nutrient-poor regions of PDAC tumors (The intracellular degradation of extracellular proteins acquired lability . Lysosomlability , and furlability . On the C tumors . Thus, alex mTORC, a key rvia macropinocytosis, and uptake of these molecules might also contribute to tumor metabolism and growth. For example, tumor cells can internalize and catabolize extracellular matrix molecules, such as fibronectin and collagen (KRAS-mutant A549 lung cancer cells can also macropinocytose extracellular ATP in order to increase the intracellular ATP pool (via macropinocytosis.Finally, molecules other than serum albumin are also internalized collagen , 68. LikATP pool . Ras-driATP pool , 71, andThe critical role that metabolic reprogramming and adaptation strategies play in supporting the growth of tumors is now widely recognized. By inducing the internalization of extracellular proteins, and other macromolecules, that can be further processed and utilized to fuel different metabolic pathways, macropinocytosis provides not only a survival mechanism under nutrient-scarce conditions but also the potential for unrestricted tumor growth in an adverse tumor microenvironment. For this reason, targeting macropinocytosis has emerged as a novel therapeutic strategy that requires further investigation. Understanding the molecular events that drive macropinocytosis in the context of different cancers might inform the design of more specific and potent inhibitors. Given that macropinocytosis is crucial to sustaining tumor growth under nutrient-deprived conditions, it is possible that macropinocytosis inhibition would be particularly beneficial in patients suffering from severely hypoxic or hypovascularized tumors, such as PDAC. Moreover, studies focused on combination therapies employing macropinocytosis inhibitors in conjunction with other metabolic pathway inhibitors could pave the way for improved therapeutic outcomes.MR and CC conceived, organized, and wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The study of feline cancers additionally may generate new insight into underexplored aspects of tumor biology that are less accessible in other species, such as the relationship between chronic inflammation and neoplasia, and the role of viruses in malignant transformation. Several factors that have hindered molecular studies of feline cancers have now been surmounted, with the most fundamental step forward coming from the development of a high-quality reference genome sequence assembly for the cat. This article reviews landmark studies that have led to our current appreciation of feline genome architecture, and outlines techniques used in cancer cytogenomics, from conventional karyotyping analysis through to the development of genomic microarrays and beyond. A summary of progress in the identification and characterization of chromosomal aberrations in feline cancers is provided using examples from studies of injection-site sarcomas, lymphomas and mammary tumors.Relative to the dog, integration of the cat into the “One Health” concept has been more restricted, particularly in the field of molecular oncology. Beyond the continual need to enhance the sophistication of feline healthcare  The domestic cat offers a diverse range of opportunities to contribute to the “One Health” concept, which capitalizes on the integration of complementary human and veterinary biomedical research efforts for synergistic gain. Cats continue to grow in popularity as family-owned pets and benefit from increasingly comprehensive medical surveillance . It has Felis catus, FCA) is organized into 18 pairs of autosomes and two sex chromosomes. Unlike in other mammals, where autosomes are numbered consecutively in descending order of size, standardized nomenclature in the cat takes into account both chromosome size and morphology. The karyotype comprises six autosomal subgroups termed A-F, with between two and four members in each. All but two autosomal pairs, cat chromosomes (FCA) F1 and F2, are biarmed, as are both sex chromosomes. There is substantial variation in chromosome size throughout the karyotype, from the largest  to the smallest  [The genome of the domestic cat  . This ma3, 43Mb) ,7. These3, 43Mb) ,10,11. TThe potential role of the domestic cat in comparative biomedical research was boosted substantially by the generation of a light (~two-fold) coverage feline genome sequence assembly of “Cinnamon”, a female Abyssinian, which captured ~65% of euchromatic sequence . This foIt is well established that the onset of a malignant phenotype is associated with the accumulation of both random and non-random genomic abnormalities ,15. Molein situ hybridization (FISH) analysis, enable both structural and numerical assessment of small numbers of discrete genomic loci  within individual cells. In contrast, indirect methods, such as comparative genomic hybridization (CGH) analysis, permit semi-quantitative measurement of mean DNA copy number within a tumor cell population at many thousands of defined intervals selected throughout the genome, but cannot detect balanced structural rearrangements. Since cancers typically exhibit a combination of both structural and numerical aberrations coupled with cell-to-cell heterogeneity, these highly complementary approaches are often used in tandem. Modified protocols allow their application to challenging specimens, such as those where the availability of clinical material is limited, or where the integrity of the sample is compromised, such as archived histologic specimens. This opens up a multitude of possibilities for retrospective studies of case materials for which detailed clinical histories are available.Techniques for molecular cytogenomic evaluation of tumors fall broadly into two categories , proIt is now 25 years since concerns were first raised over the increasing number of reports of malignant lesions arising at routine vaccination sites in cats . The masInterestingly, histopathologically similar tumors have also been described, infrequently, in dogs, ferrets and rabbits ,24. FurtRecognition of the etiology, incidence and impact of feline ISS has been challenged by difficulty in conclusive distinction between these sarcomas and those arising spontaneously without an apparent underlying injection-related origin. ISS typically are more locally invasive and prone to local recurrence than fibrosarcomas of spontaneous origin (non-ISS). Consequently, ISS generally warrant highly extensive surgical margins followed by regular and meticulous monitoring for recurrence, while non-ISS tumors may respond favorably to more conservative clinical management ,28. The Prior to the genomics era, conventional cytogenetic studies identified highly disrupted karyotypic organization in feline fibrosarcomas, with frequent hyperdiploidy and broad cell-to-cell heterogeneity ,30,31,32versus non-ISS cases. While providing support for molecular discrimination, the limited resolution capabilities of the aneuploidy array confounded the ability to refine the boundaries of these regions and in turn to assess their gene content [Towards this goal, a microarray platform was developed to permit CGH-based identification of recurrent genomic DNA copy number aberrations (CNAs) in feline tumors . This “a content .PTEN and recurrent amplifications of the KIT proto-oncogene. Elevated KIT protein expression has been reported as a persistent finding in both ISS and non-ISS tumors [KIT gene dosage, nor whether this confers potential for utilizing targeted therapies directed towards this receptor tyrosine kinase. ISS and non-ISS tumors showed grossly comparable profiles of DNA copy number imbalance; however ISS cases exhibited elevated levels of global genomic disruption, consistent with their typically more aggressive behavior and poorer outcome. Among these lay a subset of discrete regions with promise as discriminatory markers [A higher-density second-generation cat CGH microarray platform has since been developed utilizing the 14x coverage cat genome sequence assembly, comprising ~110,000 oligonucleotide probes distributed at mean intervals of ~21kb. This provides a >300-fold increase in resolution over the aneuploidy array, bringing feline resources closely in line with those of other model systems for the first time. Evaluation of the same cohort of feline fibrosarcomas with this more comprehensive platform  identifiS tumors ; however markers . With foLymphoma is the most common hematopoietic malignancy of cats and the third most common feline cancer overall, comprising approximately one third of all cancer diagnoses. Moreover, cats are considered to show the highest relative incidence of lymphoma among all species studied to date (reviewed in ), offeriMYC proto-oncogene and deletion of PTEN. Efforts to identify candidates for molecular distinction between histologic and immunophenotypic subtypes are now underway [Extensive volumes of clinically predictive cytogenomic data are accumulating for nodal lymphomas in human patients, and more recently also in dogs ,44. In cunderway . Mammary tumors represent the third most commonly diagnosed feline neoplasm, the vast majority of which (in contrast to dogs) are malignant, primarily simple carcinomas. Feline mammary carcinomas (FMCs) generally are highly aggressive, infiltrative and metastatic and show extensive heterogeneity in outcome that is not readily predicted at the time of diagnosis . FMC shaDiagnostic and prognostic descriptions for FMC frequently are limited in detail since there is little evidence to associate specific histomorphologic subtypes with outcome. In HBC histologic grade, typically defined using the Elston and Ellis scoring system, is one of the most robust prognostic factors . AlthougERBB2 gene encoding the receptor tyrosine kinase HER2/neu, of which >90% exhibit DNA copy number amplification of this region [®), a monoclonal antibody targeted against the HER2/neu receptor. Published estimates of HER2-positive FMCs by immunohistochemistry show tremendous variation between studies, largely due to the use of diverse patient cohorts and contrasting analytical techniques [Genome-wide surveys of HBC have identified discrete genomic signatures that define clinically predictive subtypes, leading to effective treatment strategies based on molecular pathogenesis . Assessms region . This abchniques . ConsequERBB2 was >3-fold more common in high-grade versus intermediate-grade tumors. The incidence of PTEN deletion was also increased in high-grade tumors, correlating with an earlier study that showed inferior outcome in FMC cases with decreased PTEN protein expression [ERBB2 and deletion of PTEN are among the most recurrent genomic imbalances in HBC and are associated with concomitant disregulation of gene expression and anti-apoptotic activity, and in turn with inferior outcome [At the genome-wide level, early conventional chromosome banding studies of FMC identified highly disrupted karyotypes, including recurrent deletions of FCA B2 and gains of FCA E3 ,66. Thespression . Gain of outcome . ContinuThe domestic cat is becoming increasingly well placed to stimulate new advances in molecular medicine. The unique range and distribution of cancers diagnosed in our feline companions fills a series of critical niches that have confounded progress in human medicine. Included among this diverse spectrum are those with direct human counterparts that have remained understudied due to their rarity in people, while others offer a means to study discrete facets of tumor biology that are largely peculiar to the cat. We are now in a position to perform rapid and comprehensive interrogation of normal and aberrant feline genomes and to interpret our observations directly in terms of gene content and potential dysfunction. In turn we have the ability to extrapolate with relative ease between the genomes of different species in the search for conserved molecular features that suggest a common disease mechanism. Given the gross similarity in the structural organization and evolutionary history of the human and cat genomes, it is conceivable that an integrated cytogenomic approach may help to reveal as yet unrecognized mechanisms of tumor-associated chromosome instability.The field of personalized medicine continues to make tremendous strides in the human field, and opportunities to integrate similar concepts into the veterinary arena are gradually becoming a reality. Cytogenomic techniques are continuing to evolve, with increases in resolution, sensitivity, flexibility, throughput and cost-effectiveness, coupled with the ability to combine related techniques into single platforms that allow synergistic acquisition of data from complementary approaches in a single experiment. In the human field, cancer cytogenomic discovery is gradually transitioning from chromosome- and microarray-based techniques toward the use of next generation sequencing approaches . These s


Eleusine coracana (L.) Gaertn.], a crop with inherently higher Ca content in its grain, is an excellent candidate for understanding genetic mechanisms associated with Ca accumulation in grain crops. Such knowledge will also contribute toward increasing Ca contents in other staple crops consumed on daily basis using plant-breeding  methods. However, developing Ca-biofortified finger millet to reach nutritional acceptability faces various challenges. These include identifying and translating the high grain Ca content to an adequately bioavailable form so as to have a positive impact on Ca malnutrition. In this review, we assess some recent advancements and challenges for enrichment of its Ca value and present possible inter-disciplinary prospects for advancing the actual impact of Ca-biofortified finger millet.Humans require more than 20 mineral elements for healthy body function. Calcium (Ca), one of the essential macromineral, is required in relatively large quantities in the diet for maintaining a sound overall health. Young children, pregnant and nursing women in marginalized and poorest regions of the world, are at highest risk of Ca malnutrition. Elderly population is another group of people most commonly affected by Ca deficiency mainly in the form of osteoporosis and osteopenia. Improved dietary intake of Ca may be the most cost-effective way to meet such deficiencies. Finger millet [ Calcium (Ca) is the fifth most abundant element present in the human body, accounting for up to 1.9% of the body weight in adults Nordin, . Its maiGiven its importance, authorities like the Food and Agriculture Organization (FAO) of the United Nations have set up a recommended daily intake (RDI) of Ca based on age, life stage and gender . The World Health Organization (WHO) has declared osteoporosis as the next main public healthcare concern globally, after cardiovascular diseases (CVDs), inflicting almost 75 million people in Europe, the United States of America and Japan alone  deficiency is almost physically undetectable and difficult to diagnose during preliminary stages  Committee to Review Dietary Reference Intakes for Vitamin D and Calcium, www.copenhagenconsensus.com). As it involves the already established agricultural systems to grow, breed and distribute nutrient-dense staple crops, it can be the most economically and socially feasible approach to integrate nutrition into the diets of the impoverished. Biofortified staple crops have been quantified to have high potential benefits and cost effectiveness as their cost per disability-adjusted life years (DALYs) saved is less than the national per capita income, when compared to other interventions, such as fortification and supplementation , regular consumption of finger millet during and after pregnancy as well as lactation can provide significant benefits to maternal and child bone health. Another advantage of Ca-enriched finger millet over expensive commercially fortified foods is its affordability to these malnourished areas. For low-income households which mostly subsist on starchy and bulky foods like rice and cassava for their calorie requirements (https://www.devex.com/news/better-crops-for-better-nutrition-86583), finger millet ensures a pragmatic solution that no family member  suffers from Ca deficiency.Therefore, integration of a naturally Ca-rich crop like finger millet in global biofortification programs can be a good starting point to alleviate Ca malnutrition  and extrinsic factors . Therefore, a better understanding of how these factors influence and impact Ca efficacy becomes essential before the biofortified finger millet can carve a path in the farmer and consumer markets.in vitro Ca solubility and dialysability corresponds to low levels of phytate, oxalate and dietary fiber , medium (2%), and high (3%) tannin sorghum diets showed that as compared to control, the Ca absorption reduced by 1.22, 1.67 and 2.22 fold, respectively  treatment was found to have no effects on the total Ca content in finger millet but lowered the Ca bioaccessibility and polyphenol content by as much as 19 and 22%, respectively , awareness of the limiting factors (enhancers and inhibitors), and prior assessment of efficacy and effectiveness of Ca biofortified food. However, currently the development in this area is extremely limited and developing more nutritious varieties is challenging. Given that millet biofortification has recently been strategized, development of research programs for Ca-biofortified finger millet will have to address some arguments as discussed below.In order to develop Ca biofortified finger millet, nutritionists must have available resources (superior Ca-rich varieties), a well-developed methodology to evaluate the bioaccessibility  and E. coracana sub-species coracana  than the domesticated E. coracana sub species coracana from Kenya (401 mg/100 g) and India (375 mg/100 g)  have proved to be another efficient breeding tool for yield and disease resistance in finger millet. These systems and their subsequent breeding can be used effectively to increase the genetic variance by creating new recombinants and segregating populations by exploiting the genetic background. Therefore, developing genetic resources for finger millet, such as mapping populations, breeding lines and male-sterile mutant lines  markers in finger millet using genotyping-by-sequencing  as well as on a chromosomal scale (synteny). For example, comparative mapping of finger millet to rice has shown a fairly high level of collinearity among these crops , two pore channel (TPC1), calmodulin (CaM), CaM-dependent protein kinases (CaMK1 and CaMK2) and 14-3-3  can aid in a better functional understanding of many candidate genes. Such advancements in gene editing technology along with well-established genetic transformation protocols for generation of transgenic finger millet  and vesicular-arbuscular mycorrhizal (VAM) fungi are generally practiced to enhance the grain yield and growth in finger millet  on Ca homeostasis in finger millet remains unknown. This is a crucial question as the soil-plant interactions substantially affect the available proportion of micronutrient to the roots. In this context, soil medium supplemented with growth promoting rhizobacterium  platforms. Extending the benefits of NGS to finger millet, a recent effort of An extension of MAS, genomic selection (GS) is an upcoming methodology in the area of genomics-assisted breeding . For trapping such useful variations, advances in the next generation sequencing technology must be utilized in generating sufficient number of markers for characterizing marker-trait associations and genomics-assisted breeding. With the implementation of such high-throughput approaches, it will be much easier to investigate the genetic architecture of this trait through comparative mapping in other millets and non-millet species. Mining of markers tightly linked to other traits governing grain Ca content and discovery of underlying genes can be an alternate strategy to develop high Ca finger millet varieties through traditional or modern breeding approaches and transformation-based methods. For example, rather than just arbitrarily increasing grain Ca content, future direction should target improvement in efficiency to mobilize, acquire, transport and store Ca in more bioavailable forms in the edible portions.in vivo Ca bioavailability needs to be cautiously attempted through designing pilot feeding studies for vulnerable groups . Although the biological effects of improved finger millet may be relatively modest, it will have potential benefits in improving healthcare cost savings by reducing the risk of osteoporotic fractures and DALYs lost.From the health perspective, at this point, we almost completely lack the understanding of interplay among grain Ca, other micronutrients and antinutrients metabolism in human body. Any potential risks of reducing the antinutrient content in the grains should be evaluated with a view of their long-term effects on human health. Therefore, the focus should be on demonstrating finger millet's bio-efficacy in order to monitor any potential negative trade-offs and unintended effects. In addition, experimental confirmation of While agronomic factors can influence acceptability of the improved finger millet varieties by farmers, other parameters are important from the view of consumer acceptance. The extent of Ca recommended dietary allowance proportion as well as the sensory satisfaction supported by finger millet is essential in terms of developing food products keeping in line with recent lifestyle changes. Even after successful Ca biofortification of finger millet, its introduction and success as a functional food still entails knowledge of adequate food processing strategies (to minimize the nutrient loss) and study of consumer preferences. Further, communication support and the creation of market demand for its value-added products will be necessary. Therefore, a multi-disciplinary research approach, incorporating nutrition, health, agriculture, along with policy and market research, is needed to ensure the impact of high Ca-biofortified finger millet. Overall, it is worthwhile to conclude that finger millet biofortification will improve the quality of life for both the rural subsistence farming families as well as the consumers.SP and RY conceptualized the manuscript. SP wrote the manuscript. JK, PS, HO, and RY assisted and edited the manuscript. SP, JK, PS, and RS contributed in critically revising the draft and updating the manuscript for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Metformin reduces maternal and neonatal weight gain in gestational diabetes mellitus; however, this effect is poorly investigated in non-diabetic women. We performed this meta-analysis to investigate the effect of metformin intake during pregnancy on maternal and neonatal outcomes in obese non-diabetic women. We searched Medline, EMBASE, and Cochrane CENTRAL for eligible randomized controlled trials addressing the efficacy of metformin in pregnant obese non-diabetic women. Data were extracted and analyzed using RevMan software (Version 5.3). Neonatal birth weight was the key outcome. Secondary outcomes included maternal weight gain, the incidence of preeclampsia, and neonatal adverse effects .Pooled data from two RCTs (n=843) showed that metformin caused a significant reduction in maternal gestational weight gain , compared to placebo. The summary effect-estimate did not favor either of the two groups in terms of reduction of neonatal birth weight Z score . Metformin was associated with 41% reduction in the risk of preeclampsia; however, this reduction was not statistically significant ). None of the neonatal adverse events including stillbirth ) and congenital anomalies  differed significantly between the two groups.For obese pregnant women, metformin could decrease gestational weight gain with no significant reduction in neonatal birth weight. In light of the current evidence, metformin should not be used to prevent poor pregnancy outcomes in obese non-diabetic women. Maternal obesity during pregnancy is a major health problem that affects 25% of pregnant women . It is aFormer studies have shown that following dietary modifications and lifestyle interventions did not significantly reduce obesity-related complications during pregnancy -15. ReceOther studies examined the effect of metformin on pregnancy outcomes in women with polycystic ovarian syndrome , 22. RecIn light of these controversial findings, we performed a pooled analysis of both RCTs to evaluate the effect of metformin on maternal and fetal outcomes in obese non-diabetic women.We followed the PRISMA statement guidelines during the preparation of this meta-analysis. Moreover, all steps were performed in a strict accordance with the Cochrane handbook for systematic reviews of interventions (updated March 2011) .Literature search strategy We searched Medline (through PubMed), EMBASE, and the Cochrane Central Register of Controlled Trials to identify relevant studies published up to April 2016. The following search terms were used: ((Metformin) AND (Obese OR Overweight) AND (Pregnant OR Pregnancy OR Gestation*) AND (Non-diabetic OR Without diabetes)). Moreover, we scanned the reference lists of retrieved articles and checked the clinical trial registry  for additional studies.Criteria for considering studies for this reviewStudies were eligible if they were controlled clinical trials, which compared metformin to placebo in obese, pregnant, non-diabetic women. Only studies reporting the results of infant birth weight, maternal weight gain, or birth consequences were included. Excluded studies comprised pharmacokinetic analysis of metformin during pregnancy, reviews, secondary data analysis, and studies with unspecified data collection and analysis methods. Three authors  independently applied the selection criteria. The abstract screening was performed first, and then the full-text articles of eligible abstracts were retrieved and screened for eligibility to meta-analysis.Data extractionet al : a) Study characteristics, including first author's name, publication year, sample size, mean age, dosage, and duration of metformin intake; b) The primary outcome measure was the neonatal birth-weight Z score, which was defined as the difference between observed and expected birth weight with adjustment for gestational age, divided by the fitted standard deviation (SD); c) Maternal secondary outcome measures included gestational weight gain, defined as the difference in maternal weight between the day of randomization and the last antenatal visit; d) Maternal adverse events including preterm birth before 37 wk of gestation, gestational DM, preeclampsia, pregnancy-induced hypertension, delivery by CS, postpartum hemorrhage, defined as blood loss of 1 L or more, and e) Neonatal adverse events including death before 24 wk of gestation, stillbirth at 24 wk of gestation or later, congenital anomalies, and neonatal death. When the mean and SD were not provided, we calculated them from the median and inter-quartile range according to Wan et al . Risk of bias assessment in included studiesThe risk of bias in retrieved RCTs was assessed according to the Cochrane handbook of systematic reviews of interventions 5.1.0 (updated March 2011). We used the risk of bias assessment table provided in  of the same book . AccordiData synthesisWe used Review Manager (RevMan) software (version 5.3 for windows) during data synthesis. Changes in the primary and secondary outcomes were pooled as mean difference (MD) in a fixed effect meta-analysis model, using the inverse variance (IV) method. Adverse events were pooled as risk ratios (RR) in a fixed effect model using Mantel-Haenszel (M-H) method. The existence of heterogeneity was assessed by Chi-Square test and measured by I-square test. In the case of a significant heterogeneity (Chi-Square p<0.1), the random effects model was used.Literature search results2 and normal glucose tolerance. The summary of included studies and their main results are shown in Our search retrieved 150 unique articles. Following the abstract screening, only 11 references were eligible for full-text screening. Nine full-text articles were excluded as follows: Two single arm/irrelevant trials, three pharmacokinetic analyses, two literature reviews, and two case reports (29-37). Finally, two RCTs were found to be eligible for the final analysis  and blinding of outcome assessors . The summary of the risk of bias assessment domains is shown in Both included studies were of a low risk of bias in all domains, except for allocation concealment .In comparison with placebo, metformin did not show a significant reduction of neonatal birth weight Z score .The overall MD favored the metformin group over the placebo group in terms of maternal gestational weight gain .The total number of reported maternal adverse events did not differ significantly between the metformin and placebo groups . The pooled RRs for individual adverse events were as follows: Postpartum hemorrhage . The pooled RRs for individual adverse events were as follows: fetal death in terms of miscarriage and stillbirth . To account for between-study variability, we re-conducted the analysis of all fixed-effect outcomes under the random-effects model with no recorded difference in our results .The results of our analysis indicate that using metformin in obese, non-diabetic pregnant women significantly reduced maternal weight gain; however, it was not associated with a significant reduction in neonatal birth weight. The incidence of gestational DM, preeclampsia, as well as other maternal and fetal adverse events, did not differ significantly between the metformin and placebo groups. Our analysis supports the results of the MOP trial regarding the effect of metformin on maternal weight gain. This can be explained by the fact that the MOP trial was designed to address the limitations of the former EMPOWaR trial by increasing adherence to treatment and using a higher metformin dose of 3 grams per day instead of 2.5 gr/day in the EMPOWaR study. Observational studies have also shown that metformin can significantly reduce maternal weight gain in women with gestational DM or polycystic ovarian syndrome , 20, 38.On the other hand, our results are in agreement with the EMPOWaR trial regarding the lack of metformin efficacy in reducing the incidence of preeclampsia. Although metformin reduced the levels of interleukin-6 (IL-6) and C-reactive protein (CRP), which are commonly elevated in preeclampsia cases, the incidence of pre-eclampsia was similar in both arms of the EMPOWaR study . SimilarFormer pharmacological analyses have shown that metformin has a similar pharmacokinetic profile in both pregnant and non-pregnant women with the ability to cross the placenta, reaching similar concentrations in the fetal plasma to those in the maternal circulation . RegardiAlthough metformin conducted its pharmacodynamic effect (lowering blood glucose and insulin concentrations) in enrolled patients of both studies, it did not significantly affect the neonatal birth weight. As confirmed by the EMPOWaR trial, these results present a challenge to the 1952 Pederson theory that states that maternal hyperglycemia stimulates fetal hyperinsulinemia and therefore, increasing fetal weight . Other fDespite the lack of effect on neonatal birth weight, follow up of these siblings should be carried out to track the effect of metformin in their adult life. A former animal study showed that prenatal intake of metformin reduced the risk of obesity and glucose intolerance during adulthood (48). Moreover, a clinical trial on metformin in women with gestational DM showed that children, born to mothers who received metformin during pregnancy, had lower visceral fat at two years of age than children, born to mothers with insulin therapy during pregnancy . The mecStrength points: The large sample size of the included studies adds to the power of our analysis and increases its potential for generalizability. Moreover, including a widely heterogenous population in the MOP trial may allow for generalizing the results of this analysis beyond the studied population.LimitationLimitations for pooling data to summarize few studies include selective reporting, inadequate accounting for heterogeneity and publication bias. The fact that the MOP trial used a higher metformin dose on a more diverse population than the EMPOWaR trial may serve as another limitation. Further studies are warranted to verify the impact of metformin on both maternal and neonatal weights. Although not significant, few adverse events, such as preeclampsia, need for caesarian section, and neonatal death had a trend towards increasing in the metformin group; therefore, future studies are needed to further assess these outcomes. We are aware of another ongoing trial on the subject (ACTRN12612001277831).RecommendationsDespite the absence of major complications in the metformin groups, minor adverse events as gastrointestinal dysregulation, commonly seen with metformin, limit participation in these clinical trials. Finding ways to improve the pharmacokinetic profile of metformin can effectively ameliorate these side effects and increase patients' participation in future studies. As stated earlier, the safety and efficacy of metformin should be followed-up in both mothers and siblings to evaluate the long term results of metformin use in pregnancy.To recapitulate, metformin intake during pregnancy in obese, non-diabetic pregnant women reduces maternal weight gain without significant reduction of neonatal birth weight. In light of the current evidence, metformin should not be used to prevent poor pregnancy outcomes in obese non-diabetic women.The authors have no conflicts of interest to declare.


Chronic pain can result from many pain syndromes including complex regional pain syndrome (CRPS), phantom limb pain and chronic low back pain, among others. On a molecular level, chronic pain syndromes arise from hypersensitization within the dorsal horn of the spinal cord, a process known as central sensitization. Central sensitization involves an upregulation of ionotropic and metabotropic glutamate receptors (mGluRs) similar to that of long-term potentiation (LTP). Regions of the brain in which LTP occurs, such as the amygdala and hippocampus, are implicated in fear- and memory-related brain circuity. Chronic pain dramatically influences patient quality of life. Individuals with chronic pain may develop pain-related anxiety and pain-related fear. The syndrome also alters functional connectivity in the default-mode network (DMN) and salience network. On a cellular/molecular level, central sensitization may be reversed through degradative glutamate receptor pathways. This, however, rarely happens. Instead, cortical brain regions may serve in a top-down regulatory capacity for the maintenance or alleviation of pain. Specifically, the medial prefrontal cortex (mPFC), which plays a critical role in fear-related brain circuits, the DMN, and salience network may be the driving forces in this process. On a cellular level, the mPFC may form new neural circuits through LTP that may cause extinction of pre-existing pain pathways found within fear-related brain circuits, the DMN, and salience network. In order to promote new LTP connections between the mPFC and other key brain structures, such as the amygdala and insula, we propose a holistic rehabilitation program including cognitive behavioral therapy (CBT) and revolving around: (1) cognitive reappraisals; (2) mindfulness meditation; and (3) functional rehabilitation. Unlike current medical interventions focusing upon pain-relieving medications, we do not believe that chronic pain treatment should focus on reversing the effects of central sensitization. Instead, we propose here that it is critical to focus on non-invasive efforts to promote new neural circuits originating from the mPFC. Recent advances in brain imaging techniques have made it possible to devise explanatory mechanisms for the development and maintenance of chronic pain. Amongst many other medical achievements, magnetic resonance imaging (MRI) is used to visualize the anatomical integrity of the spine and its associated structures. In particular, MRI has become an imaging technique used to verify clinical presentations for disc herniation. Despite this advance in the ability to visualize nervous system structures with high clarity, questions remain as to whether MRI findings coincide with clinical pain pathology and whether MRI has value in the diagnosis and therapeutic management of chronic pain  and the resulting decrease in the overall inhibitory function of GABAA receptors facilitates central sensitization , a diverse family of cell receptor proteins that are dispersed throughout the peripheral and central nervous system. GPCRs are located on the plasma membranes and nerve terminals of sensory neurons along nociceptive pain pathways  in cortical and subcortical brain regions , the amygdala and the hippocampus  phantom limb pain, complex regional pain syndrome (CRPS), musculoskeletal pain and post-surgical pain Woolf, . In ordeResearch designs utilizing functional MRI (fMRI) to examine the functional neural correlates of chronic pain can be categorized into three primary groups: (1) pain-induction trials; (2) resting state, in which no active cognitive or sensorimotor task is performed; and (3) active tasks that require perceptual, sensorimotor, or other cognitive and behavioral processes. The latter category may also include simple motor tasks, such as finger tapping, to serve as control or comparison conditions. During a resting state, researchers hope to identify underlying brain mechanisms that persist in chronic pain patients even after a painful stimulus is removed. The brain regions showing increased activation during wakeful rest, when compared with task-active states, have been described as the brain’s DMN  task-free or task-independent trials in which the subject is not assigned any stimulus or task; (2) simple sensorimotor and cognitive tasks; and (3) complex sensorimotor and cognitive tasks. By comparing either task-free designs or simple sensorimotor tasks to complex cognitive tasks, researchers can examine how brain regions in the DMN change from a resting state to a task-activated state.Structures associated with the DMN include the mPFC, medial temporal lobes (including the hippocampus) and posterior cingulate cortex  system allows for the bidirectional control of pronociception (nociceptive facilitation) and antinociception  is associated with central sensitization Woolf, . In addiInterestingly, Hashmi and associates  cognitive reappraisals; (2) mindfulness meditation; and (3) functional rehabilitation. We further hypothesize that a multimodal treatment program will lead to increased connectivity between mPFC and other cortical/subcortical regions, such as insula and amygdala, compared with any one of these therapies alone. To test this hypothesis, we suggest the implementation of an 8- to 12-week four-arm randomized clinical trial for patients experiencing chronic pain. To attain sufficient power for detecting differences among groups, 120 patients should be randomized to one of the following conditions: CBT alone, mindfulness meditation alone, functional rehabilitation alone, or combined multimodal therapy including CBT, mindfulness and functional rehabilitation. The randomization process should lead to approximately 30 patients per group, which is an adequate sample size even with the likelihood of patient drop-out and issues with image acquisition, such as participant motion or other artifact.For CBT, we recommend a program consisting of weekly 45-min sessions comprised of: psychoeducation regarding the learned mechanisms of avoidance, cognitive restructuring through reappraisal, attention diversion and self-regulatory skills. Although prior studies have utilized a group approach to CBT  scanning series, if available.We acknowledge the ambitious nature of this proposed clinical trial, requiring many hours of therapeutic services and a total of 240 MRI scanning sessions. Therefore, we recommend a multi-site approach to this intensive investigation. Despite the likelihood that such a study might take a few years to complete, the knowledge gained through this study will have lasting impacts on treatment recommendations for chronic pain.JG conceived the primary hypothesis put forward in this article. Both JG and KS contributed to searching prior literature, and both authors contributed to the writing and editing of this manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Severe chronic pain is one of the hallmarks and most debilitating manifestations of inflammatory arthritis. It represents a significant problem in the clinical management of patients with common chronic inflammatory joint conditions such as rheumatoid arthritis, psoriatic arthritis and spondyloarthropathies. The functional links between peripheral inflammatory signals and the establishment of the neuroadaptive mechanisms acting in nociceptors and in the central nervous system in the establishment of chronic and neuropathic pain are still poorly understood, representing an area of intense study and translational priority. Several well-established inducible and spontaneous animal models are available to study the onset, progression and chronicization of inflammatory joint disease, and have been instrumental in elucidating its immunopathogenesis. However, quantitative assessment of pain in animal models is technically and conceptually challenging, and it is only in recent years that inflammatory arthritis models have begun to be utilized systematically in experimental pain studies using behavioral and neurophysiological approaches to characterize acute and chronic pain stages. This article aims primarily to provide clinical and experimental rheumatologists with an overview of current animal models of arthritis pain, and to summarize emerging findings, challenges and unanswered questions in the field. Rheumatoid arthritis (RA) and spondyloarthritis are prevalent inflammatory-erosive joint diseases which affect as many as 2% of the population worldwide, causing severe, debilitating morbidity and major economic costs due to both health care expenditures and lost productivity. Inflammatory arthritides are characterized by progressive joint inflammation and destruction, deformity, loss of mobility, systemic manifestations and severe pain which ultimately hamper basic motility functions, activities of daily living and psychological health in the affected individuals , 2.Therapeutic approaches focused on the underlying inflammatory immunopathology have led to the introduction of targeted biological disease-modifying anti-rheumatic drugs (DMARDs), pioneered by anti-tumor necrosis factor (TNF) agents, which have revolutionized the clinical treatment and dramatically improved long-term outcomes of these diseases , 4.Pain, initially joint localized but often progressing to widespread in advanced stages, is a major component of inflammatory arthritis symptomatology and is typically the primary reason for initial rheumatological referrals , 2. In aThe therapeutic success of biologic DMARDs has provided new insights into the unique qualities of pain manifestations associated with chronic inflammatory arthritis. Notably, patients with a positive response to anti-TNF agents often report rapid initial therapeutic pain suppression which precedes the clinical anti-inflammatory response , 6. ThisMore problematic for the clinical management of these diseases, however, is that a significant fraction of patients fail to report long-term suppression of pain comparable to their anti-inflammatory clinical response to biologic and other DMARDs , 8–10. BFrom the patients’ perspective, effective management of arthritis-associated pain is a primary therapeutic goal, and a major component of patient-driven disease assessment often underestimated by clinical disease activity scoring tools , 10, 11.This review will first describe existing animal models of arthritis that are commonly utilized for preclinical pain studies, discussing their specific advantages and drawbacks. We will then provide key background on experimental methods for quantitative assessment of pain responses in animal models, highlighting important theoretical and practical challenges, and summarizing recent insights into the mechanisms of arthritic pain.Animal disease models have proven invaluable to unravel the pathophysiological pathways of inflammatory arthritis, and for investigational testing of therapeutic agents. The most commonly utilized animal species for this purpose are mice and rats, either as strains that spontaneously develop arthritis or as inducible models in which disease can be provoked by administration of arthritogenic stimuli. A number of comprehensive reviews have already covered the range of animal arthritis models, and their features in comparison to human disease pathophysiology and therapeutic responses –15. HereSeveral rodent strains have been reported to be susceptible to development of spontaneous arthritis, but experimental studies have primarily focused on a few genetically modified mouse strains which display full penetrance and reproducible disease progression, especially K/BxN and TNF-transgenic (TNFtg) mice , 17.7 MHC-II allele [K/BxN mice express a T-cell receptor transgene specific for a peptide derived from the ubiquitous enzyme glucose-6-phosphate isomerase (GPI), presented by the I-AgI allele , 18. AutI allele . HistoloTNFtg mice, derived in the early 1990s, express a human TNF gene lacking post-transcriptional regulatory elements, and have provided cornerstone evidence for the involvement of TNF in inflammatory arthritis . CommonlThe 3647-strain TNFtg mice display delayed onset of joint inflammation compared to multicopy transgenics (6–8 weeks of age vs 3–4 weeks), slower disease progression  and increased lifespans (over a year with appropriate husbandry) . BecauseInflammatory arthritis can be induced experimentally in many species, with well-established systems utilizing both rats and mice. Arthritogenic signals in these models can consist of nonspecific inflammatory agents, such as different types of adjuvants; of immunization procedures using specific antigens which cause self-tolerance breakdown; or of passively administered autoreactive antibodies or sera. Compared to spontaneous disease strains, advantages of inducible models include their cost-effectiveness, reduced husbandry needs and reproducibility of existing protocols. Disease typically develops rapidly, limiting the physiological windows for the study of disease onset and progression. Penetrance, persistence and chronicity of arthritis vary depending upon the model.Arthritis can be reproducibly induced in susceptible strains of rats  by intradermal injection of adjuvants, including complete or incomplete Freund’s adjuvant , pristane and squalene, or intraarticular administration of streptococcal cell wall products or antigens in presensitized rats or mice , 21. In Collagen-induced arthritis (CIA) is the most frequently utilized experimental model of arthritis. Inflammatory arthritis is induced in genetically susceptible rats, mice, rabbits and other species by immunization with type II collagen, typically of bovine origin . In a tyEvidence that serum from collagen-immunized rats and mice could passively induce arthritis in recipients provided early experimental confirmation of the direct pathogenic role of humoral immunity in arthritis. The same approach is commonly utilized to induce acute, transient arthritis in rats or mice, in models such as collagen-antibody-induced arthritis (CAIA) and K/BxN serum-transfer arthritis , 23. ArtCAIA can be induced in either rats or mice by intravenous transfer of a mixture of anti-collagen II monoclonal antibodies, most often accompanied by intraperitoneal injection of LPS to potentiate the effect. Disease onset follows rapidly after LPS injection, reaching maximal severity in 4–5 days and waning in about an additional week. Repeated injections can exacerbate and extend the response.Similarly, transfer of serum, antibodies or anti-GPI monoclonals from K/BxN mice (see earlier) can induce rapid (2–3 days) onset of polyarthritis in recipients, with almost complete penetrance and without additional inducers .Self-reported pain scores are the primary means for evaluating pain severity in patients. However, objective assessment of pain in animals represents a significant challenge for preclinical research, which has led to the development of several experimental systems which reproducibly mimic pathological pain conditions in humans, including inflammatory and neuropathic pain, and allow assessment of their outcomes. Broadly speaking, animal behavioral models of pain consist of two principal components: experimental manipulation intended to produce a pain-like state; and measurement of behavior presumably indicative of that pain state. These models can be used to experimentally assess pain as well as its relief following the administration of antinociceptive drugs.Current methods of pain assessment in animals were initially developed in the context of models of induced acute pain from different types of noxious stimuli, or of neuropathic pain following experimental nerve injury, but the same approaches are routinely applied to arthritis pain models. All of these approaches have to overcome the major conceptual hurdle of translating the subjective experience of pain in animal subjects into investigator-observable, quantifiable responses.Broadly, experimental assessment of pain in animal models relies on the quantification of pain-evoked or pain-suppressed behaviors. Pain-evoked behaviors may occur at very low rates in the absence of pain, and increase in frequency following putative nociceptive stimuli. Examples include footpad withdrawal, jumping, flinching or licking. In contrast, pain-suppressed behaviors are those that occur at high rates in the absence of noxious stimuli and decrease in magnitude or duration after exposure to a noxious stimulus. Some natural behaviors that may be suppressed in this context include locomotor activity, nesting, motor coordination/balance or feeding . In eacThe basic concept of measuring acute nociceptive pain in animals relies on the input of a normally noxious stimulus followed by the assessment of a withdrawal response. The noxious stimulus can vary in intensity and in modality, such as electrical, mechanical, thermal or chemical , 26.The advantage of these models largely rests in their simplicity, their ability to objectively measure the withdrawal response and their predictive value to assess pharmacological effectiveness of opioid analgesics in humans . HoweverClassic models of acute inflammatory pain include the injection into the hind paw of rodents of chemicals , which produce a rapid nociceptive response characterized by paw flinching and licking , and decreased response thresholds to thermal and mechanical stimuli . Neuropathic pain in animals is classically modeled by interventions that cause some degree of nerve injury, such as ligation or chronic constriction. While these systems have good predictive validity in pharmacological studies , they do not replicate the etiology of most human neuropathic states, nor some of their common clinical manifestations , 28.These experimental systems have been instrumental to unraveling basic neurophysiological pathways of nociception and for pharmacological research, but because of their limited clinical applicability they have been progressively complemented by more pathologically relevant models of chronic and/or neuropathic pain, including the inflammatory arthritis models already described.The assessment of pain in animals is naturally fraught with conceptual and experimental complexities, and significant research has been carried out in recent years to standardize protocols, identify the impact of critical variables , reduce investigator-associated subjectivity and disruption, and expand the range of testable responses from strictly sensory to include psychoaffective components , 29–31. The most frequently utilized techniques for assessment of ongoing pain in experimental animals rely on development of hyperalgesia and allodynia, which can be quantitatively measured by assessing withdrawal from non-noxious or subthreshold stimuli. Arguably the most common mechanical stimuli used for this purpose in rodents are von Frey hairs, elastic filaments of varying diameter that buckle at a defined force. Applied to the plantar surface of the paw when the animal is positioned over a wire mesh surface, von Frey hairs of increasing stiffness allow the determination of the mechanical threshold of paw withdrawal. Although less frequently utilized, electrical stimuli can also be applied briefly and quantifiably, and are reproduced easily.Lowered thresholds of evoked responses to stimuli relative to controls are considered hallmarks of allodynia and hyperalgesia. One limitation of these approaches lies in the subjective measurement of withdrawal responses, which can be obviated by use of automated electronic systems , 33. AssNeurophysiologically, the primary caveat of mechanical and electrical stimuli is that they activate both low-threshold mechanoreceptors and nociceptors, preventing a clear-cut distinction of the pathways involved. Thermal stimulation is thought to be more specific in directly activating nociceptive fibers. Commonly used methods involve applying radiant heat or immersing the distal end of the tail of a restrained animal into a thermostatic water bath. Unrestrained animals can be tested using Hargreaves’ method, during which radiant heat is applied to the plantar surface of the footpad via an infrared source to elicit paw withdrawal.Functional magnetic resonance imaging (fMRI) measures changes in paramagnetic signals secondary to oxygen extraction from oxyhemoglobin, reflecting metabolic activity in brain tissue. Human fMRI studies have demonstrated activation in specific brain structures following noxious stimuli, including in the lateral thalamus, primary and somatosensory cortex, insular cortex, anterior cingulate cortex, striatum, cerebellum, supplemental motor area and periaqueductal gray matter . Small aRecent studies have analyzed facial expressions in animals in response to painful stimuli. A 10-point facial expression (“grimace”) scale was developed in mice based on orbital tightening (closed eyelid or eye squeeze), nose bulge, cheek bulge, ear position and whisker changes following intraperitoneal administration of acetic acid . SimilarAlthough pain-evoked behaviors are most commonly utilized in animal pain studies, pain-suppressed behaviors have also been used to assess pain in animals. These are defined by a decrease of otherwise healthy behaviors that occur at high rates  following exposure to a noxious stimulus. These have clinical correlates in human chronic pain patients, where suppressed behaviors may include decreased activities of daily living or ambulation, and correlate with signs of clinical depression. Evidence of pain-suppressed behaviors in animals may be quantified by reduced feeding, reduced mating and/or reduced locomotor activity.Decreased locomotor activity has been associated with pain-like states in animals, including in rodent models of inflammatory and neuropathic pain, although with some discrepancies , 40. As The inclusion of pain-suppressed behaviors in animal testing has several advantages. First, pain-suppressed behaviors can be objectively measured using automated equipment . Second, drugs that produce motor impairment are less likely to produce false positive effects in pain-suppressed assays relative to pain-stimulated behaviors. Third, measures of pain-suppressed behaviors may be used to investigate some of the psychoaffective components of pain and may improve the translational validity of these behaviors toward the clinic. At the same time, the complexities of interpreting animal behavior from a psychoaffective standpoint cannot be understated, and important work needs to be carried out in this area to fully validate these approaches , 30.Similar to study of pain-suppressed behaviors, behavioral methods can be utilized to explore affective and/or motivational changes that occur in response to pain, bypassing some of the problems associated with pain-evoked responses. Operant procedures may require an animal to predictably emit a defined behavioral response, such as traversing a noxious stimulus , in order to obtain in a positive reinforcer  , 42. PlaAlthough animal models of arthritis have been widely utilized for decades to study not just disease pathogenesis and candidate therapeutics, only in the past 10–15 years have the biological properties of pain in these systems begun to be investigated systematically , 47.In all models studied, inflammatory disease is associated with lowered thresholds to mechanical and thermal stimulation, reflecting hyperalgesia and allodynia. However, thermal and mechanical hypersensitivity are not always closely correlated to each other, suggesting that strain-specific and method-specific differences should be considered in evaluating experimental outcomes . In addiStudies focused on the resolution end of the disease spectrum using transient antibody-induced arthritis  have shown that pain sensitization can persist for extended periods of time beyond the resolution of inflammation , 51, 52.Molecular and cellular studies of nociceptors and non-neuronal cell types in dorsal root ganglia (DRG) and spinal cord sensory pathways have begun to elucidate the neurophysiological mechanisms of hyperalgesia in models of arthritis and other inflammatory diseases , 54–56. Pharmacologically, consistent with a neurogenic mechanism, persistent pain in both CAIA and K/BxN mice appears to be alleviated by gabapentin, but not NSAIDs , 51. A rVarious forms of pain-suppressed spontaneous behaviors, including locomotor activity, as well as operant responses and place avoidance have also been studied in arthritis models, showing strong correlation with clinical disease , 60–66. Although no animal model perfectly recapitulates all aspects of human inflammatory arthritis, the diversity of existing models provides a large armamentarium for elucidating specific pathophysiological mechanisms, including the study of arthritic pain. In this respect, important criteria for model selection include relevant pathophysiology and disease kinetics, especially with regard to chronicity. Similarly, the array of systems currently utilized for the experimental evaluation of pain perception in animals—spanning from traditional quantitation of hyperalgesia and allodynia-linked responses, to behavioral studies, to the more recent neuroimaging and neurobiological approaches—offers important experimental opportunities, while also requiring thoughtful consideration of technical and interpretative caveats. Parallel progress in these two fields will greatly broaden our understanding of pain mechanisms beyond what can be achieved in human studies.Key priority targets for this research effort include the mechanisms that establish pain chronicity and the emergence of neuropathy in inflammatory arthritis, which are critical for treatment and prevention of symptom progression. Despite the recent pharmacotherapeutic advances, pain remains a major unresolved need in the management of arthritis patients. Animal models have proven instrumental in providing key insights into the inflammatory pathophysiology of arthritis, leading to the biological therapeutics revolution. Application of the same preclinical approaches has great potential for the replication of this success in treating rheumatoid pain.


The composition of ecological assemblages depends on a variety of factors including environmental filtering, biotic interactions and dispersal limitation. By evaluating the phylogenetic pattern of assemblages, we gain insight into the relative contribution of these mechanisms to generating observed assemblages. We address some limitations in the field of community phylogenetics by using simulations, biologically relevant null models, and cost distance analysis to evaluate simultaneous mechanisms leading to observed patterns of co-occurrence. Building from past studies of phylogenetic community structure, we applied our approach to hummingbird assemblages in the Northern Andes. We compared the relationship between relatedness and co-occurrence among predicted assemblages, based on estimates of suitable habitat and dispersal limitation, and observed assemblages. Hummingbird co-occurrence peaked at intermediate relatedness and decreased when a closely-related species was present. This result was most similar to simulations that included simultaneous effects of phylogenetic conservatism and repulsion. In addition, we found older sister taxa were only weakly more separated by geographic barriers, suggesting that time since dispersal is unlikely to be the sole factor influencing co-occurrence of closely related species. Our analysis highlights the role of multiple mechanisms acting simultaneously, and provides a hypothesis for the potential importance of competition at regional scales. The composition of ecological assemblages depends on diversification of clades in a region, colonization of species from outside that region and the niche overlap among species. When species diverge from a common ancestor, they tend to have a similar set of biotic and abiotic requirements, such that their niches are conserved ,2. NicheA common approach for integrating evolutionary history and community ecology is to compare the phylogenetic structure of observed assemblages to a biogeographic species pool using randomization tests ,11. ThisHere we explore patterns of hummingbird co-occurrence and relatedness in the Northern Andes combining multiple analyses including: simulations, hierarchical models of co-occurrence and relatedness, predicted assemblages based on broad-scale environment, and analysis of habitat connectivity.We simulated assemblages to simultaneously evaluate how different mechanisms influence the phylogenetic structure of assemblages. Simulated assemblages were created based on two mechanisms: phylogenetic conservatism of traits important to environmental filtering, and phylogenetic repulsion based on traits mediating competition ,16. PhylWe used a non-linear hierarchical model to simultaneously evaluate the effect of filtering and competition on patterns of co-occurrence, while allowing for species-level variation. This approach should improve upon previous studies that use randomization tests to identify assemblages as either underdispersed, overdispersed, or indistinguishable from a null expectation. Further, by using a hierarchical model we can evaluate the overall effect of relatedness on patterns of co-occurrence, while allowing for species-level variation. Rather than fitting a model to either an entire assemblage or species individually, a hierarchical model allows data-poor species to borrow strength from more commonly sampled species by allowing variance to be shared among all hummingbirds. This approach allows us to evaluate the shape of the relatedness and co-occurrence curve for each species and compare it to the curve based on predicted environmental suitability. In contrast, randomizations treat assemblages as the fundamental unit which implicitly assumes that all co-occurring species are influenced by the same mechanisms ,18.Evaluation of the phylogenetic structure of an assemblage requires a potential pool of species that could occur in a given location. While past studies have focused largely on geography in constructing species pools, we include information on environmental tolerances and dispersal limitation ,20. We uTo explore our integrated approach, we used hummingbird assemblages from the Northern Andes. This system is relatively well-developed, with community phylogenetic studies finding support for both competition and environmental filtering as potential mechanisms influencing assemblage structure. Initial work using geographic species pools found evidence for phylogenetic clustering, which was attributed to environmental filtering in high elevation assemblages ,23. AcknTo determine the patterns of relatedness and co-occurrence as a function of known mechanisms, we created a range of scenarios for species co-occurrence and phylogenetic relatedness based on the degree of environmental filtering and limiting similarity . These sWe used 230 hummingbird assemblage lists (presence/absence data) from published references, focusing on the Columbian and Ecuadorian Andes and adjacent lowlands . These lFor climate covariates, we used the Worldclim database  at the 0a priori specification of a model by using cross-validation of smaller candidate trees [To predict habitat suitability, we built ensemble models using maximum entropy (MAXENT), boosted regression trees (GBM), and generalized linear models (GLM) (R package biomod2 ). We usete trees . For ourte trees .For model evaluation we split the observed data into 80% training data and 20% testing data and computed area under the curve (AUC) and True Skill Statistic (TSS) as a measures of goodness of fit ,49. ThisWe evaluated dispersal limitation using both an elevation-weighted cost distance metric to infer the current potential for dispersal between localities and time-since-divergence to evaluate the influence of time for dispersal between current localities given historical isolation . Cost distance is an effective predictor of dispersal limitation and population genetic structure, and is a better predictor of hummingbird beta-diversity between localities than Euclidean distance . ComputiNext, for each pair of sister taxa (n = 58), we evaluated the relationship between time since divergence and the mean cost distance between their localities. As described above we used the change in elevation cost surface to calculate the least cost path between each presence locality of one sister taxa to the closest presence locality of the other sister taxa. Assemblages where both taxa co-occur had zero cost distance. We then modeled cost distance between sister taxa and modeled it as a function of time since divergence using linear regression. Given that there are relatively few early branching splits in the hummingbird phylogeny (only 3 splits greater than 10myr) and older splits could present undue leverage, we also evaluated the relationship solely among sister taxa younger than 10 myr.To provide a null hypothesis for the observed pattern of phylogenetic relatedness in the hummingbird assemblages we used biologically motivated species pools. We created two types of null assemblages: environment assemblages and environment + dispersal assemblages. Environment assemblages were based on predicted habitat suitability from bioclimatic models for each species at each geographic location for which we had observed assemblage data. Defining predicted suitable areas required turning the index of suitability returned from the bioclimatic into a statement of predicted presence or absence. Due to differences in species prevalence, taking a fixed probability cutoff across all species would bias presence towards more common species . We therth quantile of the distribution as the maximum cost distance between suitable sites.In the environment + dispersal assemblages, predicted suitable locations were considered unavailable for species’ presence if the cost distance to the closest observed location was higher than a species-specific threshold. This threshold was determined by calculating the cost distance for every observed locality of that species to the closest observed locality. To avoid letting a single assemblage drive this value, we used the 95ρ. The probability of occurrence is a logit transformed function of a species intercept (α), the linear effect of relatedness on occurrence  and the non-linear effect of relatedness on occurrence . We included a polynomial term because we believe multiple mechanisms may shape co-occurrence, leading to a non-linear relationship. We chose a hierarchical model that first fits a distribution to all species responding to the presence of closely related species (‘group level distribution’ (βi).For each of the four simulations, the two predicted assemblages, and the observed data, we compared the relationship between phylogenetic distance to the closest related species and the probability of co-occurrence. The full model reads: the presence of hummingbird species (i) at site (Y) is a Bernoulli trial with a probability of occurrence Normal with variances Gamma). All posterior probabilities for model parameters were estimated using Markov chain Monte Carlo (MCMC) in JAGS. Models were run for 50,000 draws with two chains, thinning by five draws.All model parameters had non-informative priors . Although TSS scores were more variable across modeling types , the correlation between AUC and TSS scores was high across all models and species , suggesting that our evaluation of model performance was consistent across metrics. Twenty-five of the 32 species excluded from the observed assemblage lists occurred in less than 10 localities, and five species occurred in just a single locality. On average observed assemblage lists had 91% completeness, and in 121 of the 230 assemblage lists all species were corrected predicted as present. We therefore limited our analysis of co-occurrence to species shared by all observed, environment and environment + dispersal assemblages.In both the environment and environment + dispersal assemblages, the probability of a species’ occurrence initially increased with decreasing phylogenetic distance to a species in an assemblage, followed by a decrease among closely-related species. The environment + dispersal assemblages showed a lower co-occurrence than environment suggesting that dispersal between localities was a factor in shaping predicted species lists. We compared the environment and environment + dispersal assemblages to our observed assemblage data to determine whether we would have arrived at the observed pattern based solely on environmental tolerances. Similar to the predicted assemblages, the observed assemblages showed an initial increase in the probability of co-occurrence with increasing relatedness, followed by decreased co-occurrence among closely related species . Explora2 = 0.32, df = 27, p = 0.01). For species pairs less than 10 myr, there was only a modest, non-significant, relationship between the time since divergence and average cost distance between localities of sister taxa .To estimate the importance of time on the dispersal on co-occurrence of closely species, we looked at the relationship between time since divergence and the presence of geographic barriers . We founThe relative roles of environmental filtering, dispersal, and competition in promoting the co-occurrence of related species remains a central focus of biogeography. We found the relationship between co-occurrence and relatedness was non-linear; the probability of co-occurrence was greatest for moderately related species and decreased among both distantly and closely related species. The increase in co-occurrence among moderately related species as compared to distantly related species is likely due to environmental filtering based on shared environmental tolerances.The pattern of reduced co-occurrence among closely-related species could be caused by interspecific competition, or allopatric speciation with insufficient time for re-expansion to secondary sympatry ,36,53. BPhaethornis hermits are widespread throughout the lowlands, but their elevation ranges rarely extend into the highlands [Environmental filtering, which results in co-occurrence of related species with similar traits, is well documented across multiple taxonomic groups , includiighlands .Hummingbirds compete for nectar resources and related species often use similar resources ,64. BiogAnolis lizards are less likely to occur in predicted suitable habitat when a morphologically similar species is present [Biogeographic patterns suggestive of competition have been reported in several other groups ,54. Sylv present . Recent  present , suggest present . In addi present ,73. Thes present . In our To investigate whether dispersal explains the lack of co-occurrence among very close relatives we tested the relationship between sister taxa and cost distance as a proxy for dispersal barriers. We found some evidence that older sister taxa are, on average, separated by smaller geographic barriers and tend to co-occur more frequently than younger sister taxa. This result could be interpreted as incomplete range filling , since aThere are several reasons why we hesitate to ascribe the pattern of reduced co-occurrence of closely related species wholly to insufficient time for dispersal. If dispersal limitation over evolutionary time was a driving force in community assembly, we would expect clades to be highly localized within areas surrounded by geographic barriers of unsuitable habitat . CompariThe role of niche conservatism in promoting co-occurrence of related species through environmental filtering is well documented . The mor


Remyelination therapy for multiple sclerosis (MS) is a rapidly emerging research area despite the fact that only limited success has been achieved so far in clinical trials. The extracellular matrix (ECM) is significantly altered in chronic MS lesions, which is believed to be an important remyelination-inhibiting factor. However, the ECM components have not been specifically targeted in current MS remyelinating trials. Multiple sclerosis (MS) is an autoimmune disorder of the central nervous system, characterized by inflammatory demyelination and progressive axonal loss. Demyelination has long been considered as the main pathologic feature of MS. Remyelination usually fails in chronic MS lesions despite the presence of oligodendrocyte precursor cells OPCs; . A majorThe extracellular environment, or extracellular matrix (ECM), is significantly altered inside MS plaques , which iJournal of Neuroscience has broadened our understanding of the role of fibronectin (Fn), another major ECM component, in MS de/remyelination. This study was developed based on the group’s previous investigations  OPCs. In the in vivo study, the authors demonstrated an unchanged percentage of MBP-positive cells, suggesting no effects on OPC differentiation; therefore, a similar percentagewise analysis  could potentially reveal whether GD1a affects OPC differentiation in vivo. Additionally, it remains to be confirmed whether the PLP mRNA upregulation could eventually lead to an enhanced myelin sheath formation. It is challenging to test this in vivo, Fn aggregation is only seen in the EAE model (not in the cuprizone or lysolecthin models), but the EAE model is not ideal for remyelination study because of ongoing inflammation and sporadic demyelinating lesions with unpredictable lesional site and timing; therefore, exogenous aggregated Fn had to be added in the cuprizone model in the highlighted study. Spontaneous aggregate clearance over time leads to only a transient aggregated Fn microenvironment in vivo, which makes it difficult to study ultimate remyelination. It may, therefore, be worthwhile to consider inducing EAE in a transgenic mouse line, which allows OPC labeling and lineage analysis (The recent paper  publisheigations , where tigations . By contigations  or diffeigations . Also, tanalysis .Next, As shown in In summary, the ECM comprises an extremely complex neural signaling network, with both pro- and counter-remyelinating components coexisting, and myelin-formation inhibitors being neuroprotectants. The predominant cellular signaling pathway mediating remyelination in MS is not well understood. However, recent advances in pre-clinical models have significantly improved our knowledge about the role of ECM in MS pathology, bringing us steps closer to its potential clinical applications.